Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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RNASE H-BASED ASSAYS UTILIZING MODIFIED RNA MONOMERS
CROSS-REFERENCE TO RELATED APPLICATIONS
[001] This application is a continuation in part of U.S. application No.
13/959,637 filed on
August, 5, 2013, which is a continuation in part of U.S. application No.
13/839,334, filed March
15, 2013, which is a continuation in part of U.S. application No. 12/507,142,
filed July 22, 2009,
which is a continuation in part of U.S. application No. 12/433,896, filed
April 30, 2009, which
claims benefit of priority to U.S. provisional application No. 61/049,204
filed on April 30, 2008,
which is incorporated by reference in its entirety.
SEQUENCE LISTING
[002] The instant application contains a Sequence Listing which has been
submitted in
ASCII format via EFS-Web and is hereby incorporated by reference in its
entirety. Said ASCII
copy, created on November 29, 2013, is named IDT01-004-PCT_5T25.txt, and is
101,119
bytes in size.
FIELD OF THE INVENTION
[003] This invention pertains to methods of cleaving a nucleic acid strand
to initiate,
assist, monitor or perform biological assays.
BACKGROUND OF THE INVENTION
[004] The specificity of primer-based amplification reactions, such as the
polymerase
chain reaction (PCR), largely depends on the specificity of primer
hybridization with a DNA
template. Under the elevated temperatures used in a typical amplification
reaction, the primers
ideally hybridize only to the intended target sequence and form primer
extension products to
produce the complement of the target sequence. However, amplification reaction
mixtures are
typically assembled at room temperature, well below the temperature needed to
insure primer
hybridization specificity. Under lower temperature conditions, the primers may
bind
non-specifically to other partially complementary nucleic acid sequences or to
other primers
and initiate the synthesis of undesired extension products, which can be
amplified along with
the target sequence. Amplification of non-specific primer extension products
can compete
with amplification of the desired target sequences and can significantly
decrease the efficiency
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of the amplification of the desired sequence. Non-specific amplification can
also give rise in
certain assays to a false positive result.
[005] One frequently observed type of non-specific amplification product in
PCR is a
template independent artifact of the amplification reaction known as "primer
dimers". Primer
dimers are double-stranded fragments whose length typically is close to the
sum of the two
primer lengths and are amplified when one primer is extended over another
primer. The
resulting duplex forms an undesired template which, because of its short
length, is amplified
efficiently.
[006] Non-specific amplification can be reduced by reducing the formation
of primer
extension products (e.g., primer dimers) prior to the start of the reaction.
In one method,
referred to as a "hot-start" protocol, one or more critical reagents are
withheld from the reaction
mixture until the temperature is raised sufficiently to provide the necessary
hybridization
specificity. In this manner, the reaction mixture cannot support primer
extension at lower
temperatures. Manual hot-start methods, in which the reaction tubes are opened
after the initial
high temperature incubation step and the missing reagents are added, are labor
intensive and
increase the risk of contamination of the reaction mixture.
[007] Alternatively, a heat sensitive material, such as wax, can be used to
separate or
sequester reaction components, as described in U.S. Pat. No. 5,411,876, and
Chou et al., 1992,
Nucl. Acids Res. 20(7):1717-1723. In these methods, a high temperature pre-
reaction
incubation melts the heat sensitive material, thereby allowing the reagents to
mix.
[008] Another method of reducing the formation of primer extension products
prior to the
start of PCR relies on the heat-reversible inactivation of the DNA polymerase.
U.S. Pat. Nos.
5,773,258 and 5,677,152, both incorporated herein by reference, describe DNA
polymerases
reversibly inactivated by the covalent attachment of a modifier group.
Incubation of the
inactivated DNA polymerase at high temperature results in cleavage of the
modifier-enzyme
bond, thereby releasing an active form of the enzyme. Non-covalent reversible
inhibition of a
DNA polymerase by DNA polymerase-specific antibodies is described in U.S. Pat.
Nos.
5,338,671, incorporated herein by reference.
[009] One objective of the present invention can be used, for example, to
address the
problem of carry-over cross contamination which is a significant concern in
amplification
reactions, especially PCR wherein a large number of copies of the amplified
product are
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produced. In the prior art, attempts have been made to solve this problem in a
number of ways.
For example, direct UV irradiation can effectively remove contaminating DNA
(Rys & Persing,
1993, J Clin Microbiol. 31(9):2356-60 and Sarkar & Sommer, 1990 Nature.
343(6253):27) but
the irradiation of the PCR reagents must take place before addition of
polymerase, primers, and
template DNA. Furthermore, this approach may be inefficient because the large
numbers of
mononucleotides present in the reaction will absorb much of the UV light. An
alternative, the
"TING method", incorporates dUTP into the amplified fragments to alter the
composition of
the product so that it is different from native, naturally occurring DNA
(Longo et al. 1990,
Gene, 93(1): 125-128). The enzyme Uracil-N-Glycosylase (TING) is added
together with the
other components of the PCR mixture. The TING enzyme will cleave the uracil
base from
DNA strands of contaminating amplicons before amplification, and render all
such products
unable to act as a template for new DNA synthesis without affecting the sample
DNA. The
TING enzyme is then heat-inactivated and PCR is then carried out. The
requirement for dUTP
and the TING enzyme adds significantly to the cost of performing PCR.
[010] Another objective of the present invention is to provide PCR assays
in which a
hot-start reaction is achieved through a coupled reaction sequence with a
thermostable RNase
H.
Ribonuclease Enzymes
[011] Ribonucleases (RNases) are enzymes that catalyze the hydrolysis of
RNA into
smaller components. The enzymes are present internally; in bodily fluids; on
the surface of
skin; and on the surface of many objects, including untreated laboratory
glasswear.
Double-stranded RNases are present in nearly all intracellular environments
and cleave
RNA-containing, double-stranded constructs. Single-stranded RNases are
ubiquitous in
extracellular environments, and are therefore extremely stable in order to
function under a wide
range of conditions.
[012] The RNases H are a conserved family of ribonucleases which are
present in all
organisms examined to date. There are two primary classes of RNase H: RNase H1
and RNase
H2. Retroviral RNase H enzymes are similar to the prokaryotic RNase Hi. All of
these
enzymes share the characteristic that they are able to cleave the RNA
component of an
RNA:DNA heteroduplex. The human and mouse RNase H1 genes are 78% identical at
the
amino acid level (Cerritelli, et al., (1998) Genomics, 53, 300-307). In
prokaryotes, the genes
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are named rnha (RNase H1) and rnhb (RNase H2). A third family of prokaryotic
RNases has
been proposed, rnhc (RNase H3) (Ohtani, et al. (1999) Biochemistry, 38, 605-
618).
[013] Evolutionarily, "ancient" organisms (archaeal species) in some cases
appear to
have only a single RNase H enzyme which is most closely related to the modern
RNase H2
enzymes (prokaryotic) (Ohtani, et al., J Biosci Bioeng, 88, 12-19). Exceptions
do exist, and the
archaeal Halobacterium has an RNase H1 ortholog (Ohtani, et al., (2004)
Biochem J, 381,
795-802). An RNase H1 gene has also been identified in Thermus thermophilus
(Itaya, et al.,
(1991) Nucleic Acids Res, 19, 4443-4449). RNase H2 enzymes appear to be
present in all
living organisms. Although all classes of RNase H enzymes hydrolyze the RNA
component of
an RNA:DNA heteroduplex, the substrate and co-factor requirements are
different. For
example, the Type II enzymes utilize Mg', Mn", Co" (and sometimes Ni") as
cofactor,
while the Type I enzymes require Mg" and can be inhibited by Mn" ions. The
reaction
products are the same for both classes of enzymes: the cleaved products have a
3'-OH and
5'-phosphate (See Figure 1). RNase III class enzymes which cleave RNA:RNA
duplexes (e.g.,
Dicer, Ago2, Drosha) result in similar products and contain a nuclease domain
with similarity
to RNase H. Most other ribonucleases, and in particular single stranded
ribonucleases, result in
a cyclic 2',3'-phosphate and 5'-OH products (see Figure 2).
Type I RNase H
[014] E. coli RNase H1 has been extensively characterized. A large amount
of work on
this enzyme has been carried out, focusing on characterization of substrate
requirements as it
impacts antisense oligonucleotide design; this has included studies on both
the E. coli RNase
H1 (see Crooke, et al., (1995) Biochem J, 312 (Pt 2), 599-608; Lima, et al.,
(1997) J Biol Chem,
272, 27513-27516; Lima, et al., (1997) Biochemistry, 36, 390-398; Lima, et
al., (1997) J Biol
Chem, 272, 18191-18199; Lima, et al., (2007) Mol Pharmacol, 71, 83-91; Lima,
et al., (2007)
Mol Pharmacol, 71, 73-82; Lima, et al., (2003) J Biol Chem, 278, 14906-14912;
Lima, et al.,
(2003) J Biol Chem, 278, 49860-49867) and the Human RNase H1 (see Wu, et al.,
(1998)
Antisense Nucleic Acid Drug Dev, 8, 53-61; Wu, et al., (1999) J Biol Chem,
274, 28270-28278;
Wu, et al., (2001) J Biol Chem, 276, 23547-23553). In tissue culture,
overexpression of human
RNase H1 increases potency of antisense oligos (AS0s) while knockdown of RNase
H1 using
either siRNAs or ASOs decreases potency of antisense oligonucleotides.
[015] Type I RNase H requires multiple RNA bases in the substrate for full
activity. A
DNA/RNA/DNA oligonucleotide (hybridized to a DNA oligonucleotide) with only 1
or 2
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RNA bases is inactive. With E. coil RNase H1 substrates with three consecutive
RNA bases
show weak activity. Full activity was observed with a stretch of four RNA
bases (Hogrefe, et
al., (1990) J Biol Chem, 265, 5561-5566). An RNase H1 was cloned from Thermus
thermophilus in 1991 which has only 56% amino acid identity with the E. coil
enzyme but
which has similar catalytic properties (Itaya, et al., (1991) Nucleic Acids
Res, 19, 4443-4449).
This enzyme was stable at 65 C but rapidly lost activity when heated to 80 C.
[016] The human RNase H1 gene (Type I RNase H) was cloned in 1998
(Genomics, 53,
300-307 and Antisense Nucleic Acid Drug Dev, 8, 53-61). This enzyme requires a
5 base RNA
stretch in DNA/RNA/DNA chimeras for cleavage to occur. Maximal activity was
observed in
1 mM Mg buffer at neutral pH and Mn" ions were inhibitory (J Biol Chem, 274,
28270-28278). Cleavage was not observed when 2'-modified nucleosides (such as
2'-0Me,
2'-F, etc.) were substituted for RNA.
[017] Three amino acids (Asp-10, Glu-48, and Asp-70) make up the catalytic
site of E.
coli RNase H1 which resides in the highly conserved carboxy-terminal domain of
the protein
(Katayanagi, et al., (1990) Nature, 347, 306-309); this domain has been
evaluated by both site
directed mutagenesis and crystal structure determination. The same amino acids
are involved
in coordination of the divalent ion cofactor.
[018] Interestingly, 2'-modification of the substrate duplex alters the
geometry of the
helix and can adversely affect activity of RNase Hi. 2'-0-(2-methoxy)ethyl
(MOE)
modifications flanking the RNA segment reduce cleavage rates, presumably due
to alterations
in the sugar conformation and helical geometry. Locked nucleic acid (LNA)
bases perturb
helical geometry to a greater degree and impacted enzyme activity to a greater
extent (Mol
Pharmacol, 71, 83-91 and Mol Pharmacol, 71, 73-82). Damha (McGill University)
has
studied the effects of 2'-F modified nucleosides (2'-deoxy-2'-fluoro-b-D-
ribose) when present
in the substrate duplex and finds that this group cannot be cleaved by RNase
H1 (Yazbeck, et
al., (2002) Nucleic Acids Res, 30, 3015-3025). Formulas A and B illustrate the
two different
mechanisms that have been proposed for RNase H1 cleavage, both of which
require
participation of the 2'0H group.
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A
0--,
õ a- -14 "
.`"
S.
tf
Cr
.4:;* .......................................
(7.
e.
Formulas A and B
[019] Damha's studies are consistent with the known active site of the
enzyme, wherein
the reaction mechanism involves the 2' -OH group. The enzyme active site
resides within a
cluster of lysine residues which presumably contribute to electrostatic
binding of the duplex.
Interaction between the binding surface and negatively charged phosphate
backbone is
believed to occur along the minor grove of the RNA:DNA heteroduplex (Nakamura,
et al.,
(1991) Proc Natl Acad Sci USA, 88, 11535-11539); changes in structure that
affect the minor
groove should therefore affect interactions between the substrate and the
active site. For
example, the minor groove width is 7.5 A in a B-form DNA:DNA duplex, is 11 A
in a pure
A-form RNA:RNA duplex, and is 8.5 A in the hybrid A-form duplex of an RNA:DNA
duplex
(Fedoroff et al., (1993) J Mol Biol, 233, 509-523). 2' -modifications protrude
into the minor
groove, which may account for some of the behavior of these groups in reducing
or eliminating
activity of modified substrates for cleavage by RNase Hl. Even a 2'-F
nucleoside, which is the
most "conservative" RNA analog with respect to changing chemical structure,
adversely
affects activity.
Type II RNase H
[020] The human Type II RNase H was first purified and characterized by
Eder and
Walder in 1991 (Eder, et al., (1991) J Biol Chem, 266, 6472-6479). This enzyme
was initially
designated human RNase H1 because it had the characteristic divalent metal ion
dependence of
what was then known as Class I RNases H. In the current nomenclature, it is a
Type II RNase
H enzyme. Unlike the Type I enzymes which are active in Mg but inhibited by
Mn" ions, the
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Type II enzymes are active with a wide variety of divalent cations. Optimal
activity of human
Type II RNase H is observed with 10 mM Mg', 5 mM Co', or 0.5 mM Mn'.
[021] Importantly, the substrate specificity of the Type II RNase H
(hereafter referred to
as RNase H2) is different from RNase Hi. In particular, this enzyme can cleave
a single
ribonucleotide embedded within a DNA sequence (in duplex form) (Eder, et al.,
(1993)
Biochimie, 75, 123-126). Interestingly, cleavage occurs on the 5' side of the
RNA residue (See
Figure 3). See a recent review by Kanaya for a summary of prokaryotic RNase H2
enzymes
(Kanaya (2001) Methods Enzymol, 341, 377-394).
[022] The E. coli RNase H2 gene has been cloned (Itaya, M. (1990) Proc Natl
Acad Sci U
SA, 87, 8587-8591) and characterized (Ohtani, et al., (2000) J Biochem
(Tokyo), 127, 895-899).
Like the human enzyme, the E. coli enzyme functions with Mn" ions and is
actually more
active with manganese than magnesium.
[023] RNase H2 genes have been cloned and the enzymes characterized from a
variety of
eukaryotic and prokaryotic sources. The RNase H2 from Pyrococcus kodakaraensis
(KOD1)
has been cloned and studied in detail (Haruki, et al., (1998) J Bacteriol,
180, 6207-6214;
Mukaiyama, et al., (2004) Biochemistry, 43, 13859-13866). The RNase H2 from
the related
organism Pyrococcus furious has also been cloned but has not been as
thoroughly
characterized (Sato, et al., (2003) Biochem Biophys Res Commun, 309, 247-252).
[024] The RNase H2 from Methanococcus jannaschii was cloned and
characterized by
Lai (Lai, et al., (2000) Structure, 8, 897-904; Lai et al., (2003)
Biochemistry, 42, 785-791).
Isothermal titration calorimetry was used to quantitatively measure metal ion
binding to the
enzyme. They tested binding of Mn", Mg', Ca', and Bo" and in all cases
observed a 1:1
molar binding ratio, suggesting the presence of only a single divalent metal
ion cofactor in the
enzyme's active site. The association constant for Mn was 10-fold higher than
for Mg'.
Peak enzyme activity was seen at 0.8 mM MnC12.
[025] Nucleic acid hybridization assays based on cleavage of an RNA-
containing probe
by RNase H such as the cycling probe reaction (Walder et al., U.S. Pat. No.
5,403,711) have
been limited in the past by background cleavage of the oligonucleotide by
contaminating
single-stranded ribonucleases and by water catalyzed hydrolysis facilitated by
Mg2+ and other
divalent cations. The effect of single-stranded ribonucleases can be mitigated
to a certain
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degree by inhibitors such as RNasin that block single-stranded ribonucleases
but do not
interfere with the activity of RNase H.
[026] Single-stranded ribonucleases cleave 3' of an RNA residue, leaving a
cyclic
phosphate group at the 2' and 3' positions of the ribose (See FIG. 2). The
same products are
produced by spontaneous water catalyzed hydrolysis. In both cases, the cyclic
phosphate can
hydrolyze further forming a 3 '-monophosphate ester in the enzyme catalyzed
reaction, or a
mixture of the 3'- and 2'-monophosphate esters through spontaneous hydrolysis.
The
difference between the cleavage products formed by RNase H (FIG. 1) and those
formed by
nonspecific cleavage of the probe (FIG. 2) provides a basis for distinguishing
between the two
pathways. This difference is even more pronounced when comparing cleavage by
RNase H2
and single-stranded ribonucleases with substrates having only a single RNA
residue. In that
case, RNase H2 and single-stranded ribonucleases attack at different positions
along the
phosphate backbone (See Figure 3).
[027] RNase H has been used as a cleaving enzyme in cycling probe assays,
in PCR
assays (Han et al., U.S. Patent No. 5,763,181; Sagawa et al., U.S. Pat. No.
7,135,291; and
Behlke and Walder, U.S. Pat. App. No. 20080068643) and in polynomial
amplification
reactions (Behlke et al., U.S. Patent No. 7,112,406). Despite improvements
offered by these
assays, there remain considerable limitations. The PCR assays utilize a hot-
start DNA
polymerase which adds substantially to the cost. Moreover, each time an
alternative DNA
polymerase is required a new hot-start version of the enzyme must be
developed. In addition,
the utility of these various assays has been limited by undesirable cleavage
of the
oligonucleotide probe or primer used in the reaction, including water and
divalent metal ion
catalyzed hydrolysis 3' to RNA residues, hydrolysis by single-stranded
ribonucleases and
atypical cleavage reactions catalyzed by Type II RNase H enzymes at positions
other than the
5'-phosphate of an RNA residue. The present invention overcomes these
limitations and offers
further advantages and new assay formats for use of RNase H in biological
assays.
[028] The current invention provides novel biological assays that employ
RNase H
cleavage in relation to nucleic acid amplification, detection, ligation,
sequencing, and synthesis.
Additionally, the invention provides new assay formats to utilize cleavage by
RNase H and
novel oligonucleotide substrates for such assays. The compounds, kits, and
methods of the
present invention provide a convenient and economic means of achieving highly
specific
primer-based amplification reactions that are substantially free of
nonspecific amplification
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impurities such as primer dimers. The methods and kits of the present
invention avoid the need
for reversibly inactivated DNA polymerase and DNA ligase enzymes.
BRIEF SUMMARY OF THE INVENTION
[029] One objective of the present invention is to enable hot start
protocols in nucleic acid
amplification and detection assays including but not limited to PCR, OLA
(oligonucleotide
ligation assays), LCR (ligation chain reaction), polynomial amplification and
DNA sequencing,
wherein the hot start component is a thermostable RNase H or other nicking
enzyme that gains
activity at the elevated temperatures employed in the reaction. Such assays
employ a modified
oligonucleotide of the invention that is unable to participate in the reaction
until it hybridizes to
a complementary nucleic acid sequence and is cleaved to generate a functional
5'- or 3'-end.
Compared to the corresponding assays in which standard unmodified DNA
oligonucleotides
are used the specificity is greatly enhanced. Moreover the requirement for
reversibly
inactivated DNA polymerases or DNA ligases is eliminated.
[030] In the case of assays involving primer extension (e.g., PCR,
polynomial
amplification and DNA sequencing) the modification of the oligonucleotide
inhibiting activity
is preferably located at or near the 3'-end. In some embodiments where the
oligonucleotides
are being used as primers, the oligonucleotide inhibiting activity may be
positioned near the 3'
end of the oligonucleotide, e.g., up to about 10 bases from the 3' end of the
oligonucleotide of
the invention. In other embodiments, the oligonucleotide inhibiting activity
may be positioned
near the 3' end, e.g., about 1-6 bases from the 3' end of the oligonucleotide
of the invention. In
other embodiments, the oligonucleotide inhibiting activity may be positioned
near the 3' end,
e.g., about 1-5 bases from the 3' end of the oligonucleotide of the invention.
In other
embodiments, the oligonucleotide inhibiting activity may be positioned near
the 3' end,
e.g., about 1-3 bases from the 3' end of the oligonucleotide of the invention.
In other
embodiments, the precise position (i.e., number of bases) from the 3' end
where the
oligonucleotide inhibiting activity may be positioned will depend upon factors
influencing the
ability of the oligonucleotide primer of the invention to hybridize to a
shortened complement of
itself on the target sequence (i.e., the sequence for which hybridization is
desired). Such
factors include but are not limited to Tm, buffer composition, and annealing
temperature employed in the reaction(s).
[031] For ligation assays (e.g., OLA and LCR) the modification inhibiting
activity may be
located at or near either the 3'- or 5'-end of the oligonucleotide. In other
embodiments, for
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ligation assays, modification inhibitory activity, if used, is preferably
placed within the domain
that is 3' to the cleavable RNA base in the region that is removed by probe
cleavage. In other
embodiments, for ligation assays, C3 spacers may be positioned close to the
RNA base in the
oligonucleotide probes of the invention to improve specificity that is helpful
for improving
mismatch discrimination. In other embodiments, in an OLA assay, where readout
depends
upon a PCR assay to amplify the product of a ligation event, any blocking
group may be placed
in the domain of the oligonucleotide of the invention that is removed by RNase
H cleavage. In
such embodiments, in an OLA assay where readout depends upon a PCR assay to
amplify the
product of a ligation event, the precise position of the blocking group in the
RNase H cleavable
domain may be adjusted to alter specificity for cleavage and precise placement
of the blocking
group relative to the cleavable RNA bases may alter the amount of enzyme
needed to achieve
optimal cleavage rates.
[032] Yet a further objective of the present invention is to provide novel
modifications of
oligonucleotides to interfere with primer extension and ligation.
[033] Yet a further objective of the present invention is to provide
modifications of
oligonucleotides that prevent the oligonucleotide from serving as a template
for DNA synthesis
and thereby interfere with PCR.
[034] Yet a further objective of the invention is to provide modified
oligonucleotide
sequences lacking RNA that are cleaved by RNase H. In one such embodiment, the
oligonucleotide contains a single 2'-fluoro residue and cleavage is mediated
by a Type II
RNase H enzyme. In a more preferred embodiment the oligonucleotide contains
two adjacent
2'-fluoro residues.
[035] Yet a further objective of the present invention is to provide
oligonucleotides for
use in the above mentioned assays that are modified so as to inhibit undesired
cleavage
reactions including but not limited to water and divalent metal ion catalyzed
hydrolysis 3' to
RNA residues, hydrolysis by single-stranded ribonucleases and atypical
cleavage reactions
catalyzed by Type II RNase H enzymes at positions other than the 5'-phosphate
of an RNA
residue (see Figure 3). In one such embodiment the 2'-hydroxy group of an RNA
residue is
replaced with an alternative functional group such as fluorine or an alkoxy
substituent (e.g.,
0-methyl). In another such embodiment the phosphate group 3' to an RNA residue
is replaced
with a phosphorothioate or a dithioate linkage. In yet another embodiment the
oligonucleotide
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is modified with nuclease resistant linkages further downstream from the 3'-
phosphate group
of an RNA residue or on the 5'-side of an RNA residue to prevent aberrant
cleavage by RNase
H2. Nuclease resistant linkages useful in such embodiments include
phosphorothioates,
dithioates, methylphosphonates, and abasic residues such as a C3 spacer.
Incorporation of
such nuclease resistant linkages into oligonucleotide primers used in PCR
assays of the present
invention has been found to be particularly beneficial (see Examples 25, 27
and 28).
[036] Yet a further objective of the invention is to provide
oligonucleotides for use in the
above-mentioned assays that are modified at positions flanking the cleavage
site to provide
enhanced discrimination of variant alleles. Such modifications include but are
not limited to
2'-0-methyl RNA residues and secondary mismatch substitutions (see Example
23).
[037] Yet a further objective is to provide oligonucleotides and assay
formats for use in
the present invention wherein cleavage of the oligonucleotide can be measured
by a change in
fluorescence. In one such embodiment a primer cleavable by RNase H is labeled
with a
fluorophore and a quencher and the assay is monitored by an increase in
fluorescence (see
Examples 19-21).
[038] Yet a further objective of the invention is to provide RNase H
compositions and
protocols for their use in which the enzyme is thermostable and has reduced
activity at lower
temperatures.
[039] In yet a further embodiment a Type II RNase H is employed in a
cycling probe
reaction in which the RNA residue in the probe is replaced with a 2'-fluoro
residue. In a more
preferred embodiment a probe with two adjacent 2'-fluoro residues is used.
[040] Many of the aspects of the present invention relating to primer
extension assays,
ligation assays and cycling probe reactions are summarized in Tables 1, 2, and
3, respectively.
[041] In yet a further embodiment of the invention Type II RNase H enzymes
are used in
novel methods for DNA sequencing.
[042] In yet a further embodiment of the invention Type II RNase H enzymes
are used in
novel methods for DNA synthesis.
[043] Yet a further objective is to increase the ability of the assays of
the present invention
to distinguish the presence of a base mismatch between the primer sequence and
the target
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nucleic acid by providing sets of overlapping blocked primers. In one
embodiment of the
present invention, the RNA base of the blocked-cleavable primer is positioned
at the site of a
single base polymorphism (the SNP). It is readily appreciated by one with
skill in the art that a
primer which overlays a polymorphic site can be made specific to the top or
bottom (sense or
antisense) strand of a duplex DNA target nucleic acid. In one embodiment of
the present
invention a single blocked-cleavable primer is employed having the RNA residue
positioned
directly at the site of the SNP (single nucleotide polymorphism) so that
hybridization to a target
having a perfect match with the primer results in efficient cleavage by RNase
H2 whereas
hybridization to a target having a mismatch at this site results in
inefficient cleavage by RNase
H2. When paired with a second unmodified primer that is positioned a suitable
distance from
the first primer, an allele-specific amplification reaction can be performed
using PCR where a
significant delay is observed in detection of product when using a mismatched
target compared
with a matched target. In another embodiment of the invention, two blocked-
cleavable primers
are paired, one corresponding to the top strand and the second corresponding
to the bottom
strand, with the SNP site positioned at the RNA base. The two primers overlap
and, following
activation by RNase H2 cleavage, function as a PCR primer pair and
preferentially amplify a
matched target over a mismatched target. By incorporating upstream and
downstream blocked
primers that overlap at the mutation site, the selectivity of the assay is
further enhanced.
[044] In
another object of the invention, a method of improving specificity during
amplification of a target DNA sequence is provided. The method includes four
steps. The first
step includes providing a reaction mixture. The reaction mixture includes: (i)
an
oligonucleotide primer having a cleavage domain, which is cleavable by an
RNase H enzyme,
positioned 5' of a blocking group, said blocking group linked at or near the
3'-end of the
oligonucleotide primer wherein said blocking group prevents primer extension
and/or inhibits
the oligonucleotide primer from serving as a template for DNA synthesis; (ii)
a sample nucleic
acid that may or may not the target sequence; (iii) a DNA polymerase, and
(iv) an RNase H enzyme wherein said RNase H enzyme is thermostable and has at
least a
10-fold decrease in activity at 30 C as compared to 70 C. The second step
includes
hybridizing the oligonucleotide primer to the target DNA sequence to form a
double-stranded
substrate. The third step includes cleaving the hybridized oligonucleotide
primer with said
RNase H enzyme at a cleavage site within or adjacent to the cleavage domain to
remove the
blocking group from the oligonucleotide primer. The fourth step includes
extending the
oligonucleotide primer with the DNA polymerase. Th reaction mixture includes a
concentration of a divalent cation such that a ACp of at least about 5.0 or
greater or about 50%
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or greater is obtained for the amplification of the target DNA as adjudged by
a 3 '-mismatch
discrimination assay.
[045] In another object of the invention, a method of amplifying a target
DNA sequence is
provided. The method includes four steps. The first step includes providing a
reaction mixture.
The reaction mixture includes: (i) an oligonucleotide primer having a cleavage
domain, which
is cleavable by an RNase H enzyme, positioned 5' of a blocking group, said
blocking group
linked at or near the 3'-end of the oligonucleotide primer wherein said
blocking group prevents
primer extension and/or inhibits the oligonucleotide primer from serving as a
template for
DNA synthesis; (ii) a sample nucleic acid that may or may not the target
sequence; (iii) a
DNA polymerase, and (iv) an RNase H enzyme wherein said RNase H enzyme is
thermostable
and has at least a 10 -fold decrease in activity at 30 C as compared to 70 C.
The second step
includes hybridizing the oligonucleotide primer to the target DNA sequence to
form a
double-stranded substrate. The third step includes cleaving the hybridized
oligonucleotide
primer with said RNase H enzyme at a cleavage site within or adjacent to the
cleavage domain
to remove the blocking group from the oligonucleotide primer. The fourth step
includes
extending the oligonucleotide primer with the DNA polymerase. The reaction
mixture includes
a divalent cation and a non-ionic detergent comprising polyethylene glycol
hexadecyl ether.
[046] In another object of the invention, a kit for performing
amplification of a target
DNA sequence is provided. The kit has a reaction buffer that includes a metal
salt comprising a
divalent cation and associated counterion. The divalent cation comprises Mg',
and the
reaction buffer provides a final concentration of the metal salt no greater
than about 2.0 mM
free Mg in the reaction mixture for performing amplification of the target
DNA.
BRIEF DESCRIPTION OF THE FIGURES
[047] Figure 1 depicts the cleavage pattern that occurs with an RNase H
enzyme on a
substrate containing multiple RNA bases.
[048] Figure 2 depicts the cleavage pattern that occurs with a single-
stranded
ribonuclease enzyme or through water catalyzed hydrolysis, wherein the end-
product results in
a cyclic phosphate group at the 2' and 3' positions of the ribose.
[049] Figure 3 depicts the cleavage sites for RNase H2 and single-stranded
ribonucleases
on a substrate containing a single RNA base.
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[050] Figures 4A and 4B are photographs of SDS 10% polyacrylamide gels that
illustrate
the induced protein produced from five Archaeal RNase H2 synthetic genes.
Figure 4A shows
induced protein for Pyrococcus furiosus and Pyrococus abyssi. Figure 4B shows
induced
protein for Methanocaldococcus jannaschii, Sulfolobus solfataricus, Pyrococcus
kodadarensis.
[051] Figure 5 shows a Coomassie Blue stained protein gel showing pure,
single bands
after purification using nickel affinity chromatography of recombinant His tag
RNase H2
proteins.
[052] Figure 6 shows a Western blot done using anti-His tag antibodies
using the protein
gel from FIG. 5.
[053] Figure 7 is a photograph of a gel that shows the digestion of a
duplex, containing a
chimeric 11 DNA ¨ 8 RNA ¨ 11 DNA strand and a complementary DNA strand, by
recombinant RNase H2 enzymes from Pyrococcus kodakaraensis, Pyrococcus
furiosus, and
Pyrococcus abyssi.
[054] Figures 8A and 8B are photographs of gels that show the digestion of
a duplex,
containing a chimeric 14 DNA ¨ 1 RNA ¨ 15 DNA strand and a complementary DNA
strand,
by recombinant RNase H2 enzymes from Pyrococcus abyssi, Pyrococcus furiosus,
and
Methanocaldococcus jannaschii (FIG. 8A) and Pyrococcus kodakaraensis (FIG.
8B).
[055] Figure 9 shows the effects of incubation at 95 C for various times
on the activity of
the Pyrococcus abyssi RNase H2 enzyme.
[056] Figure 10 is a photograph of a gel that shows the relative amounts of
cleavage of a
single ribonucleotide-containing substrate by Pyrococcus abyssi RNase H2 at
various
incubation temperatures.
[057] Figure 11 is a graph showing the actual quantity of substrate cleaved
in the gel from
FIG. 10.
[058] Figure 12 is a photograph of a gel that shows cleavage by Pyrococcus
abysii RNase
H2 of various single 2' modified substrates in the presence of different
divalent cations.
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[059] Figure 13 is a photograph of a gel that shows cleavage by Pyrococcus
abyssi RNase
H2 of single 2'-fluoro or double 2'-fluoro (di-fluoro) modified substrates.
The divalent cation
present was Mn".
[060] Figure 14 is a graph quantifying the relative cleavage by Pyrococcus
abyssi RNase
H2 of all 16 possible di-fluoro modified substrates.
[061] Figure 15 is a graph quantifying the relative cleavage by Pyrococcus
abyssi RNase
H2 of rN substrates with a variable number of 3' DNA bases (i.e., number of
DNA bases on the
3' side of the RNA residue).
[062] Figure 16 is a graph quantifying the relative cleavage by Pyrococcus
abyssi RNase
H2 of rN substrates with a variable number of 5' DNA bases (i.e., number of
DNA bases on the
5' side of the RNA residue).
[063] Figure 17 is a graph quantifying the relative cleavage by Pyrococcus
abyssi RNase
H2 of di-fluoro substrates with a variable number of 3' DNA bases (i.e.,
number of DNA bases
on the 3' side of the fUfC residues).
[064] Figure 18 is a reaction schematic of RNase H2 activation of blocked
PCR primers.
[065] Figure 19 is a photograph of a gel that shows the products of an end
point PCR
reaction performed with a single rU-containing blocked primer. The suffix 2D,
3D, etc.
represents the number of DNA bases between the rU residues and the 3 '-end of
the primer. The
primer is blocked with a dideoxy C residue.
[066] Figures 20A-B are PCR amplification plots for a 340 bp amplicon
within the human
HRAS gene, using both unmodified and blocked rN primers, without RNase H2
(20A) and
with RNase H2 (20B). Cycle number is shown on the X-axis and relative
fluorescence
intensity is shown on the Y-axis.
[067] Figures 21A-B are PCR amplification plots for a 184 bp amplicon
within the human
ETS2 gene, using both unmodified and blocked rN primers, without RNase H2
(21A) and with
RNase H2 (21B). Cycle number is shown on the X-axis and relative fluorescence
intensity is
shown on the Y-axis.
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[068] Figures 22A-B are PCR amplification plots for a synthetic 103 bp
amplicon, using
both unmodified and 3'-fN modified primers, without RNase H2 (22A) and with
RNase H2
(22B).
[069] Figure 23A shows HPLC traces of a rN primer containing a single
phosphorothioate intemucleoside modification (SEQ ID NO. 192). The top panel
shows the
original synthesis product demonstrating resolution of the two isomers. The
middle panel is
the purified Rp isomer and the bottom panel is the purified Sp isomer.
[070] Figure 24 shows the relationship between RNase H2 versus RNase A
enzymatic
cleavage with substrates having (SEQ ID NOS 302 and 103, respectively, in
order of
appearance) having a single RNA base and different phosphorothioate
stereoisomers.
[071] Figure 25 shows a photograph of a polyacrylamide gel used to separate
products
from PCR reactions done using standard and blocked/cleavable primers on a HCV
amplicon
showing that use of standard primers results in formation of undesired small
primer-dimer
species while use of blocked primers results in specific amplification of the
desired product.
The nucleic acids were imaged using fluorescent staining and the image was
inverted for
clarity.
[072] Figure 26 is a graph quantifying the relative cleavage by Pyrococcus
abyssi RNase
H2 of a radiolabeled rC containing substrate in buffer containing different
detergents at
different concentrations (expressed as % vol:vol).
[073] Figure 27 is a reaction schematic of RNase H2 activation of
fluorescence-quenched
(F/Q) blocked PCR primers.
[074] Figure 28 is an amplification plot showing the fluorescence signal
resulting from
use of unblocked primers with a fluorescence-quenched dual-labeled probe (DLP)
compared
with a blocked fluorescence-quenched cleavable primer for a 103 base synthetic
amplicon.
Cycle number is shown on the X-axis and relative fluorescence intensity is
shown on the
Y-axis.
[075] Figure 29 is an amplification plot showing the fluorescence signal
resulting from
use of a F/Q configuration blocked fluorescence-quenched cleavable primer
compared with a
Q/F configuration blocked fluorescence-quenched cleavable primer for a 103
base synthetic
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amplicon. Cycle number is shown on the X-axis and relative fluorescence
intensity is shown
on the Y-axis.
[076] Figure 30 is an amplification plot showing the fluorescence signal
resulting from
use of F/Q configuration blocked fluorescence-quenched cleavable primers to
distinguish
DNA templates that differ at a single base within the SMAD 7 gene. Panel (A)
shows results
from the FAM channel where the FAM-labeled "C" allele probe was employed.
Panel "B"
shows results from the HEX channel wherein the HEX-labeled "T" allele probe
was employed.
Cycle number is shown on the X-axis and relative fluorescence intensity is
shown on the
Y-axis.
[077] Figure 31 is a reaction schematic of RNase H2 cleavage of
fluorescence-quenched
(FQT) primer used in a primer probe assay. The Primer Domain is complementary
to the target
nucleic acid and serves to primer DNA synthesis. The
Reporter Domain is
non-complementary to target and contains a RNA base positioned between a
reporter dye and a
quencher group. The Reporter Domain remains single-stranded until conversion
to
double-stranded form during PCR where this domain now serves as template.
Conversion to
double-stranded form converts the Reporter Domain into a substrate for RNase
H2; cleavage
by RNase H2 separates reporter from quencher and is a detectable event.
[078] Figure 32 shows amplification plots of qPCR reactions done with
primers specific
for the human Drosha gene using HeLa cell cDNA. A) Reactions performed using
unmodified
primers and a fluorescence-quenched dual-labeled probe (DLP), 5'-nuclease
assay format.
The reaction was performed with or without template (HeLa cDNA) as indicated.
B) Reactions
performed using a fluorescence-quenched FQT For primer and an unmodified Rev
primer in a
primer-probe assay format. Reactions were performed with or without the
addition of RNase
H2 as indicated. Cycle number is shown on the X-axis and relative fluorescence
intensity is
shown on the Y-axis.
[079] Figure 33 shows the sequences of cleavable-blocked primers that are
either perfect
match or contain a mismatch at position +2 relative to the single RNA base (2
bases 3'- to the
ribonucleotide). SMAD7 target sequences at SNP site rs4939827 are aligned
below the
primers to indicate how this strategy results in the presence of a single
mismatch when primers
hybridize with one allele vs. a double mismatch when hybridize with the second
allele. DNA
bases are uppercase, RNA bases are lowercase, and SpC3 is a Spacer C3
modification. Figure
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33 discloses SEQ ID NOS 231-232, 234, 233, 303, 235-236, 238, 237 and 303,
respectively, in
order of appearance.
[080] Figure 34 is a graph that shows the relative functional activity of
different
oligonucleotide compositions to prime DNA synthesis in a linear primer
extension reaction.
[081] Figure 35 shows the scheme for performing cycles of DNA sequencing by
ligation
using RNase H2 cleavable ligation probes Figure 35 discloses the "3'-
AGTCCAGGTCA"
sequence as SEQ ID NO: 304.
[082] Figure 36 shows the scheme for hybridization, ligation, and
subsequent cleavage by
RNase H2 of RNA-containing cleavable ligation probes of a set of specific
exemplary
synthetic sequences (SEQ ID NOS 253, 255, 254, 256, 257-259, 305, 258, 306 and
258,
respectively, in order of appearance).
[083] Figure 37 shows a photograph of a polyacrylamide gel used to separate
products
from ligation reactions done using cleavable ligation probes on a synthetic
template showing
that the 9mer probes are efficiently ligated to the acceptor nucleic acid
(ANA) and that the
ligation product is efficiently cleaved by RNase H2, leaving an ANA species
that is lengthened
by one base. The nucleic acids were imaged using fluorescent staining and the
image was
inverted for clarity.
[084] Figure 38 shows the scheme for hybridization and ligation of RNA-
containing
cleavable ligation probes containing either three or four 5-nitroindole
residues. Figure 38
discloses SEQ ID NOS. 257, 260, 307, 258, 308 and 258, respectively, in order
of appearance.
[085] Figure 39 shows a photograph of a polyacrylamide gel used to separate
products
from ligation reactions done using cleavable ligation probes on a synthetic
template showing
that an 8mer probe containing three 5-nitroindole (3x5NI) bases is efficiently
ligated to an
acceptor nucleic acid (ANA) whereas an 8mer probe containing four 5-
nitroindole (4x5NI)
bases is not. The nucleic acids were imaged using fluorescent staining and the
image was
inverted for clarity.
[086] Figure 40 shows a photograph of a polyacrylamide gel used to separate
ligation
products from reactions done using cleavable ligation probes on a synthetic
template showing
that an 8mer probe containing a single fixed DNA base at the 5'-end, four
random bases, and 3
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universal base 5-nitroindoles can specifically ligate to the target as
directed by the single fixed
DNA base.
[087] Figure 41 shows the scheme for a traditional oligonucleotide ligation
assay (OLA).
Panel A shows the three oligonucleotides needed to interrogate a two allele
target system.
Panel B shows the steps involved in making a ligation product.
[088] Figure 42 shows the scheme for the RNase H2 cleavable oligonucleotide
ligation
assay (OLA) of the present invention. Panel A shows the four oligonucleotides
needed to
interrogate a two allele target system. Panel B shows the steps involved in
making a ligation
product using the RNase H2 method. Panel C illustrates how this method tests
the identity of
the base polymorphism twice.
[089] Figure 43 shows alignment of sequences (SEQ ID NOS 268, 309-310, 309,
300,
310, 309, 311, 309 and 311-312, respectively, in order of appearance) used in
the present
Example during each step of the RNase H2 cleavable probe OLA using
fluorescence
microbeads and a Luminex L100 system to detect the ligation products.
[090] Figure 44 is a chart that shows the resulting fluorescent signal
detected by a
Luminex L100 system to assess identity of the reaction products generated from
the RNase H2
allelic discrimination OLA shown in Figure 43.
[091] Figure 45 is a set of schematic figures outlining the single blocked-
cleavable primer
approach for the "For" orientation is shown in Figure 45A and for the "Rev"
orientation in
Figure 45B. Figure 45C is a schematic outlining the dual blocked-cleavable
primer approach.
DETAILED DESCRIPTION OF THE INVENTION
[092] The current invention provides novel nucleic acid compounds having a
cleavage
domain and a 3' or 5' blocking group. These compounds offer improvements to
existing
methods for nucleic acid amplification, detection, ligation, sequencing and
synthesis. New
assay formats comprising the use of these novel nucleic acid compounds are
also provided.
Definitions
[093] To aid in understanding the invention, several terms are defined
below.
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[094] The terms "nucleic acid" and "oligonucleotide," as used herein, refer
to
polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides
(containing
D-ribose), and to any other type of polynucleotide which is an N glycoside of
a purine or
pyrimidine base. There is no intended distinction in length between the terms
"nucleic acid",
"oligonucleotide" and "polynucleotide", and these terms will be used
interchangeably. These
terms refer only to the primary structure of the molecule. Thus, these terms
include double-
and single-stranded DNA, as well as double- and single-stranded RNA. For use
in the present
invention, an oligonucleotide also can comprise nucleotide analogs in which
the base, sugar or
phosphate backbone is modified as well as non-purine or non-pyrimidine
nucleotide analogs.
[095] Oligonucleotides can be prepared by any suitable method, including
direct
chemical synthesis by a method such as the phosphotriester method of Narang et
al., 1979,
Meth. Enzymol. 68:90-99; the phosphodiester method of Brown et al., 1979,
Meth. Enzymol.
68:109-151; the diethylphosphoramidite method of Beaucage et al., 1981,
Tetrahedron Lett.
22:1859-1862; and the solid support method of U.S. Pat. No. 4,458,066, each
incorporated
herein by reference. A review of synthesis methods of conjugates of
oligonucleotides and
modified nucleotides is provided in Goodchild, 1990, Bioconjugate Chemistry
1(3): 165-187,
incorporated herein by reference.
[096] The term "primer," as used herein, refers to an oligonucleotide
capable of acting as
a point of initiation of DNA synthesis under suitable conditions. Such
conditions include those
in which synthesis of a primer extension product complementary to a nucleic
acid strand is
induced in the presence of four different nucleoside triphosphates and an
agent for extension
(e.g., a DNA polymerase or reverse transcriptase) in an appropriate buffer and
at a suitable
temperature. Primer extension can also be carried out in the absence of one or
more of the
nucleotide triphosphates in which case an extension product of limited length
is produced. As
used herein, the term "primer" is intended to encompass the oligonucleotides
used in
ligation-mediated reactions, in which one oligonucleotide is "extended" by
ligation to a second
oligonucleotide which hybridizes at an adjacent position. Thus, the term
"primer extension",
as used herein, refers to both the polymerization of individual nucleoside
triphosphates using
the primer as a point of initiation of DNA synthesis and to the ligation of
two oligonucleotides
to form an extended product.
[097] A primer is preferably a single-stranded DNA. The appropriate length
of a primer
depends on the intended use of the primer but typically ranges from 6 to 50
nucleotides,
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preferably from 15-35 nucleotides. Short primer molecules generally require
cooler
temperatures to form sufficiently stable hybrid complexes with the template. A
primer need
not reflect the exact sequence of the template nucleic acid, but must be
sufficiently
complementary to hybridize with the template. The design of suitable primers
for the
amplification of a given target sequence is well known in the art and
described in the literature
cited herein.
[098] Primers can incorporate additional features which allow for the
detection or
immobilization of the primer but do not alter the basic property of the
primer, that of acting as
a point of initiation of DNA synthesis. For example, primers may contain an
additional nucleic
acid sequence at the 5' end which does not hybridize to the target nucleic
acid, but which
facilitates cloning or detection of the amplified product. The region of the
primer which is
sufficiently complementary to the template to hybridize is referred to herein
as the hybridizing
region.
[099] The terms "target, "target sequence", "target region", and "target
nucleic acid," as
used herein, are synonymous and refer to a region or sequence of a nucleic
acid which is to be
amplified, sequenced or detected.
[0100] The term
"hybridization," as used herein, refers to the formation of a duplex
structure by two single-stranded nucleic acids due to complementary base
pairing.
Hybridization can occur between fully complementary nucleic acid strands or
between
"substantially complementary" nucleic acid strands that contain minor regions
of mismatch.
Conditions under which hybridization of fully complementary nucleic acid
strands is strongly
preferred are referred to as "stringent hybridization conditions" or "sequence-
specific
hybridization conditions". Stable duplexes of substantially complementary
sequences can be
achieved under less stringent hybridization conditions; the degree of mismatch
tolerated can be
controlled by suitable adjustment of the hybridization conditions. Those
skilled in the art of
nucleic acid technology can determine duplex stability empirically considering
a number of
variables including, for example, the length and base pair composition of the
oligonucleotides,
ionic strength, and incidence of mismatched base pairs, following the guidance
provided by the
art (see, e.g., Sambrook et al., 1989, Molecular Cloning¨A Laboratory Manual,
Cold Spring
Harbor Laboratory, Cold Spring Harbor, New York; Wetmur, 1991, Critical Review
in
Biochem. and Mol. Biol. 26(3/4):227-259; and Owczarzy et al., 2008,
Biochemistry, 47:
5336-5353, which are incorporated herein by reference).
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[0101] The term
"amplification reaction" refers to any chemical reaction, including an
enzymatic reaction, which results in increased copies of a template nucleic
acid sequence or
results in transcription of a template nucleic acid. Amplification reactions
include reverse
transcription, the polymerase chain reaction (PCR), including Real Time PCR
(see U.S. Pat.
Nos. 4,683,195 and 4,683,202; PCR Protocols: A Guide to Methods and
Applications (Innis et
al., eds, 1990)), and the ligase chain reaction (LCR) (see Barany et al., U.S.
Pat. No. 5,494,810).
Exemplary "amplification reactions conditions" or "amplification conditions"
typically
comprise either two or three step cycles. Two step cycles have a high
temperature denaturation
step followed by a hybridization/elongation (or ligation) step. Three step
cycles comprise a
denaturation step followed by a hybridization step followed by a separate
elongation or ligation
step.
[0102] As used
herein, a "polymerase" refers to an enzyme that catalyzes the
polymerization of nucleotides. Generally, the enzyme will initiate synthesis
at the 3'-end of the
primer annealed to a nucleic acid template sequence. "DNA polymerase"
catalyzes the
polymerization of deoxyribonucleotides. Known DNA polymerases include, for
example,
Pyrococcus furiosus (Pfu) DNA polymerase (Lundberg et al., 1991, Gene, 108:1),
E. coil DNA
polymerase I (Lecomte and Doubleday, 1983, Nucleic Acids Res. 11:7505), T7 DNA
polymerase (Nordstrom et al., 1981, J. Biol. Chem. 256:3112), Thermus
thermophilus (Tth)
DNA polymerase (Myers and Gelfand 1991, Biochemistry 30:7661), Bacillus
stearothermophilus DNA polymerase (Stenesh and McGowan, 1977, Biochim Biophys
Acta
475:32), Thermococcus litoralis (Tli) DNA polymerase (also referred to as Vent
DNA
polymerase, Cariello et al., 1991, Nucleic Acids Res, 19: 4193), Thermotoga
maritima (Tma)
DNA polymerase (Diaz and Sabino, 1998 Braz J. Med. Res, 31:1239), Thermus
aquaticus (Taq)
DNA polymerase (Chien et al., 1976, J. Bacteoriol, 127: 1550), Pyrococcus
kodakaraensis
KOD DNA polymerase (Takagi et al., 1997, Appl. Environ. Microbiol. 63:4504),
JDF-3 DNA
polymerase (Patent application WO 0132887), and Pyrococcus GB-D (PGB-D) DNA
polymerase (Juncosa-Ginesta et al., 1994, Biotechniques, 16:820). The
polymerase activity of
any of the above enzymes can be determined by means well known in the art.
[0103] As used
herein, a primer is "specific," for a target sequence if, when used in an
amplification reaction under sufficiently stringent conditions, the primer
hybridizes primarily
to the target nucleic acid. Typically, a primer is specific for a target
sequence if the
primer-target duplex stability is greater than the stability of a duplex
formed between the
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primer and any other sequence found in the sample. One of skill in the art
will recognize that
various factors, such as salt conditions as well as base composition of the
primer and the
location of the mismatches, will affect the specificity of the primer, and
that routine
experimental confirmation of the primer specificity will be needed in many
cases.
Hybridization conditions can be chosen under which the primer can form stable
duplexes only
with a target sequence. Thus, the use of target-specific primers under
suitably stringent
amplification conditions enables the selective amplification of those target
sequences which
contain the target primer binding sites.
[0104] The term
"non-specific amplification," as used herein, refers to the amplification of
nucleic acid sequences other than the target sequence which results from
primers hybridizing to
sequences other than the target sequence and then serving as a substrate for
primer extension.
The hybridization of a primer to a non-target sequence is referred to as "non-
specific
hybridization" and is apt to occur especially during the lower temperature,
reduced stringency,
pre-amplification conditions, or in situations where there is a variant allele
in the sample
having a very closely related sequence to the true target as in the case of a
single nucleotide
polymorphism (SNP).
[0105] The term
"3'-mismatch discrimination" refers to a property of a DNA polymerase to
distinguish a fully complementary sequence from a mismatch-containing (nearly
complementary) sequence where the nucleic acid to be extended (for example, a
primer or
other oligonucleotide) has a mismatch at the 3' end of the nucleic acid
compared to the template
to which the nucleic acid hybridizes. In some embodiments, the nucleic acid to
be extended
comprises a mismatch at the 3' end relative to the fully complementary
sequence.
[0106] The term
"3'-mismatch discrimination assay" refers to an assay to discern the
present of improved specificity in amplification of a target DNA sequence when
the target
DNA sequence is interrogated with two primers having substantially identical
sequence except
for the occurrence of one of more nucleotide residue having different base
composition at or
near their respective 3' -ends. For example, a
first primer having
3'-end sequences with perfect complementarity to the target DNA sequence is
considered a
3'-matched primer and a second primer having a 3'-end sequences having at
least one
nucleotide base non-complementarity to the target DNA sequence is considered a
3 '-mismatched primer. An example of a 3 '-mismatch discrimination assay is
provided in many
of the examples, such as EXAMPLES 36 and 37, among others.
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[0107] The term
"primer dimer," as used herein, refers to a template-independent
non-specific amplification product, which is believed to result from primer
extensions wherein
another primer serves as a template. Although primer dimers frequently appear
to be a
concatamer of two primers, i.e., a dimer, concatamers of more than two primers
also occur.
The term "primer dimer" is used herein generically to encompass a template-
independent
non-specific amplification product.
[0108] The term
"reaction mixture," as used herein, refers to a solution containing reagents
necessary to carry out a given reaction. An "amplification reaction mixture",
which refers to a
solution containing reagents necessary to carry out an amplification reaction,
typically contains
oligonucleotide primers and a DNA polymerase or ligase in a suitable buffer. A
"PCR reaction
mixture" typically contains oligonucleotide primers, a DNA polymerase (most
typically a
thermostable DNA polymerase), dNTP's, and a divalent metal cation in a
suitable buffer. A
reaction mixture is referred to as complete if it contains all reagents
necessary to enable the
reaction, and incomplete if it contains only a subset of the necessary
reagents. It will be
understood by one of skill in the art that reaction components are routinely
stored as separate
solutions, each containing a subset of the total components, for reasons of
convenience, storage
stability, or to allow for application-dependent adjustment of the component
concentrations,
and that reaction components are combined prior to the reaction to create a
complete reaction
mixture. Furthermore, it will be understood by one of skill in the art that
reaction components
are packaged separately for commercialization and that useful commercial kits
may contain
any subset of the reaction components which includes the blocked primers of
the invention.
[0109] For the
purposes of this invention, the terms "non-activated" or "inactivated," as
used herein, refer to a primer or other oligonucleotide that is incapable of
participating in a
primer extension reaction or a ligation reaction because either DNA polymerase
or DNA ligase
cannot interact with the oligonucleotide for their intended purposes. In some
embodiments
when the oligonucleotide is a primer, the non-activated state occurs because
the primer is
blocked at or near the 3 ' -end so as to prevent primer extension. When
specific groups are
bound at or near the 3'-end of the primer, DNA polymerase cannot bind to the
primer and
extension cannot occur. A non-activated primer is, however, capable of
hybridizing to a
substantially complementary nucleotide sequence.
[0110] For the
purposes of this invention, the term "activated," as used herein, refers to a
primer or other oligonucleotide that is capable of participating in a reaction
with DNA
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polymerase or DNA ligase. A primer or other oligonucleotide becomes activated
after it
hybridizes to a substantially complementary nucleic acid sequence and is
cleaved to generate a
functional 3'- or 5'-end so that it can interact with a DNA polymerase or a
DNA ligase. For
example, when the oligonucleotide is a primer, and the primer is hybridized to
a template, a
3 '-blocking group can be removed from the primer by, for example, a cleaving
enzyme such
that DNA polymerase can bind to the 3' end of the primer and promote primer
extension.
[0111] The term
"cleavage domain" or "cleaving domain," as used herein, are synonymous
and refer to a region located between the 5' and 3' end of a primer or other
oligonucleotide that
is recognized by a cleavage compound, for example a cleavage enzyme, that will
cleave the
primer or other oligonucleotide. For the purposes of this invention, the
cleavage domain is
designed such that the primer or other oligonucleotide is cleaved only when it
is hybridized to a
complementary nucleic acid sequence, but will not be cleaved when it is single-
stranded. The
cleavage domain or sequences flanking it may include a moiety that a) prevents
or inhibits the
extension or ligation of a primer or other oligonucleotide by a polymerase or
a ligase, b)
enhances discrimination to detect variant alleles, or c) suppresses undesired
cleavage reactions.
One or more such moieties may be included in the cleavage domain or the
sequences flanking
it.
[0112] The term
"RNase H cleavage domain," as used herein, is a type of cleavage domain
that contains one or more ribonucleic acid residue or an alternative analog
which provides a
substrate for an RNase H. An RNase H cleavage domain can be located anywhere
within a
primer or oligonucleotide, and is preferably located at or near the 3 ' -end
or the 5' -end of the
molecule.
[0113] An
"RNase H1 cleavage domain" generally contains at least three residues. An
"RNase H2 cleavage domain" may contain one RNA residue, a sequence of
contiguously
linked RNA residues or RNA residues separated by DNA residues or other
chemical groups.
In one embodiment, the RNase H2 cleavage domain is a 2'-fluoronucleoside
residue. In a more
preferred embodiment the RNase H2 cleavable domain is two adjacent 2' -fluoro
residues.
[0114] The
terms "cleavage compound," or "cleaving agent" as used herein, refers to any
compound that can recognize a cleavage domain within a primer or other
oligonucleotide, and
selectively cleave the oligonucleotide based on the presence of the cleavage
domain. The
cleavage compounds utilized in the invention selectively cleave the primer or
other
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oligonucleotide comprising the cleavage domain only when it is hybridized to a
substantially
complementary nucleic acid sequence, but will not cleave the primer or other
oligonucleotide
when it is single stranded. The cleavage compound cleaves the primer or other
oligonucleotide
within or adjacent to the cleavage domain. The term "adjacent," as used
herein, means that the
cleavage compound cleaves the primer or other oligonucleotide at either the 5'-
end or the 3'
end of the cleavage domain. Cleavage reactions preferred in the invention
yield a 5'-phosphate
group and a 3'-OH group.
[0115] In a
preferred embodiment, the cleavage compound is a "cleaving enzyme." A
cleaving enzyme is a protein or a ribozyme that is capable of recognizing the
cleaving domain
when a primer or other nucleotide is hybridized to a substantially
complementary nucleic acid
sequence, but that will not cleave the complementary nucleic acid sequence
(i.e., it provides a
single strand break in the duplex). The cleaving enzyme will also not cleave
the primer or other
oligonucleotide comprising the cleavage domain when it is single stranded.
Examples of
cleaving enzymes are RNase H enzymes and other nicking enzymes.
[0116] The term
"nicking," as used herein, refers to the cleavage of only one strand of the
double-stranded portion of a fully or partially double-stranded nucleic acid.
The position
where the nucleic acid is nicked is referred to as the "nicking site" (NS). A
"nicking agent"
(NA) is an agent that nicks a partially or fully double-stranded nucleic acid.
It may be an
enzyme or any other chemical compound or composition. In certain embodiments,
a nicking
agent may recognize a particular nucleotide sequence of a fully or partially
double-stranded
nucleic acid and cleave only one strand of the fully or partially double-
stranded nucleic acid at
a specific position (i.e., the NS) relative to the location of the recognition
sequence. Such
nicking agents (referred to as "sequence specific nicking agents") include,
but are not limited to,
nicking endonucleases (e.g., N.BstNB).
[0117] A
"nicking endonuclease" (NE), as used herein, thus refers to an endonuclease
that
recognizes a nucleotide sequence of a completely or partially double-stranded
nucleic acid
molecule and cleaves only one strand of the nucleic acid molecule at a
specific location relative
to the recognition sequence. In such a case the entire sequence from the
recognition site to the
point of cleavage constitutes the "cleavage domain".
[0118] The term
"blocking group," as used herein, refers to a chemical moiety that is
bound to the primer or other oligonucleotide such that an amplification
reaction does not occur.
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For example, primer extension and/or DNA ligation does not occur. Once the
blocking group
is removed from the primer or other oligonucleotide, the oligonucleotide is
capable of
participating in the assay for which it was designed (PCR, ligation,
sequencing, etc). Thus, the
"blocking group" can be any chemical moiety that inhibits recognition by a
polymerase or
DNA ligase. The blocking group may be incorporated into the cleavage domain
but is
generally located on either the 5'- or 3'-side of the cleavage domain. The
blocking group can
be comprised of more than one chemical moiety. In the present invention the
"blocking group"
is typically removed after hybridization of the oligonucleotide to its target
sequence.
[0119] The term "fluorescent generation probe" refers either to a) an
oligonucleotide
having an attached fluorophore and quencher, and optionally a minor groove
binder or to b) a
DNA binding reagent such as SYBR Green dye.
[0120] The terms "fluorescent label" or "fluorophore" refers to compounds
with a
fluorescent emission maximum between about 350 and 900 nm. A wide variety of
fluorophores can be used, including but not limited to: 5-FAM (also called
5-carboxyfluorescein; also called Spiro(isobenzofuran-1(3H), 9'-(9H)xanthene)-
5-carboxylic
acid,3',6'-dihydroxy-3-oxo-6-carboxyfluorescein); 5 -Hexachloro-F luorescein;
([4,7,2',4',5 ',7'-hexachloro-(3 ',6'-dipivaloyl-fluoresceiny1)-6-carboxylic
acid]);
6-Hexachloro-F luorescein;
([4,7,2',4',5 ',7'-hexachloro-(3 ',6'-dipivaloylfluoresceiny1)-5-carboxylic
acid]);
5-Tetrachloro-Fluorescein; ([4,7,2',7'-tetra-chloro-(3',6'-
dipivaloylfluoresceiny1)-5-carboxylic
acid]); 6-Tetrachloro-Fluorescein;
([4,7,2',7'-tetrachloro-(3',6'-dipivaloylfluoresceiny1)-6-carboxylic acid]); 5-
TAMRA
(5-carboxytetramethylrhodamine); Xanthylium,
9-(2,4-dicarboxypheny1)-3,6-bis(dimethyl-amino); 6-TAMRA
(6-carboxytetramethylrhodamine); 9-(2,5-dicarboxypheny1)-3,6-
bis(dimethylamino); EDANS
(5-((2 -amino ethyl)amino)naphthalene-l-sulfonic acid); 1,5 -IAEDANS
(5-((((2 -io do ac etyl)amino)ethyl)amino)naphthalene-1 -sulfonic acid); Cy5
(Indodicarbocyanine-5); Cy3 (Indo-dicarbocyanine-3); and BODIPY FL
(2,6-dibromo-4,4-difluoro-5,7-dimethy1-4-bora-3a,4a-diaza-s-indacene-3-
proprionic acid);
Quasar -670 dye (Biosearch Technologies); Cal Fluor Orange dye (Biosearch
Technologies);
Rox dyes; Max dyes (Integrated DNA Technologies), as well as suitable
derivatives thereof
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[0121] As used
herein, the term "quencher" refers to a molecule or part of a compound,
which is capable of reducing the emission from a fluorescent donor when
attached to or in
proximity to the donor. Quenching may occur by any of several mechanisms
including
fluorescence resonance energy transfer, photo-induced electron transfer,
paramagnetic
enhancement of intersystem crossing, Dexter exchange coupling, and exciton
coupling such as
the formation of dark complexes. Fluorescence is "quenched" when the
fluorescence emitted
by the fluorophore is reduced as compared with the fluorescence in the absence
of the quencher
by at least 10%, for example, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,
95%, 98%,
99%, 99.9% or more. A number of commercially available quenchers are known in
the art, and
include but are not limited to DABCYL, Black H01eTM Quenchers (BHQ-1, BHQ-2,
and
BHQ-3), Iowa Black FQ and Iowa Black RQ. These are so-called dark quenchers.
They
have no native fluorescence, virtually eliminating background problems seen
with other
quenchers such as TAMRA which is intrinsically fluorescent.
[0122] The term
"ligation" as used herein refers to the covalent joining of two
polynucleotide ends. In various embodiments, ligation involves the covalent
joining of a 3'
end of a first polynucleotide (the acceptor) to a 5' end of a second
polynucleotide (the donor).
Ligation results in a phosphodiester bond being formed between the
polynucleotide ends. In
various embodiments, ligation may be mediated by any enzyme, chemical, or
process that
results in a covalent joining of the polynucleotide ends. In certain
embodiments, ligation is
mediated by a ligase enzyme.
[0123] As used
herein, "ligase" refers to an enzyme that is capable of covalently linking the
3' hydroxyl group of one polynucleotide to the 5' phosphate group of a second
polynucleotide.
Examples of ligases include E. coli DNA ligase, T4 DNA ligase, etc.
[0124] The
ligation reaction can be employed in DNA amplification methods such as the
"ligase chain reaction" (LCR), also referred to as the "ligase amplification
reaction" (LAR),
see Barany, Proc. Natl. Acad. Sci., 88:189 (1991); and Wu and Wallace,
Genomics 4:560
(1989) incorporated herein by reference. In LCR, four oligonucleotides, two
adjacent
oligonucleotides which uniquely hybridize to one strand of the target DNA, and
a
complementary set of adjacent oligonucleotides, that hybridize to the opposite
strand are mixed
and DNA ligase is added to the mixture. In the presence of the target
sequence, DNA ligase
will covalently link each set of hybridized molecules.
Importantly, in LCR, two
oligonucleotides are ligated together only when they base-pair with sequences
without gaps.
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Repeated cycles of denaturation, hybridization and ligation amplify a short
segment of DNA.
A mismatch at the junction between adjacent oligonucleotides inhibits
ligation. As in other
oligonucleotide ligation assays this property allows LCR to be used to
distinguish between
variant alleles such as SNPs. LCR has also been used in combination with PCR
to achieve
enhanced detection of single-base changes, see Segev, PCT Public. No.
W09001069 (1990).
Novel oligonucleotides and compounds of the present invention.
[0125] In one
embodiment, the novel oligonucleotides of the present invention are primers
for DNA replication, as for example in PCR, DNA sequencing and polynomial
amplification,
to name a few such applications. In this embodiment, the primers have an
inactive
configuration wherein DNA replication (i.e., primer extension) is blocked, and
an activated
configuration wherein DNA replication proceeds. The inactive configuration of
the primer is
present when the primer is either single-stranded, or the primer is hybridized
to the DNA
sequence of interest and primer extension remains blocked by a chemical moiety
that is linked
at or near to the 3' end of the primer. The activated configuration of the
primer is present when
the primer is hybridized to a nucleic acid sequence of interest and
subsequently acted upon by
RNase H or other cleaving agent to remove the blocking group and allow for an
enzyme (e.g., a
DNA polymerase) to catalyze primer extension.
[0126] A number
of blocking groups are known in the art that can be placed at or near the
3' end of the oligonucleotide (e.g., a primer) to prevent extension. A primer
or other
oligonucleotide may be modified at the 3 '-terminal nucleotide to prevent or
inhibit initiation of
DNA synthesis by, for example, the addition of a 3' deoxyribonucleotide
residue (e.g.,
cordycepin), a 2',3'-dideoxyribonucleotide residue, non-nucleotide linkages or
alkane-diol
modifications (U.S. Pat. No. 5,554,516). Alkane diol modifications which can
be used to
inhibit or block primer extension have also been described by Wilk et al.,
(1990, Nucleic Acids
Res., 18 (8):2065), and by Arnold et al., (U.S. Pat. No. 6,031,091).
Additional examples of
suitable blocking groups include 3' hydroxyl substitutions (e.g., 3'-
phosphate, 3 '-triphosphate
or 3'-phosphate diesters with alcohols such as 3- hydroxypropyl), a 2'3'-
cyclic phosphate, 2'
hydroxyl substitutions of a terminal RNA base (e.g., phosphate or sterically
bulky groups such
as triisopropyl silyl (TIPS) or tert-butyl dimethyl silyl (TBDMS)). 2'-alkyl
silyl groups such as
TIPS and TBDMS substituted at the 3 '-end of an oligonucleotide are described
by Laikhter et
al., U.S. Pat. App. Serial No. 11/686,894 which is incorporated herein by
reference. Bulky
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substituents can also be incorporated on the base of the 3'-terminal residue
of the
oligonucleotide to block primer extension.
[0127] Blocking
groups to inhibit primer extension can also be located upstream, that is 5',
from the 3 '-terminal residue. Sterically bulky substituents which interfere
with binding by the
polymerase can be incorporated onto the base, sugar or phosphate group of
residues upstream
from the 3 '-terminus. Such substituents include bulky alkyl groups like t-
butyl, triisopropyl
and polyaromatic compounds including fluorophores and quenchers, and can be
placed from
one to about 10 residues from the 3'-terminus. Alternatively abasic residues
such as a C3
spacer may be incorporated in these locations to block primer extension. In
one such
embodiment two adjacent C3 spacers have been employed (see Examples 27 and
28).
[0128] In the
case of PCR, blocking moieties upstream of the 3'-terminal residue can serve
two functions: 1) to inhibit primer extension, and 2) to block the primer from
serving as a
template for DNA synthesis when the extension product is copied by synthesis
from the reverse
primer. The latter is sufficient to block PCR even if primer extension can
occur. C3 spacers
placed upstream of the 3'-terminal residue can function in this manner (see
Examples 26 and
27).
[0129] A
modification used as a blocking group may also be located within a region 3'
to
the priming sequence that is non-complementary to the target nucleic acid
sequence.
[0130] The
oligonucleotide further comprises a cleavage domain located upstream of the
blocking group used to inhibit primer extension. An RNase H cleavage domain is
preferred.
An RNase H2 cleavage domain comprising a single RNA residue or replacement of
the RNA
base with one or more alternative nucleosides is most preferred.
[0131] In one
embodiment, RNase H2 can be used to cleave duplexes containing a single
2'-fluoro residue. Cleavage occurs on the 5' side of the 2'-fluoro residue. In
a preferred
embodiment, an RNase H2 cleavage domain comprising two adjacent 2'-fluoro
residues is
employed (see Example 6). The activity is enhanced when two consecutive 2'-
fluoro
modifications are present. In this embodiment cleavage occurs preferentially
between the
2'-fluoro residues. Unlike
oligonucleotides containing unmodified RNA residues,
oligonucleotides with 2'-fluoro groups are not cleaved by single-stranded
ribonucleases and
are resistant to water catalyzed cleavage and completely stable at high
temperatures. Enhanced
cleavage has also been found when a 2'-fluoro modified RNA residue is used
with a 2' LNA
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modified RNA residue. 2'-fluoro-containing oligonucleotides have been found to
be further
advantageous in certain applications compared to RNA-containing
oligonucleotides in offering
greater discrimination with respect to mismatches between the oligonucleotide
and the target
sequence.
[0132]
Alternatives to an RNA residue that can be used in the present invention
wherein
cleavage is mediated by an RNase H enzyme include but are not limited to 2'-0-
alkyl RNA
nucleosides, preferably 2'-0-methyl RNA nucleosides, 2'-fluoronucleosides,
locked nucleic
acids (LNA), 2'-ENA residues (ethylene nucleic acids), 2'-alkyl nucleosides,
2'-aminonucleosides and 2'-thionucleosides. The RNase H cleavage domain may
include one
or more of these modified residues alone or in combination with RNA bases. DNA
bases and
abasic residues such as a C3 spacer may also be included to provide greater
performance.
[0133] If the
cleaving agent is an RNase H1 enzyme a continuouse sequence of at least
three RNA residues is preferred. A continuous sequence of four RNA residues
generally leads
to maximal activity. If the cleaving agent is an RNase H2 enzyme a single RNA
residue or 2
adjacent 2'-fluoro residues are preferred.
[0134] One
objective of incorporating modified residues within an RNase H cleavage
domain is to suppress background cleavage of a primer or probe due to water
catalyzed
hydrolysis or cleavage by single stranded ribonucleases. Replacement of the 2'-
hydroxyl
group with a substituent that cannot attack the adjacent phosphate group of an
RNA residue can
accomplish this goal. Examples of this approach include the use of the 2'-
substituted
nucleosides listed above, such as 2'-fluoro and 2'-0-methyl nucleosides. This
is particularly
advantageous when cleavage is mediated by RNase H2 and there is a single RNA
residue
within the cleavage domain. As shown in Figure 3, in this case cleavage by
single stranded
ribonucleases or water catalyzed hydrolysis occurs at a different position
than cleavage by
RNase H2.
[0135] Other
examples of modifications that can be used to suppress cleavage by single
stranded ribonucleases and water catalyzed hydrolysis at RNA residues include
substitution of
the 5' oxygen atom of the adjacent residue (3'- to the RNA base) with an amino
group, thiol
group, or a methylene group (a phosphonate linkage). Alternatively one or both
of the
hydrogen atoms on the 5' carbon of the adjacent residue can be replaced with
bulkier
substituents such as methyl groups to inhibit background cleavage of a
ribonucleotide residue.
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In another such embodiment, the phosphate group at the 3'-side of an RNA
residue can be
replaced with a phosphorothioate, phosphorodithioates or boronate linkage. In
the case of a
phosphorothioate the S stereoisomer is preferred. Combinations of these
various modifications
may also be employed.
[0136] It
should be noted that background cleavage at RNA residues by single stranded
ribonucleases or water catalyzed hydrolysis leads to a blocked 3'-end (see
Figure 3) that cannot
serve as a primer for DNA synthesis. This mitigates the occurrence of false
positive results
even if such cleavage does occur.
[0137] The
cleavage domain may include the blocking group provided that cleavage
occurs on the 5'-side of the blocking group and generates a free 3'-OH.
Generally however the
cleavage domain and the blocking group are separated by one to about 15 bases.
After
cleavage takes place the portion of the primer 3' from the cleavage site
containing the blocking
group dissociates from the template and a functional 3'-hydroxyl group is
exposed, capable of
being acted on by a polymerase enzyme. The optimal distance between the
cleavage site and
the blocking group will depend on the cleaving agent and the nature of the
blocking group.
When cleavage of the oligonucleotide is mediated by RNase H2 at a single RNA
residue a
distance of 3 to about 8 bases between the cleavage site and the blocking
group is preferred. If
the blocking group is sterically small, for example a phosphodiester at the 3'
terminal
nucleotide as in the following structure
- D - P =0
(CH2)3 -OH
bases from the 3?-end is gene
.oup is larger it is advantageot
uther from it.
'referred embodiment, a then
stilized to cleave the oligonuc
d embodiment, a thermoph
a cleavage site 5 bases from the 3 '-end is generally optimal. If the blocking
group is larger it is
advantageous to position the cleavage site further from it.
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[0138] In a
preferred embodiment, a thermophilic RNase H2 enzyme is utilized to cleave
the oligonucleotide. In yet a more preferred embodiment, a thermophilic RNase
H2 enzyme is
used which is less active at room temperature than at elevated temperatures.
This allows a
hot-start type of reaction to be achieved in PCR and other primer extension
assays using the
blocked primers of the present invention without actually requiring a hot
start, i.e., reversibly
inactivated, DNA polymerase. Standard less expensive DNA polymerase
polymerases such as
Taq polymerase can be used instead of the much more expensive hot start
versions of the
enzyme. Moreover, for different applications alternative DNA polymerases may
be preferred.
Utilizing RNase H as the hot start component of the assay obviates the need to
develop a new
reversibly inactivated analog of each different DNA polymerase.
[0139] Hot
start properties of the enzyme may be intrinsic to the protein as in the case
of
Pyrococcus abysii RNase H2 (see Example 4). Alternatively the enzyme may be
reversibly
inactivated by chemical modification using, for example, maleic acid anhydride
analogs such
as citroconic anhydride. These compounds react with amino groups of the
protein and at high
temperature are released restoring activity. In yet another embodiment
antibodies against an
RNase H which block the enzyme may be employed which are denatured at elevated
temperatures.
[0140] In yet
another embodiment, the oligonucleotide of the present invention has a
cleavage domain that is recognized and cleaved by a sequence specific nicking
agent, e.g., a
nicking enzyme. The nicking agent also can be designed to cleave an
oligonucleotide (e.g., a
primer) at a modified nucleic acid or grouping of modified nucleic acids. In
this embodiment,
the oligonucleotide is designed to be recognized by a nicking agent upon
hybridization with the
target nucleic acid, and the nicking of the oligonucleotide/target duplex can
be used to remove
a blocking group and allow for oligonucleotide extension. The nicking site
(NS) is preferably
located at or near the 3'-end of the oligonucleotide, specifically, one to
about 15 bases from the
3'-end of the oligonucleotide.
[0141]
Exemplary nicking agents include, without limitation, single strand nicking
restriction endonucleases that recognize a specific sequence such as N.BstNBI;
or repair
enzymes such as Mut H, MutY (in combination with an AP endonuclease), or
uracil-N-glycosylase (in combination with an AP Lyase and AP endonucleases);
and the geneII
protein of bacteriophage fl.
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[0142] The
blocked primers of the present invention minimize non-specific reactions by
requiring hybridization to the target followed by cleavage before primer
extension. If a primer
hybridizes incorrectly to a related sequence, cleavage of the primer is
inhibited especially when
there is a mismatch that lies at or near the cleavage site. This reduces the
frequency of false
priming at such locations and thereby increases the specificity of the
reaction. It should be
noted that with Pyrococcus abysii Type II RNase H and other RNase H enzymes
used in the
present invention some cleavage does occur even when there is a mismatch at
the cleavage site.
Reaction conditions, particularly the concentration of RNase H and the time
allowed for
hybridization and extension in each cycle, can be optimized to maximize the
difference in
cleavage efficiencies between the primer hybridized to its true target and
when there is a
mismatch. This allows the methods of the present invention to be used very
effectively to
distinguish between variant alleles, including SNPs (see Examples 12-14, 22-
25).
[0143] As noted
above, background cleavage of the primer does not lead to false-positive
priming when RNA residues are incorporated into the oligonucleotide, because
the 2',3' -cyclic
phosphate (or 2' or 3' -phosphate) formed at the 3' end of the cleaved primer
blocks primer
extension. A freely accessible 3' OH group is needed to form a substrate for
DNA polymerase.
The formation of primer-dimers, a common side reaction occurring in PCR, can
also be
inhibited using the 3' blocked primers of the present invention. This allows
for a greater degree
of multiplexing in PCR (e.g., detecting multiple target sequences in the case
of a DNA
detection/amplification assay).
[0144] Without
being bound by any theory, it has been observed that atypical cleavage can
occur at a low frequency 3' to an RNA residue when there is a mismatch,
presumably catalyzed
by RNase H2, to generate a free 3' -OH and lead to primer extension. This can
result in a
decrease in the specificity of the reaction. To mitigate this effect nuclease
resistant residues
can be incorporated into the primer 3' to the RNA residue (see Example 22, 25
and 28). Such
groups include but are not limited to one or more phosphorothioates,
phosphorodithioates,
methyl phosphonates and abasic residues such as a C3 spacer.
[0145] Other
substitutions both 5' and 3' to the RNA residue can also be utilized to
enhance the discrimination and detection of variant alleles in the methods of
the present
invention. Such substitutions include but are not limited to 2' -0-methyl RNA
and secondary
mismatches (see Example 23).
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[0146] The
nature of the blocking group which prevents primer extension is not critical.
It
can be placed at the 3'-terminal residue or upstream from it. Labeling groups
can be
incorporated within the blocking group or attached at other positions on the
3'-segment of the
oligonucleotide primer which dissociates from the template after cleavage
occurs. Such
labeling groups include, but are not limited to, fluorophores, quenchers,
biotin, haptens such as
digoxigenin, proteins including enzymes and antibodies, mass tags which alter
the mass of the
cleavage fragment for detection by mass spectrometry, and radiolabels such as
14C, 3H, "S, 32P
and 33P. These labeling groups can also be attached to the primer 5' to the
cleavage site, in
which case they will be incorporated within the extension product.
[0147] In one
embodiment, the blocking group at or near the 3'-end of the oligonucleotide
can be a fluorescent moiety. In this case, release of the fluorescent molecule
can be used to
monitor the progress of the primer extension reaction. This is facilitated if
the oligonucleotide
also contains a quencher moiety on the 5'-side of the cleavage site. Cleavage
of the
oligonucleotide during the reaction separates the fluorophore from the
quencher and leads to an
increase in fluorescence. If the quencher is itself a fluorophore, such as
Tamra, a decrease in its
fluorescence may also be observed.
[0148] In yet a
further embodiment, the oligonucleotide is labeled with a fluorescent
molecule on the 5'-side of the cleavage domain, and the blocking group located
at or near the
3'-end of the molecule is a quencher such as Iowa Black , Black H01eTM, or
Tamra to name a
few. Again, cleavage of the quencher from the oligonucleotide (e.g., a primer)
leads to an
increase in fluorescence which can be used to monitor the progress of the
oligonucleotide
extension reaction. Moreover, in this case, the primer extension product is
fluorescently
labeled.
[0149] In yet a
further embodiment, the blocked primers of the present invention are used
for nucleic acid sequencing. As in the case of DNA amplification reactions,
the specificity of
primer extension for DNA sequencing is also increased when using the
oligonucleotides of the
present invention. In one sequencing embodiment, 2',3' dideoxynucleotide
triphosphates that
are fluorescently labeled and used as chain terminators and the nested
fragments produced in
the reaction are separated by electrophoresis, preferably capillary
electrophoresis.
[0150] In yet
another embodiment, an oligonucleotide primer of the present invention is
labeled with a fluorescent group and the 3' dideoxynucleotide triphosphate
chain terminators
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are unlabeled. In this embodiment, the blocking group can be a quencher, in
which case
background fluorescence is reduced because the primer itself is not
fluorescent. Only the
extension products are fluorescent.
[0151] Another
aspect of the invention includes the incorporation of alternative divalent
cations such as Mn2+, Ni2+ or Co2+, with or without Mg2+, into the assay
buffer. In certain
embodiments of the invention, when such alternative divalent cations are
present, the
effectiveness of the particular assay is increased due to enhanced cleavage by
RNase H2. In
one embodiment, when two adjacent 2'-fluoronucleoside residues constitute the
RNase H2
cleavable domain, 0.3-1 mM MnC12 with 2-4 mM MgC12 gave optimal performance in
the
assay (see Example 3).
The Methods of the Present Invention
[0152] The
primers, probes and other novel oligonucleotides described herein can be
utilized in a number of biological assays. Although the following list is not
comprehensive, the
majority of the methods of the present invention fall into six general
categories: (1) primer
extension assays (including PCR, DNA sequencing and polynomial amplification),
(2)
oligonucleotide ligation assays (OLA), (3) cycling probe reactions, (4)
sequencing by ligation,
(5) sequencing by generation of end-labeled fragments using RNase H enzymes,
and (6)
synthesis by ligation.
[0153] The
primers, probes and other novel oligonucleotides described herein can be
utilized in a number of primer extension assays.
Primer Extension Assays
[0154] In one
embodiment of the present invention, a method of amplifying a target DNA
sequence of interest is provided. The method comprises the steps of:
(a) providing a reaction mixture comprising a primer having a cleavage
domain and a
blocking group linked at or near to the 3' end of the primer which prevents
primer extension, a
sample nucleic acid having the target DNA sequence of interest, a cleaving
enzyme and a
polymeras e;
(b) hybridizing the primer to the target DNA sequence to form a double-
stranded substrate;
(c) cleaving the hybridized primer with the cleaving enzyme at a point
within or adjacent to
the cleavage domain to remove the blocking group from the primer; and
(d) extending the primer with the polymerase.
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PCR in General
[0155] When
used in PCR, a 3'-blocked primer containing a cleavage domain first
hybridizes to the target sequence. In this embodiment, the primer cannot
extend until cleavage
of the 3' blocking group occurs after hybridization to the complementary DNA
sequence. For
example, when an RNase H cleavage domain is present in the primer, an RNase H
enzyme will
recognize the double-stranded substrate formed by the primer and target and
cleave the primer
within or adjacent to the cleavage domain. The primer can then extend and
amplification of the
target can then occur. Because the primer needs to be recognized and cleaved
by RNase H
before extension, non-specific amplification is reduced.
[0156] In
conventional PCR, a "hot start" polymerase is often used to reduce primer
dimers
and decrease non-specific amplification. Blocked primers of the present
invention requiring
cleavage by RNase H can confer the same advantage. A thermophilic RNase H
enzyme with
little or no activity at lower temperatures is preferred. Activation of the
primers occurs only
after hybridization to the target sequence and cleavage at elevated
temperatures. Advantages
of this approach compared to the use of a hot start reversibly inactivated DNA
polymerase have
been described above. Of course a hot start RNase H enzyme and a hot start DNA
polymerase
can be used in conjunction, if desired.
[0157] Three
types of hot start RNase H enzymes are described here (see Tables 1, 2, and
3): 1) a thermostable RNase H enzyme that has intrinsically little or no
activity at reduced
temperatures as in the case of Pyrococcus abysii RNase H2; 2) a thermostable
RNase H
reversibly inactivated by chemical modification; and 3) a thermostable RNase H
reversibly
inactivated by a blocking antibody. In addition, through means well-known in
the art, such as
random mutagenesis, mutant versions of RNase H can be synthesized that can
further improve
the traits of RNase H that are desirable in the assays of the present
invention. Alternatively,
mutant strains of other enzymes that share the characteristics desirable for
the present invention
could be used.
[0158] In one
embodiment, the cleavage domain within the primer is cleavable by RNase
H. In yet a further embodiment, the RNase H cleavage domain consists of a
single RNA
residue and cleavage of the primer is mediated by a Type II RNase H enzyme,
preferably by a
thermophilic Type II RNase H enzyme, and even more preferably a thermophilic
Type II
RNase H enzyme which is less active at room temperature than at elevated
temperatures. In yet
a further embodiment, the RNase H2 cleavage domain consists of two adjacent 2'-
fluoro
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nucleoside residues. In yet a more preferred embodiment of the present
invention in which the
cleavage domain consists of two adjacent 2'-fluoro nucleoside residues, the
PCR is carried out
in buffers containing alternative divalent cations, including but not limited
to, Mn2+, Ni2+ or
Co2+ in addition to Mg2+. In an additional embodiment, the novel 3'-blocked
primers of the
present invention comprising a cleavage domain can be utilized in a variation
of hot start PCR
in which a thermophilic nicking enzyme is used and the cleavage domain is a
nicking site.
[0159]
Alternatively, a cleavage enzyme that lacks hot start characteristics can be
used in
the present invention with traditional hot-start methods such as adding the
enzyme at an
elevated temperature, encasing a necessary reagent or enzyme in wax, or with a
hot start
reversibly inactivated DNA polymerase.
[0160] The
increased specificity of the present invention, when used in amplification
reactions, enables real-time PCR applications to achieve more specific
results, as compared to
conventional real-time PCR with standard DNA primers. For example, double-
stranded
DNA-binding dye assays, such as SYBR Green assays, have a disadvantage in
that a signal is
produced once the dye binds to any double-stranded product produced by PCR
(e.g., a primer
dimer) and can thereby give rise to a false positive result. But when a primer
of the current
invention is used, non-specific amplification and primer-dimer formation is
reduced, and the
intensity of the signal of the double-stranded DNA-binding dye will reflect
amplification only
of the desired target (see Example 17).
[0161] The
reagent concentrations and reaction conditions of the assay can be varied to
maximize its utility. The relative efficiency of PCR using the blocked primers
of the present
invention relates to the concentration of the unblocking enzyme and the dwell
time at the
anneal/extend reaction temperature (where unblocking proceeds). With low
amounts of
enzyme and short dwell times, cleavage can be incomplete and the reactions
with blocked
primers have lower efficiency than those with unblocked primers. As either
enzyme
concentration or dwell time increases, the reaction efficiency with blocked
primers increases
and becomes identical to unblocked primers. The use of even more enzyme or
longer dwell
times can decrease the specificity of the assay and lessen the ability of the
system to
discriminate mismatches at the cleavage site or within the surrounding
sequence (see Example
4). This results because there is an increase in the efficiency of cleavage of
the primer when it
is hybridized to a mismatch sequence. Cleavage at the true target site cannot
be further
increased because it is already at 100% each cycle. Thus the assay can be
tuned for SNP assays
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requiring higher specificity, or for quantitation of expression levels of mRNA
requiring less
specificity. Specificity can also be adjusted by varying composition of the
reaction buffer. For
example, a Mg ion concentration of 3 mM may support a very robust, high
efficiency assay;
lowering Mg' ion concentration to 2.5 mM, 2.0 mM, or lower may increase
specificity of SNP
discrimination, but may also lead to a slightly lower reaction efficiency (See
Examples 36 and
37). Certain DNA polymerase enzymes may require use of higher Mg' ion
concentrations.
Therefore optimization of Mg' ion concentration can be used to adjust
performance as the
assay to suit specific needs, and this process is familiar to all with skill
in the art. Some DNA
polymerase enzymes perform better when small amounts of detergent are present
in the buffer.
Pyrococcus abyssi RNase H2, and likely other RNase H2 enzymes, similarly has
higher
reaction rates when small amounts of detergent are present in the buffer.
Detergent content can
be adjusted within a wide range and still be compatible with PCR
amplification, using
non-ioninc detergents (e.g., Triton-X-100, Brij-58, etc.) or select ionic
detergents (e.g., CTAB)
(see Examples 18 and 38).
[0162] In
another embodiment, a primer pair having one blocked primer and one
unblocked primer, can be used. In another embodiment, an enzyme can be
selected that has
less sequence specificity and can cleave various sequences. In yet another
embodiment, an
additional mismatch flanking the cleavage site can be added to increase the
ability to
discriminate variant alleles. Modified bases such as 2'-0-methyl nucleosides
can also be
introduced into the primer on either side of the cleavage site to increase
specificity (see
Example 23).
[0163] The
reactions of the various assays described herein can be monitored using
fluorescent detection, detection by mass tags, enzymatic detection, and via
labeling the probe
or primer with a variety of other groups including biotin, haptens,
radionucleotides and
antibodies to name a few. In one embodiment, the progress of PCR using the
modified primers
of the present invention is monitored in real time using a dye intercelating
assay with, for
example, SYBR Green. In yet a further embodiment, the progress of PCR using
the modified
primers of the present invention is monitored using a probe labeled with a
fluorophore and a
quencher such as a molecular beacon or, as in the 5' nuclease assay where
cleavage of the probe
occurs. Alternatively, a dual labeled probe which is cleavable by RNase H2 may
be employed.
In the latter case, cleavage of both the hybridized primers and the probe can
be mediated by the
same enzyme. The RNase H cleavage domain within the probe may comprise only
RNA
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residues. In general, all of the combinations of residues useful in the
cleavage domain of the
blocked primers of the present invention can be used as the cleavage domain
within the probe.
In particular, when RNase H2 is used as the cleavage enzyme, a single RNA
residue or two
adjacent 2'-F residues are preferred as the cleavage domain within the probe.
Such a modified
oligonucleotide probe is particularly useful in real-time PCR and can be
employed with
standard DNA primers or with the blocked primers of the present invention. In
such real-time
PCR assays, thermophilic versions of RNase H2 are preferred, especially
thermophilic RNase
H2 enzymes having lower activity at reduced temperatures. In the examples
provided herein, a
number of thermophilic RNase H2 enzymes have been isolated and have shown to
be stable
under thermocycling conditions and useful in PCR. When used with the blocked
primers of the
present invention, the need for a specific hot-start DNA polymerase can be
eliminated. This
results in a significant decrease in assay cost.
[0164] In
another embodiment, the blocked primers of the present invention can be used
in
the primer-probe assay format for PCR described in U.S. Patent App.
2009/0068643. In this
case, the primer also contains a label domain on the 5' end of the
oligonucleotide which may or
may not be complementary to the target nucleic acid. The product generated by
extension of
the primer serves as a template for synthesis by the reverse primer in the
next cycle of PCR.
This converts the label domain into a double stranded structure. In one such
embodiment a
fluorophore and a quencher are attached to the label domain and the reaction
is monitored by an
increase in fluorescence resulting from an increase in the distance between
the fluorophore and
quencher in the double stranded form compared to the single stranded state. In
yet another such
embodiment the label domain contains a cleavage domain located between the
fluorophore and
quencher. Cleavage occurs only when the cleavage domain is double stranded.
Again the
reaction is monitored by an increase in fluorescence. In this instance the
cleaving agent may be
one that cleaves both strands, the primer and its complement, such as a
restriction enzyme.
Alternativley the cleaving agent may be a nicking agent that cleaves only the
primer, preferably
an RNase H enzyme, and even more preferably a thermostable RNase H2 enzyme.
There are
two cleavage domains within the primer in this assay format: one 5' of the
blocking group at
which cleavage occurs to activate the primer and allow extension and the
second within the
label domain. Cleavage at both sites can be mediated by the same cleaving
agent. The label
domain may also contain other labeling groups including but not limited to
biotin, haptens and
enzymes to name a few. Alternatively the 5' fragment released by cleavage
within the label
domain may serve as a mass tag for detection by mass spectrometry.
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[0165] In yet
another embodiment, the blocked primers of the present invention can be
used in the template-probe assay format for PCR described in U.S. Patent App.
2009/0068643.
[0166] In
another embodiment of the invention, RNase H2 cleavable blocked
oligonucleotides are used to detect 5-methylcytosine residues by PCR analysis
of sodium
bisulfite treated nucleic acids, including but not limited to DNA and RNA.
Previous work has
established that treatment of nucleic acid template with bisulfite will
rapidly deaminate
cytosines that are not methylated on the 5' carbon of the base. This
deamination reaction
converts the unmethylated cytosines into uracil, resulting in a functional C-
>T transition
mutation in the nucleic acid sequence. It is also known that 5-methylcytosine
is highly
resistant to this deamination, resulting in preservation of the 5-
methylcytosine nucleotide as a
cytosine, rather than conversion to a thymine. Numerous methods have been
employed to
detect 5' cytosine methylation modifications following the bisulfite
conversion technique.
Examples include, but are not limited to, standard mismatch-specific
quantitative and
non-quantitative PCR methods, as well as subcloning and sequencing of the
generated sodium
bisulfite reaction products.
[0167] In the
present invention, the template is bisulfite treated by methods that are well
known to those in the art. If the starting template was RNA, a complementary
cDNA strand is
generated by any well known reverse transcription method. Blocked
cleavable
oligonucleotides that will either match or discriminate against the target
template cytosines
(now converted to uracils) or 5-methylcytosines are added to a PCR reaction
containing the
RNase H2 enzyme and the bisulfite treated template. Amplification of the
mismatched
(converted cytosine>uracil or unconverted 5-methylcytosine>5-methylcytosine)
base
containing template is highly reduced relative to the matched base template
due to the
mismatch discrimination of RNase H2 cleavage reaction. Incomplete bisulfite
conversion of
cytosines to uracils, a consistent concern with the sodium bisulfite
conversion technique, can
be detected by the designing blocked cleavable oligonucleotides that target
known
non-5'-methylated cytosines in the bisulfite converted template. PCR
amplification of
unconverted cytosines with these primers should display greater discrimination
relative to
standard unblocked primers. The present invention is expected to significantly
increase the
discrimination of the methylated and unmethylated cytosines.
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Allele Specific PCR
[0168] The
blocked primers of the present invention can also be used in allele-specific
PCR (AS-PCR). In general, AS-PCR is used to detect variant alleles of a gene,
especially
single base mutations such as SNPs (see for example U.S. Patent No.
5,496,699). SNP
locations in the genome, as well as sequences of mutated oncogenes, are known
in the art and
PCR primers can be designed to overlap with these regions.
[0169]
Detection of single base mismatches is a critical tool in diagnosing and
correlating
certain diseases to a particular gene sequence or mutation. Although AS-PCR
has been known
in the biological arts for more than a decade (Bottema et al., 1993, Methods
Enzymol., 218, pp.
388-402), tools are still needed to more accurately discriminate between
particular mismatches
and fully complementary sequences. The present invention addresses this need.
[0170] In AS-
PCR a primer is utilized which overlaps the variant locus. Generally the
primer is designed such that the 3'-terminal nucleotide is positioned over the
mutation site.
Alternatively, the mutation site is sometimes located over one or two bases
from the 3'-end. If
there is a mismatch at or near the 3 '-end, primer extension and hence PCR are
inhibited. The
difference between the efficiency of amplification when there is an exact
match with the
primers versus an allelic variant where there is one or more mismatches can in
some cases be
measured by end point PCR in which case the final amplification products are
analyzed by, for
example, gel electrophoresis. More commonly real time PCR is used to determine
the
efficiency of amplification. A fluorescence based method of detection of the
amplicon in real
time such as a DNA dye binding assay or a dual labeled probe assay is most
often used. The
PCR cycle where fluorescence is first detectable above background levels (the
Cp, or crossing
point) provides a measure of amplification efficiency. If there is a mismatch
between the
primer and the target DNA, amplification efficiency is reduced and the Cp is
delayed.
Generally an increase in Cp of 4 to 5 cycles is sufficient for discrimination
of SNPs.
[0171] In one
AS-PCR embodiment of the present invention, the primer contains a single
RNA residue, and the mismatch can be aligned directly over the RNA residue of
the primer.
The difference in crossing point (Cp) values between a perfect match and a
mismatch,
correlating to a cleavage differential, is readily apparent (see Example 13).
In some instances,
aligning the mismatch one base to either the 5' side or the 3' side of the RNA
residue increases
the difference in Cp values. When the mismatch is located on the 5' side of
the RNA residue,
the subsequent RNase H2 cleavage would leave the mismatch as the last base of
the 3' end of
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the cleaved primer. Surprisingly, having the mismatch directly on top of the
RNA residue is
more effective in most cases than locating the mismatch to the 5' side of the
RNA residue.
[0172] In
another embodiment, the primer contains multiple RNA residues or two adjacent
2'-fluoro residues and detection of the mismatch follows the same principles
as with a primer
containing one RNA residue; the mismatch preferably is located near or on top
of the expected
point of cleavage.
[0173] In
another embodiment, a second mismatch is used to increase the sensitivity of
the
assay. In yet a further embodiment, the second mismatch is placed to the 3'
side of the
mismatch directly over the SNP site. In yet a further embodiment, the second
mismatch is
placed one or two bases from the mismatch directly over the SNP site (see
Example 23).
[0174] In yet
another embodiment, modified residues are incorporated into the primer on
the 5'- or 3 '-side of the base located over the mutation site. In one such
embodiment of the
present invention a 2'-0-methyl ribonucleoside is placed immediately 5' to the
RNA base
within the primer (see Example 22).
[0175] The
sensitivity of the assay can also be increased through incorporation of
nuclease
resistant analogs into the primer on the 3 '-side of the base over the
mutation site. Such
nuclease resistant analogs include, but are not limited to, phosphorothioates,
phosphorodithioates, methylphosphonates and abasic residues such as a C3
spacer. In one such
embodiment of the present invention, phosphorothioate intemucleotide linkages
are
incorporated at each position from the RNA base over the mutation site to the
3 '-end of the
primer. In yet another such embodiment phosphorothioate linkages or
phosphoroditioate are
incorporated at all positions from the base on the 3'-side of the RNA residue
to the 3'-end of
the primer. In yet another such embodiment a single phosphorothioate or
phosphorodithioates
is introduced on the 3 '-side of the residue immediately downstream from the
RNA base within
the primer. In one embodiment, the phosphorothioate bonds are placed between
each
monomer 3' to the RNA monomer directly over the SNP site, as well as between
the RNA
monomer and the base 3' to the RNA base (see Example 25).
[0176] The
assay sensitivity can also be improved by optimizing the placement of the 3'
blocking group or groups. In one embodiment, a blocking group is placed
internal to the 3' end
of the oligonucleotide. In a further embodiment, more than on blocking group
is placed
internal to the 3' terminus. In yet a further embodiment, an RNA monomer sits
directly over
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the SNP site, with a DNA monomer 3' to the RNA monomer, followed by two C3
spacers, and
finally followed by a 3' terminal base (see Example 28). The assay sensitivity
and specificity
also be improved by optimization of magnesium ion concentration in the
reaction buffer,
wherein lower magnesium levels (e.g., 1.5 mM or 2.0 or 2.5 mM, etc.) may
confer higher
specificity and higher magnesium levels (e.g., 3.0 mM, 3.5 mM, etc.) may
confer lower
specificity but with higher amplification efficiency (see Examples 36 and 37).
[0177] One
convenient, quantitative measure of improved specificity during amplification
of a target DNA sequence is the observed change in Cp value (ACp) for
amplifying a target
DNA sequence with a matched primer vs. a mismatched primer. A preferred ACp of
at least
about 5 or greater or a relative increase of ACp of at least about 50% or
greater is indicative of
improved specificity by optimization of magnesium ion concentration. More
typically,
however, as revealed by the Examples, preferred ACp can be much greater, such
as ACp values
of at least about 10-20 or greater or a relative increase of ACp of at least
about 100% or greater.
The observed ACp indicateive of improved specificity by optimization of
magnesium ion
concentration can depend upon primer design and the particular target DNA
sequence being
interrogated.
[0178] In one
embodiment of the allele-specific PCR, the primers can be designed to detect
more than one mismatch. For example, the forward primer can detect a first
mismatch, and the
reverse primer could detect a second mismatch. In this embodiment, the assay
can be used to
indicate whether two mismatches occur on the same gene or chromosome being
analyzed. This
assay would be useful in applications such as determining whether a bacterium
of interest is
both pathogenic and antibiotic resistant.
[0179] In
another embodiment, the forward and reverse primers are both blocked and
overlap at the mismatch. In a further embodiment, the blocking groups are
internal to the 3'
end of the oligonucleotide. In yet a further embodiment, for one or both the
forward and
reverse primers, an RNA monomer sits directly over the SNP site, with a DNA
monomer 3' to
the RNA monomer, followed by two C3 spacers, and finally followed by a 3'
terminal base.
Reverse transcriptase PCR (RT-PCR)
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[0180] In yet
another embodiment the methods of the present invention can be used in
coupled reverse transcription-PCR (RT-PCR). In one such embodiment reverse
transcription
and PCR are carried out in two disctinct steps. First a cDNA copy of the
sample mRNA is
synthesized using either an oligo dT primer or a sequence specific primer.
Random hexamers
and the like can also be used to prime cDNA synthesis. The resulting cDNA is
then used as the
substrate for PCR employing the blocked primers and methods of the present
invention.
[0181]
Alternatively reverse transcription and PCR can be carried out in a single
closed
tube reaction. In one such embodiment three primers are employed, one for
reverse
transcription and two for PCR. The primer for reverse transcription binds to
the mRNA 3' to
the position of the PCR amplicon. Although not essential, the reverse
transcription primer can
include RNA residues or modified analogs such as 2'-0-methyl RNA bases which
will not
form a substrate for RNase H when hybridized to the mRNA. Preferably an RNase
H2 enzyme
which has decreased activity at lower temperatures is used as the cleaving
agent.
[0182] In the
three primer RT-PCR assay it is desirable to inhibit the RT-primer from
participating in the PCR reaction. This can be accomplished by utilizing an RT-
primer having
a lower Tm than the PCR primers so it will not hybridize under the PCR
conditions.
Alternatively, a non-replicable primer incorporating, for example, two
adjacent C3 spacers can
be used as the RT-primer (as in polynomial amplification, see U.S. Pat. No.
7,112,406). In this
case when the cDNA is copied by extension of the forward PCR primer it will
not include the
binding site for the RT-primer.
[0183] In one
embodiment, only the reverse PCR primer is blocked utilizing the
compositions and methods of the present invention. In yet another embodiment
both the
forward and reverse PCR primers are blocked. The reverse PCR primer is blocked
in the 3
primer RT-PCR assay to prevent it from being utilized for reverse
transcription. If desired,
modified bases such as 2'-0-methyl RNA residues can be incorporated in the
reverse PCR
primer although any such modification must allow the primer sequence to serve
as a template
for DNA synthesis and be copied.
[0184] In the
two primer RT-PCR assays of the present invention, only the forward PCR is
blocked. The reverse PCR primer also serves as the RT-primer and therefore can
not be
blocked.
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[0185] While
not comprehensive, Table 1 illustrates how variations in the blocking groups,
labeling groups, cleavage site embodiments, modifications to the cleavage site
or other regions
of the oligonucleotide, buffer conditions and enzyme can further optimize
assay formats
depending on their particular application. Examples of assay formats and
applications include
PCR; real-time PCR utilizing double-stranded DNA-binding dyes such as SYBR
Green, 5'
nuclease assays (TaqmanTm assays) or molecular beacons; primer-probe and
template-probe
assays (see U.S. Patent Application 2009/0068643); polynomial or linked linear
amplification
assays; gene construction or fragment assembly via PCR; allele-specific PCR
and other
methods used to detect single nucleotide polymorphisms and other variant
alleles; nucleic acid
sequencing assays; and strand displacement amplification. In these various
assays, cleavage of
the primers of the present invention can be used to enhance the specificity of
the particular
reaction.
46
0
Table 1: PCR/Primer Extension/Polyamp
t.)
o
Primer Blocking Labeling RNase H Cleavage Flanking
Divalent DNA RNase H Sample Use Assay Format
.6.
Group Group Site sequence cation Polymerase
1-,
modifications
.6.
c...)
None None RNA None mg2+ Hot Start
RNase H1 Genomic Sample Prep No additional probe t.)
t.)
1. Single RNA
1. Ab DNA 1. Detection of primer oe
residue 2.Chemi-
cleavage
Fluorophore
2. Multiple RNA Nuclease-
catty RNase H2
Coupled
A. Fluorescence
resistant linkages 1. Non-
amplification
residues modified
B. Mass Spec
1. thermostable to reverse
C. Electrophoresis
Phosphorothioate 2.
Thermostable transcription 2. Dye-binding assay
2. Dithioate A. Hot Start
A. Sybr Green
Fluorophore/ 3. Methyl- i.
Intrinsic
Quencher phosphonate ii. Ab
4. Non-nucleotide iii.
Chemically
spacers modified
B. Non-Hot Start
P
Enzyme
Mitochon- Quantification
IV
___________ 1 . Horseradish drial/
___________________________________________ of target
0
0
0,
Modification of 3'- peroxidase Modified residues:
Alter- Non-Hot RNase H3 and chloroplast nucleic acid With
an internal probe µ.0
0,
terminal residue 2. Alkaline 1. 2 adjacent 2' F
native Start other catalysts that DNA sequence
1. Taqman 0,
1. C3 spacer phosphatase residues divalent
cleave RNA/DNA 1. Chromoso- 2. Fluorescence- 0
1-
cation heteroduplexes
mal copy quenched
0
+1_ mg2+
number
linear probe ,0
,
2. mRNA
3. Molecular beacon 1-
0.
4. RNase H-cleavable
Biotin 2'0Me
cDNA Detection of probe
variant allele
Hapten
1. Deoxigenin
Upstream Antibody Secondary RNase H
mutants RNA Gene/Fragment Tempro Assay
modification mismatches having altered
1. mRNA construction 1. RNase H2-
1. Adjacent to the Mass Tag
cleavage cleavable probe
IV
3'-terminal specificity
n
residue Radiolabel 1. Enhanced
1-3
2. Further upstream 32P, 24C, 3H, cleavage of 2'-F
(4
355, etc. substrates
t.)
o
1-,
c...,
-a-,
-..,
t..,
c7,
=
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Cyc1in2 Probe Reactions
[0186] Cycling probe reactions are another technique for detecting specific
nucleic
acid sequences (see U.S. Pat. No. 5,403,711). The reaction operates under
isothermal
conditions or with temperature cycling. Unlike PCR products accumulate in a
linear
fashion.
[0187] Table 2 illustrates a non-comprehensive set of possible elements of
the
current invention to improve assays based on the cycling probe reaction. New
features
of the invention include 1) use of a hot start RNase H enzyme; 2) cleavage of
novel
sequences by RNase H enzymes (e.g., cleavage of substrates containing
2'-fluoronucleosides by Type II RNases H); and 3) introduction of
modifications and
secondary mismatches flanking an RNase H cleavage domain to enhance
specificity
and/or suppress nonspecific cleavage reactions. Such modifications and
secondary
mismatches are particularly useful when cleavage is mediated by a Type II
RNase H
and the cleavage domain is a single RNA residue or two adjacent 2'-fluoro
residues.
48
0
Table 2: Cycling Probe Reaction
t..)
o
Primer Labeling RNase H Flanking Divalent RNase H
Sample Use Assay Format
.6.
Extension Group Cleavage Site sequence mods cation
.6.
Blocking Group
c,.)
n.)
n.)
oe
None None RNA None mg2+ RNase H1
Genomic Quantification of Stand-alone
1. Single RNA DNA
____________________________________________________________ target nucleic
acid 1. Isothermal
Modification of Fluorophore residue Nuclease-resist
RNase H2 sequence 2. Temperature cycling
3'- terminal 2. Multiple ant linkages 1. Non-thermostable
1. Chromosomal
residue RNA residues 1.
2. Thermostable copy number
1. C3 spacer Phos-phorothi A. Hot Start
2. mRNA
Fluorophore/ oate i. Intrinsic
Quencher 2. Dithioate ii. Ab Mito-
chondr
3. iii. Chemically
ial/
Methyl-phosph modified
chloroplast P
_________________________________ onate B. Non-Hot Start
DNA
Upstream Enzyme Modified 4. Altemativ RNase H3 and other
Detection of variant Coupled to .
,..
modification 1. Horseradish residues: Non-nucleotid e
divalent catalysts that cleave allele Amplification u,
r.,
1. Adjacent to peroxidase 1. 2 adjacent 2' e spacers
cation +/- RNA/DNA 1. PCR o
the 2. 2. Alkaline F residues
mg2+ heteroduplexes 2. LCR
3 '-terminal phosphatase
3. Polyamp .
,
1-
residue
2. Further Biotin 2'0Me RNase H mutants having
cDNA
upstream altered cleavage
Hapten specificity
1. Deoxigenin 1. Enhanced cleavage of
Antibody Secondary 2'-F substrates
mismatches
Mass Tag
IV
n
Radiolabel
ei
32p, 14C, 3H,
CP
35S, etc.
n.)
o
1¨,
-1
--.1
n.)
o
o
o
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DNA Li2ation Assays
[0188] The present invention can also serve to increase the specificity of
DNA
ligation assays. Donor and/or acceptor oligonucleotides of the present
invention can be
designed which bind adjacent to one another on a target DNA sequence and are
modified to prevent ligation. Blocking groups on the acceptor oligonucleotide
useful to
inhibit ligation are the same as those used to prevent primer extension.
Blocking the
donor oligonucleotide can be readily accomplished by capping the 5'-OH group,
for
example as a phosphodiester, e.g..:
0
HO-(H2C)30 -P-0
0 0
Other 5' blocking groups include 5'-0-alkyl substituents such as 5'-0-methyl
or
' -0-trityl groups, 5 '-0-heteroalkyl groups such as 5 ' -OCH2CH2OCH3, 5 ' -0-
aryl
groups, and 5'-0-sily1 groups such as TIPS or TBDMS. A 5' deoxy residue can
also be
used to block ligation.
[0189] Sterically bulky groups can also be placed at or near the 5'-end of
the
oligonucleotide to block the ligation reaction. A 5'-phosphate group cannot be
used to
block the 5'-OH as this is the natural substrate for DNA ligase. Only after
hybridization to the target DNA sequence are the blocking groups removed by,
for
example cleavage at an RNase H cleavable domain, to allow ligation to occur.
Preferably cleavage is mediated by an RNase H Type II enzyme, and even more
preferably a thermophilic Type II RNase H enzyme. More preferably, a
thermophilic
Type II RNase H enzyme which is less active at room temperature than at
elevated
temperature is utilized to mediate cleavage and thereby activation of the
acceptor
and/or donor oligonucleotide. Alternatively, a sequence specific nicking
enzyme, such
as a restriction enzyme, may be utilized to mediate cleavage of the donor
and/or
acceptor oligonucleotide.
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[0190] In a further embodiment, the cleaving reaction is first carried out
at a higher
temperature at which only one of the two oligonucleotides hybridizes to the
target
sequence. The temperature is then lowered, and the second oligonucleotide
hybridizes
to the target, and the ligation reaction then takes place.
[0191] In yet a further embodiment in which there is a cleavage domain
located in
the donor oligonucleotide, this oligonucleotide is not blocked at or near the
5'-end, but
simply has a free 5'-OH. This oligonucleotide cannot serve as a donor in the
ligation
reaction; to do so requires a 5'-phosphate group. Thus, the 5'-end is
functionally
blocked. Cleavage by RNase H generates a 5'-phosphate group allowing the donor
oligonucleotide to participate in the ligation reaction.
[0192] An important advantage of the present invention is that it allows
double
interrogation of the mutation site, and hence greater specificity, than
standard ligation
assays. There is an opportunity for discrimination of a variant allele both at
the
cleavage step and the ligation step.
[0193] Table 3 illustrates a non-comprehensive set of possible elements of
the
current invention to improve oligonucleotide ligation assays.
51
0
Table 3: Oligonucleotide Ligation Assay
b..)
Donor Acceptor Labeling RNase H Flanking sequence
Divalent DNA Ligase RNase H Sample Use Reaction Assay Format
C,
1¨)
Oligonucleotide Oligonucleotide Group Cleavage Site
mods cation Conditions .P.
--...
Blocking Group
Blocking Group 1¨)
.P.
(....)
b..)
b..)
None None None RNA None me( Hot Start RNase
HI Genomic Quantification RNase H cleavage Stand-alone C'e
(5 ' -phosphate) 1. Single 1. Ab
DNA of target and DNA ligation 1. Single cycle
RNA residue 2. Chem-ically
nucleic acid at single 2. Linear
5'-OH Modification of Fluorophore 2. Multiple
Nuclease-resistant modified RNase H2 sequence
temperature Amplification
(Functional block) 3'-terminal residue RNA
residues linkages 1. Non-thermostable 1.Chromosomal
3. LCR
1. C3 spacer 1.2. Thermostable
copy number
Phos-phorothioate A. Hot
Start 2. mRNA
2. Dithioate i.
Intrinsic
Modification of 5'- Fluorophore/ 3. ii. Ab
residue Quencher Methyl-phosphona
iii. Chemically Mito-chondri
1. C3 spacer te
modified al/
4. Non-nucleotide B. Non-
Hot Start chloroplast
P
Downstream Upstream Enzyme Modified spacers
Altemativ Non-Hot Start RNase H3 and other DNA Detection of RNase H
cleavage Coupled to
modification modification 1. residues:
e divalent catalysts that cleave variant at elevated
primer extension 2
1. Adjacent to the 1. Adjacent to the Horse-radish 1. 2
adjacent 2' cation +/- RNA/DNA allele temperature 1. PCR 0
o
5'-terminal 3'-terminal peroxidase F residues mg2+
heteroduplexes (reduced 2. Reverse t.
o
residue residue 2. Alkaline
temperature for transcription u)
n,
2. Further 2. Further
phosphatase DNA ligation) 3. Polyamp o
r
downstream upstream
T
.,
Biotin 2'0Me RNase H
mutants cDNA r,
A.
having altered
Hapten cleavage
specificity
1. Deoxi- 1.
Enhanced cleavage
genin of 2'-F
substrates
Antibody Secondary
mismatches
Mass Tag
Radiolabel
32p, 14C, 3H,
.0
35S, etc.
n
cp
k...)
L.
,
-4
k...)
c.,
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DNA Sol uencin2 Reactions
[0194] In one embodiment, a method of sequencing a target DNA of interest
is
provided. The method entails
(a) providing a reaction mixture comprising a primer having a cleavage
domain and
a blocking group linked at or near to the 3' end of the primer which prevents
primer
extension, a sample nucleic acid comprising the target DNA sequence of
interest, a
cleaving enzyme, nucleotide triphosphate chain terminators (e.g., 3'
dideoxynucleotide
triphosphates) and a polymerase,
(b) hybridizing the primer to the target nucleic acid to form a double-
stranded
substrate;
(c) cleaving the hybridized primer with the cleaving enzyme at a point
within or
adjacent to the cleavage domain to remove the blocking group from the primer;
and
(d) extending the primer with the polymerase.
[0195] In one embodiment, the invention is used in a "next generation"
sequencing
platform. One type of next generation sequencing is "sequencing by synthesis",
wherein genomic DNA is sheared and ligated with adapter oligonucleotides or
amplified by gene-specific primers, which then are hybridized to complementary
oligonucleotides that are either coated onto a glass slide or are placed in
emulsion for
PCR. The subsequent sequencing reaction either incorporates dye-labeled
nucleotide
triphosphates or is detected by chemiluminescence resulting from the reaction
of
pyrophosphate released in the extension reaction with ATP sulfurylase to
generate ATP
and then the ATP-catalyzed reaction of luciferase and its substrate luciferin
to generate
oxyluciferin and light.
[0196] A second type of next generation sequencing is "sequencing by
ligation",
wherein four sets of oligonucleotides are used, representing each of the four
bases. In
each set, a fluorophore-labeled oligonucleotide of around 7 to 11 bases is
employed in
which one base is specified and the remaining are either universal or
degenerate bases.
If, for example, an 8-base oligonucleotide is used containing 3 universal
bases such as
inosine and 4 degenerate positions, there would be 44 or 256 different
oligonucleotides
in each set each with a specified base (A, T, C or G) at one position and a
fluorescent
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label attached to either the 5'- or 3'-end of the molecule or at an internal
position that
does not interfere with ligation. Four different labels are employed, each
specific to
one of the four bases. A mixture of these four sets of oligonucleotides is
allowed to
hybridize to the amplified sample DNA. In the presence of DNA ligase the
oligonucleotide hybridized to the target becomes ligated to an acceptor DNA
molecule.
Detection of the attached label allows the determination of the corresponding
base in
the sample DNA at the position complementary to the base specified within the
oligonucleotide.
[0197] In one
embodiment of the present invention, a donor oligonucleotide of
about 7-11 bases contains a specified base at the 5' end of the
oligonucleotide. The
remaining bases are degenerate or universal bases, and a label specific to the
specified
base is incorporated on the 3' side of the specified base. The 3' end of the
probe is
irreversibly blocked to prevent the donor oligonucleotide from also acting as
an
acceptor. In some cases this may be accomplished by the labeling group. The
second
base from the 5' end of the oligonucleotide, i.e., the residue next to the
specified base is
a degenerate mixture of the 4 RNA bases. Alternatively, any anaolog recognized
by
RNase H2, such as a 2'-fluoronucleoside may be substituted at this position. A
universal base such as riboinosine or ribo-5-nitroindole, may also be
incorporated at
this location. The probe first hybridizes to the target sequence and becomes
ligated to
the acceptor DNA fragment as in the standard sequencing by ligation reaction.
After
detection of the specified base, RNase H2 is added which cleaves the probe on
the
5'-side of the RNA residue leaving the specified base attached to the 3' end
of the
acceptor fragment. The end result is that the acceptor fragment is elongated
by one
base and now is in position to permit the determination of the next base
within the
sequence. The cycle is repeated over and over, in each case moving the
position of
hybridization of the donor oligonucleotide one base 3' down the target
sequence. The
specificity is increased compared to traditional sequencing by ligation
because the
specified base is always positioned at the junction of the ligation reaction.
[0198] The donor
oligonucleotide probe can optionally contain universal bases
including, but not limited to, 5-nitroindole, ribo-5'-nitroindole, 2'-0-
methy1-
5-nitroindole, inosine, riboinosine, 2'-0-methylriboinosine and 3-
nitropyrrole. This
reduces the number of different oligonucleotides in each set required for the
assay by a
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factor of four for every degenerate position on the probe substituted with a
universal
base. The method can also include a capping step between the ligation reaction
and the
RNase H2 cleaving step. The capping reaction can be performed by introducing a
DNA
polymerase and a chain terminator, thereby capping any of the acceptor
fragment
molecules that did not ligate with a donor oligonucleotide probe in the
previous step.
[0199] In the above example the ligation reactions and hence the sequencing
readout proceeds in the 5 ' - to 3 ' -direction one base at a time.
Alternatively the donor
oligonucleotide can be designed so that two bases are determined in each
cycle. In this
case the first two bases on the 5 ' -end of the donor oligonucleotide are
specified (for
example, pA CRNNN I-I-X, where R = a degenerate mixture of all 4 RNA bases,
N
= a degenerate DNA base, I = inosine, and X is a fluorophore). As in all cases
there is a
5' -phosphate (p) to permit ligation of the donor oligonucleotide to the
acceptor.
Sixteen such oligonucleotide sets are required, one for each of the sixteen
possible
dinucleotides. Each of the sixteen can be labeled with a different
fluorophore.
Alternatively ligation reactions can be carried out with 4 separate pools each
having
four such sets of oligonucleotides. In that case, only four different
fluorophores are
required.
[0200] In another embodiment for sequencing in the 5'- to 3 ' -direction a
donor
oligonucleotide of the following type can be used: pA NRNNN I-I-X wherein p,
N,
R, I and X are as defined in the previous example. One base is determined at
each cycle
but at alternate positions: 1, 3, 5, etc. This may be adequate for
identification of the
sequence if compared to a reference database. If desired, the remaining bases
(positions 2, 4, 6, etc.) can be determined by repeating the sequencing
reaction on the
same template with the original acceptor oligonucleotide shifted one base
upstream or
downstream. In a related example a donor oligonucleotide of the following type
can be
used: p-A-F-FN-N-N-I-I-X wherein p, N, I and X are as defined above and F is a
degenerate mixture of all four 2' -fluoronucleosides. Following ligation,
cleavage by
RNase H2 results in the addition of two bases to the 3 ' -end of the acceptor
(i.e., AF).
After the next ligation reaction, the sequence at the 3 '-end of the acceptor
would
be ...A F SF F N-N --- I-I-X where S is the specified base at position 3,
and X would
be a different fluorophore from the previous cycle if the specified base were
not A.
Cleavage with RNase H2 next occurs between the two 2' -fluororesidues.
Cleavage by
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RNase H2 at the isolated 2'-fluororesidue occurs much more slowly and can be
avoided
by adjusting the RNase H2 concentration and reaction time.
[0201] A variant of the above method can be performed in which sequencing
proceeds in the 3'- to 5'-direction. In this case an acceptor oligonucleotide
is added at
each cycle as in the following structure: X-I-I NNNF F-S-OH wherein the
specified
base (S) is at the 3'-end of the oligonucleotide. The 5'-end is blocked to
prevent the
oligonucleotide from acting as a donor. Cleavage by RNase H2 leaves the
sequence
pF-S at the 5'-end of the donor fragment which is prepared for the next
sequencing
cycle. A capping step can be included in the cycle before the cleavage
reaction using a
phosphatase to remove the 5'-phosphate of the donor oligonucleotide if
ligation to the
acceptor failed to occur.
[0202] In a further embodiment, the invention provides an improvement for
DNA
sequencing using ribotriphosphates (or alternative analogs which provide a
substrate
for RNase H2, such as 2'-fluoronucleoside triphosphates) in conjunction with a
fluorescently labeled primer. Similar to traditional sequencing methods known
in the
art, the triphosphate residue would be incorporated by a DNA polymerase. The
concentration of the ribo triphosphate, or the alternative analog providing a
substrate
for RNase H2, is adjusted to a concentration such that on average one such
base is
incorporated randomly within each extension product produced by the
polymerase.
The nested family of fragments originating from the primer is generated by
cleavage
with RNase H2 and then separated by electrophoresis as in standard DNA
sequencing
methods. Alternatively, multiple RNA residues or modified nucleosides such as
2'-fluoronucleosides may be incorporated into the extension product and the
subsequent digestion with RNase H2 is limited so that on average each strand
is cut
only once. Four separate reactions are run, each substituting one of the bases
with a
different ribotriphosphate (A, C, T or G) or other RNase H2 cleavable analog.
In this
assay, use of expensive fluorescently labeled dideoxy triphosphate chain
terminators is
obviated.
Next Generation DNA Sequencing (NGS)
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[0203] Modern next
generation sequencing (NGS) methods typically involve PCR
amplification of sub-fragments of a target nucleic acid sample prior to
performing the
actual sequencing reactions and base identity interrogation. Selective
amplification of
regions adjacent to SNP sites could be achieved using the blocked-cleavable
primers
with RNase H2 method of the present invention, enriching those sequences for
input
into NGS analysis. Further, NGS methods can involve the need for multiplex PCR
reactions to enrich for many specific sequences of interest, and primer-dimer
and other
unwanted reactions can occur which conbritue unwanted fragemtns into the
sequencing
workflow, decreasing useful information content, increasing cost, and lowering
throughput. These
unwanted side reactions could be suppressed using the
blocked-cleavable primers with RNase H2 method of the present invention.
Tyically,
PCR amplification preformed as part of the work flow of NGS employs high
fidelity
DNA polymerases, which can have 10-100 fold lower base incorporation error
rate than
Taq DNA polymerase. Many high fidelity DNA polymerases possess a 3 '-
exonuclease
proofreading activity, which can in some cases remove a non-nucleotide
blocking
group from the 3' -end of a modified primer. Certain designs of blocked-
cleavable
primers, particularly those with internal template blocking groups (e.g.,
internal C3
spacers) having an unmodified DNA 3' -end are not recognized by the 3' -
exonuclease
activity of the high fidelity polymerase and permit linked use of blocked-
cleavable
primers and RNase H2 with amplification reactions employing a high fidelity
DNA
polymerase (see Examples 39 and 40).
Oligonucleotide Synthesis
[0204] In another
embodiment of the present invention, an improved method for
oligonucleotide synthesis is provided. Using similar techniques as described
above, a
composition acting as a donor oligonucleotide can be ligated to an acceptor
fragment in
order to add additional bases to the 3' -end of the acceptor fragment. It is
the acceptor
fragment that is the growing polynucleotide undergoing synthesis. In this
case, the
composition of the donor fragment is preferably a single-stranded
oligonucleotide that
forms a hairpin to provide a double-stranded region with an overhang of about
1-8
bases on the 3'-end. The base at the 5' end would be the desired base to add
to the
growing acceptor fragment. For synthesis of a polynucleotide containing all
four bases
(A, C, T and G), four different donor fragments are employed which can have
the
identical sequence except varying in the 5' base. Preferably the donor is
blocked at the
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3' -end so it cannot react as an acceptor. The blocking group placed at or
near the 3'-end
of the donor can be a label to allow monitoring of the reaction. Four
different labels can
be used corresponding to the four different bases at the 5' -end of the donor.
The base
adjacent to the desired base at the 5' -end is a RNA base or an alternative
analog such as
a 2' -fluoronucleoside which provides a substrate for RNase H2. The overhang
at the
3' -end can be random (degenerate) bases or universal bases or a combination
of both.
The donor fragment binds to the acceptor fragment, through hybridization of
the 3 ' -end
of the acceptor to the 3 '-overhang of the donor oligonucleotide. A DNA ligase
enzyme
is then used to join the two fragments. Next a Type II RNase H is used to
cleave the
product on the 5'-side of the RNase H2 cleavage site, transferring the 5' base
of the
donor to the 3 '-end of the acceptor. Optionally, a third step can be included
in the cycle
between the ligase and RNase H2 cleavage reactions in which molecules of the
growing
polynucleotide chain which may have failed to ligate are capped by reaction
with a
dideoxynucleotide triphosphate (or other chain terminator) catalyzed by a DNA
polymerase. In one embodiment the DNA polymerase is a deoxynucleotide terminal
transferase. The cycle is repeated, and the acceptor fragment can continue to
be
extended in a 5' to 3' direction. To facilitate isolation of the growing
polynucleotide at
each step the acceptor can be attached to a solid support such as controlled
pore glass or
polystyrene
[0205] Similar to the sequence method described above, a donor
oligonucleotide
can be used to add two bases to the 3' -end of the acceptor oligonucleotide at
each cycle.
In this case the RNase H2 cleavable residue would be positioned 3' from the 5'
end of
the donor. This enzymatic synthesis method is particularly advantageous for
synthesis
of longer DNA molecules. The hairpin reagents corresponding to each base can
be
collected for reuse in further cycles or additional syntheses. Because the
system does
not use organic solvents, waste disposal is simplified.
Kits of the Present Invention
[0206] The present invention also provides kits for nucleic acid
amplification,
detection, sequencing, ligation or synthesis that allow for use of the primers
and other
novel oligonucleotides of the present invention in the aforementioned methods.
In
some embodiments, the kits include a container containing a cleavage compound,
for
example a nicking enzyme or an RNase H enzyme; another container containing a
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DNA polymerase and/or a DNA ligase and preferably there is an instruction
booklet for
using the kits. In certain embodiments, the kits include a container
containing both a
nicking enzyme or an RNase H enzyme combined with a DNA polymerase or DNA
ligase. Optionally, the modified oligonucleotides used in the assay can be
included
with the enzymes. The cleavage enzyme agent, DNA polymerase and/or DNA ligase
and oligonucleotides used in the assay are preferably stored in a state where
they
exhibit long-term stability, e.g., in suitable storage buffers or in a
lyophilized or freeze
dried state. In addition, the kits may further comprise a buffer for the
nicking agent or
RNase H, a buffer for the DNA polymerase or DNA ligase, or both buffers.
Alternatively, the kits may further comprise a buffer suitable for both the
nicking agent
or RNase H, and the DNA polymerase or DNA ligase. Buffers may include RNasin
and
other inhibitors of single stranded ribonucleases. Descriptions of various
components
of the present kits may be found in preceding sections related to various
methods of the
present invention.
[0207] Optionally, the kit may contain an instruction booklet providing
information
on how to use the kit of the present invention for amplifying or ligating
nucleic acids in
the presence of the novel primers and/or other novel oligonucleotides of the
invention.
In certain embodiments, the information includes one or more descriptions on
how to
use and/or store the RNase H, nicking agent, DNA polymerase, DNA ligase and
oligonucleotides used in the assay as well as descriptions of buffer(s) for
the nicking
agent or RNase H and the DNA polymerase or DNA ligase, appropriate reaction
temperature(s) and reaction time period(s), etc.
[0208] Accordingly, in one embodiment, a kit for the selective
amplification of a
nucleic acid from a sample is provided. The kit comprises
(a) a first and a second oligonucleotide primer, each having a 3' end and
5' end,
wherein each oligonucleotide is complementary to a portion of a nucleic acid
to be
amplified or its complement, and wherein at least one oligonucleotide
comprises a
RNase H cleavable domain, and a blocking group linked at or near to the 3' end
of the
oligonucleotide to prevent primer extension and/or to prevent the primer from
being
copied by DNA synthesis directed from the opposite primer;
(b) an RNase H enzyme; and
(c) an instruction manual for amplifying the nucleic acid.
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The kit may optionally include a DNA polymerase.
[0209] In a further embodiment, the kit for selective amplification of a
nucleic acid
includes an oligonucleotide probe having a 3' end and a 5' end comprising an
RNase H
cleavable domain, a fluorophore and a quencher, wherein the cleavable domain
is
positioned between the fluorophore and the quencher, and wherein the probe is
complementary to a portion of the nucleic acid to be amplified or its
complement.
[0210] In yet another embodiment, the present invention is directed to a
kit for the
ligation of an acceptor oligonucleotide and a donor oligonucleotide in the
presence of a
target nucleic acid sequence. The kit comprises
(a) a donor oligonucleotide and an acceptor oligonucleotide in which one or
both of
the oligonucleotides comprise an RNase H cleavable domain and a blocking group
preventing ligation;
(b) an RNase H enzyme; and
(c) an instruction manual for ligating the acceptor and donor
oligonucleotides in the
presence of a target nucleic acid sequence.
[0211] In a further embodiment, the kit may optionally include a DNA ligase
enzyme.
[0212] In a further ligation kit embodiment, the donor oligonucleotide
contains an
RNase H cleavage domain, but lacks a blocking group at or near the 5'-end and
instead
has a free 5'-OH.
Blocking Group Designs
[0213] In several embodiments, oligonucleotide primers are provided that
include
a cleavage domain, which is cleavable by an RNase H enzyme, positioned 5' of a
blocking group. The blocking group can be linked at or near the 3'-end of the
oligonucleotide primer. The blocking group prevents primer extension and/or
inhibits
the oligonucleotide primer from serving as a template for DNA synthesis. The
blocking
group can encompass one of several designs selected from the group consisting
of
RDDDDx, RDDDDMx, RDxxD, RDxxDM, RDDDDxxD, RDDDDxxDM and DxxD,
wherein R is an RNA residue, D is a DNA residue, M is a mismatched residue and
x is
a C3 spacer. When an oligonucleotide that includes a blocking group having an
M (for
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examples, RDDDDMx, RDxxDM, RDDDDxxDM) is hybrized to the target DNA
sequence to form a double-stranded substrate, a mismatch exists at the
location of the
M in the blocking group oligonucleotide and its pairing partner in the target
DNA
sequence.
Examples
[0214] The present invention is further illustrated by reference to the
following
Examples. However, it should be noted that these Examples, like the
embodiments
described above, are illustrative and are not to be construed as restricting
the enabled
scope of the invention in any way.
EXAMPLE 1 ¨ Cloning of codon optimized RNase H2 enzymes from
thermophilic organisms
[0215] This example describes the cloning of codon optimized RNase H2
enzymes
from thermophilic organisms.
[0216] To search for functional novel RNase H2 enzymes with potentially new
and
useful activities, candidate genes were identified from public nucleotide
sequence
repositories from Archaeal hyperthermophilic organisms whose genome sequences
had
previously been determined. While RNase H2 enzymes do share some amino acid
homology and have several highly conserved residues present, the actual
homology
between the identified candidate genes was low and it was uncertain if these
represented functional RNase H2 enzymes or were genes of unknown function or
were
non-functional RNase H2 genes. As shown in Table 4, five genes were selected
for
study, including two organisms for which the RNase H2 genes have not been
characterized and three organisms to use as positive controls where the RNase
H2
genes (rnhb) and functional proteins have been identified and are known to be
functional enzymes. Although two uncharacterized predicted rnhb genes were
selected
for this initial study, many more Archaeal species have had their genome
sequences
determined whose rnhb genes are uncharacterized which could similarly be
studied.
Table 4. Five candidate RNase H2 (rnhb) genes from thermophilic bacteria
Organism Accession # Length Comments
Pyrococcus AB012613 687 bp, 228 See References (1-3) below
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Table 4. Five candidate RNase H2 (rnhb) genes from thermophilic bacteria
Organism Accession # Length Comments
kodakaraensis AA
Pyrococcus 675 bp, 224 See Reference (4) below and
AE010276
furiosus AA UA20040038366A1
Methanocaldococcus 693 bp, 230
U67470 See References (5,6) below
jannaschii AA
Pyrococcus 675 bp, 224
AJ248284 uncharacterized
abyssi AA
Sulfolobus 639 bp, 212
AE006839 uncharacterized
solfataricus AA
Bp = base pairs; AA = amino acids
References 1-6: 1) Haruki, M., Hayashi, K., Kochi, T., Muroya, A., Koga, Y.,
Morikawa, M., Imanaka, T. and Kanaya, S. (1998) Gene cloning and
characterization
of recombinant RNase HIT from a hyperthermophilic archaeon. J Bacteriol, 180,
6207-6214; 2) Haruki, M., Tsunaka, Y., Morikawa, M. and Kanaya, S. (2002)
Cleavage
of a DNA-RNA-DNA/DNA chimeric substrate containing a single ribonucleotide at
the DNA-RNA junction with prokaryotic RNases HIT. FEBS Lett, 531, 204-208; 3)
Mukaiyama, A., Takano, K., Haruki, M., Morikawa, M. and Kanaya, S. (2004)
Kinetically robust monomeric protein from a hyperthermophile. Biochemistry,
43,
13859-13866 4) Sato, A., Kanai, A., Itaya, M. and Tomita, M. (2003)
Cooperative
regulation for Okazaki fragment processing by RNase HIT and FEN-1 purified
from a
hyperthermophilic archaeon, Pyrococcus furiosus. Biochem Biophys Res Commun,
309,
247-252; 5) Lai, B., Li, Y., Cao, A. and Lai, L. (2003) Metal ion binding and
enzymatic
mechanism of Methanococcus jannaschii RNase HIT. Biochemistry, 42, 785-791;
and 6)
Lai, L., Yokota, H., Hung, L.W., Kim, R. and Kim, S.H. (2000) Crystal
structure of
archaeal RNase HIT: a homologue of human major RNase H. Structure, 8, 897-904.
[0217] The predicted physical properties of the proteins encoded by the
rnhb genes
listed above are shown in Table 5 (Pace, C.N. et al., (1995) Protein Sci., 4,
p.2411).
Table 5. Characteristics of five RNase H2 enzymes
# residues
Molecules/fig e 280 nm
Organism Mol. weightTrp, Tyr,
protein m-icm-i
Cys
Pyrococcus
25800.5 2.3E13 1, 7, 0 15930
kodakarensis
Pyrococcus
25315.2 2.4E13 2, 8, 0 22920
furiosus
Methanocaldococcus
26505.8 2.3E13 1, 9, 3 19285
jannaschii
Pyrococcus
25394.2 2.4E13 3, 7, 0 26930
abysii
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Table 5. Characteristics of five RNase H2 enzymes
# residues
Molecules/lug e 280 nm
Organism Mol. weightTrp, Tyr,
protein M- cmCys
Sulfolobus
23924.8 2.5E13 3, 10, 0 31400
solfataricus
[0218] The amino acid similarity between RNase H2 enzymes (or candidate
enzymes) from different Archaeal species within this set of 5 sequences ranges
from
34% to 65%. An amino-acid identity matrix is shown in Table 6 below.
Table 6. Amino acid identity between five Archaeal RNase H2 proteins
P. kod. P. fur. M. jann. P. ab. S. solf:
P. kodakarensis 0.570 0.595 0.358 0.333
P. furiosus 0.570 0.654 0.410 0.362
M. jannasehll 0.595 0.654 0.380 0.363
P. abysii 0.358 0.410 0.380 0.336
S. solfatarieus 0.333 0.362 0.363 0.336
[0219] Codons of the native gene sequence were optimized for expression in
E. colt
using standard codon usage tables. The following sequences were assembled and
cloned into plasmids as artificial genes made from synthetic oligonucleotides
using
standard methods. DNA sequence identity was verified on both strands.
Sequences of
the artificial DNA constructs are shown below. Lower case letters represents
linker
sequences, including a Bam HI site on the 5'-end and a Hind III site on the 3'-
end.
Upper case letters represents coding sequences and the ATG start codons are
underlined.
[0220] SEQ ID NO: 1 ¨ codon optimized mhb gene from Pyrococcus
kodakaraensis
ggat ccgATGAAGATTGCTGGCATCGATGAAGCCGGCCGTGGCCCGGTAATTGGTCCAATGGTTATCGC
TGCGGTAGTCGTGGACGAAAACAGCCTGCCAAAACTGGAAGAGCTGAAAGTGCGTGACTCCAAGAAACT
GACCCCGAAGCGCCGTGAAAAGCTGTTTAACGAAATTCTGGGTGTCCTGGACGATTATGTGATCCTGGA
GCTGCCGCCTGATGTTATCGGCAGCCGCGAAGGTACTCTGAACGAGTTCGAGGTAGAAAACTTCGCTAA
AGCGCTGAATTCCCTGAAAGTTAAACCGGACGTAATCTATGCTGATGCGGCTGACGTTGACGAGGAACG
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TTT TGCCCGCGAGC TGGGTGAACGTC TGAAC TT TGAAGCAGAGGT TGTTGCCAAACACAAGGCGGACGA
TATCTTCCCAGTCGTGTCCGCGGCGAGCATTCTGGCTAAAGTCACTCGTGACCGTGCGGTTGAAAAACT
GAAGGAAGAATACGGTGAAATCGGCAGCGGTTATCCTAGCGATCCTCGTACCCGTGCGTTTCTGGAGAA
CTACTACCGTGAACACGGTGAATTCCCGCCGATCGTACGTAAAGGTTGGAAAACCCTGAAGAAAATCGC
GGAAAAAGTTGAATCTGAAAAAAAAGCTGAAGAACGTCAAGCAACTCTGGACCGTTATTTCCGTAAAGT
Gaagct t
[0221] SEQ ID NO: 2 ¨ codon optimized rnhb gene from Pyrococcus furiosus
ggat ccgATGAAGATTGGTGGCATCGACGAAGCCGGCCGTGGTCCGGCGATCGGTCCGCTGGTAGTAGC
TACTGTTGTAGTGGATGAAAAAAACATCGAAAAACTGCGTAACATCGGCGTAAAAGACTCCAAACAGCT
GACGCCGCACGAACGTAAAAACCTGTTTTCCCAGATCACCTCCATTGCGGATGATTACAAGATCGTAAT
CGTGTCTCCGGAAGAAATTGACAACCGTAGCGGTACCATGAACGAGCTGGAAGTTGAAAAATTCGCGCT
GGCGCTGAACTCTCTGCAGATCAAGCCGGCTCTGATCTACGCAGACGCAGCAGATGTTGATGCAAACCG
CTTCGCATCCCTGATCGAACGTCGCCTGAACTATAAAGCCAAAATCATCGCGGAACACAAAGCAGACGC
AAAGTACCCGGTCGTTTCTGCGGCGAGCATTCTGGCGAAGGTTGTGCGTGACGAAGAAATCGAAAAGCT
GAAAAAGCAATATGGCGACTTTGGCAGCGGTTACCCGAGCGACCCGAAAACGAAGAAATGGCTGGAGGA
GTATTACAAGAAACATAACAGCTTCCCACCGATCGTTCGTCGTACGTGGGAAACTGTCCGCAAAATTGA
AGAGTCCATCAAAGCCAAAAAGTCCCAGCTGACCCTGGATAAATTCTTCAAGAAACCGaagct t
[0222] SEQ ID NO: 3 ¨ codon optimized rnhb gene from Methanocaldococcus
jannaschii
ggat ccgATGATTATCATTGGTATCGATGAAGCTGGCCGTGGTCCTGTACTGGGCCCGATGGTTGTATG
TGCGTTCGCTATCGAGAAGGAACGTGAAGAAGAACTGAAAAAGCTGGGCGTTAAAGATTCTAAAGAACT
GACGAAGAATAAACGCGCGTACCTGAAAAAGCTGCTGGAGAACCTGGGCTACGTGGAAAAGCGCATCCT
GGAGGCTGAGGAAATTAACCAGCTGATGAACAGCATTAACCTGAACGACATTGAAATCAACGCATTCAG
CAAGGTAGCTAAAAACCTGATCGAAAAGCTGAACATTCGCGACGACGAAATCGAAATCTATATCGACGC
TTGT TC TACTAACACCAAAAAGTTCGAAGAC TC TT TCAAAGATAAAATCGAAGATATCAT TAAAGAACG
CAATCTGAATATCAAAATCATTGCCGAACACAAAGCAGACGCCAAGTACCCAGTAGTGTCTGCGGCGAG
CAT TATCGCGAAAGCAGAACGCGACGAGATCATCGAT TAT TACAAGAAAATC TACGGTGACATCGGCTC
TGGC TACCCATC TGACCCGAAAACCATCAAATTCC TGGAAGAT TACT T TAAAAAGCACAAGAAACTGCC
GGATATCGCTCGCACTCACTGGAAAACCTGCAAACGCATCCTGGACAAATCTAAACAGACTAAACTGAT
TATCGAAaagct t
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[0223] SEQ ID NO: 4 ¨ codon optimized rnhb gene from Pyrococcus abysii
ggat ccgATGAAAGTTGCAGGTGCAGATGAAGCTGGTCGTGGTCCAGTTATTGGTCCGCTGGTTATTGT
TGCTGCTGTTGTGGAGGAAGACAAAATCCGCTCTCTGACTAAGCTGGGTGTTAAAGACTCCAAACAGCT
GACCCCGGCGCAACGTGAAAAACTGTTCGATGAAATCGTAAAAGTACTGGATGATTACTCTGTGGTCAT
TGTGTCCCCGCAGGACATTGACGGTCGTAAGGGCAGCATGAACGAACTGGAGGTAGAAAACTTCGTTAA
AGCCCTGAATAGCCTGAAAGTTAAGCCGGAAGTTATTTACATTGATTCCGCTGATGTTAAAGCTGAACG
TTTCGCTGAAAACATTCGCAGCCGTCTGGCGTACGAAGCGAAAGTTGTAGCCGAACATAAAGCGGATGC
GAAGTATGAGATCGTATCCGCAGCCTCTATCCTGGCAAAAGTTATCCGTGACCGCGAGATCGAAAAGCT
GAAAGCCGAATACGGTGATTTTGGTTCCGGTTACCCGTCTGATCCGCGTACTAAGAAATGGCTGGAAGA
ATGGTATAGCAAACACGGCAATTTCCCGCCGATCGTGCGTCGTACTTGGGATACTGCAAAGAAAATCGA
AGAAAAATTCAAACGTGCGCAGCTGACCCTGGACAACTTCCTGAAGCGTTTTCGCAACaagct t
[0224] SEQ ID NO: 5 ¨ codon optimized rnhb gene from Sulfolobus
solfataricus
ggat ccgATGCGCGTTGGCATCGATGAAGCGGGTCGCGGTGCCCTGATCGGCCCGATGATTGTTGCTGG
TGTTGTAATCTCTGACACTAAACTGAAGTTTCTGAAAGGCATCGGCGTAAAAGACTCTAAACAGCTGAC
TCGCGAGCGTCGTGAAAAGCTGTTTGATATTGTTGCTAACACTGTGGAAGCATTCACTGTCGTTAAAGT
TTTCCCTTATGAAATCGACAACTATAACCTGAATGACCTGACCTACGACGCAGTTTCTAAAATCATCCT
GAGCCTGTCTAGCTTTAACCCAGAAATTGTAACGGTTGATAAAGTGGGCGATGAGAAACCGGTTATCGA
ACTGATTAATAAGCTGGGCTACAAAAGCAACGTCGTACACAAGGCAGATGTACTGTTTGTAGAAGCCTC
CGC TGC TAGCATCATTGCGAAAGT TAT TCGTGATAAC TACATTGACGAACTGAAACAAGTATACGGTGA
CTTTGGTAGCGGTTACCCAGCTGATCCTCGCACTATCAAATGGCTGAAATCTTTCTACGAAAAGAATCC
GAATCCGCCGCCAATCATTCGTCGTTCCTGGAAGATTCTGCGTTCTACCGCCCCGCTGTATTACATTTC
CAAAGAAGGTCGCCGTCTGTGGaagct t
EXAMPLE 2 ¨ Expression of recombinant RNase H2 peptides
[0225] The following example demonstrates the expression of recombinant
RNase
H2 peptides.
[0226] The five synthetic gene sequences from Example 1 were subcloned
using
unique Bam HI and Hind III restriction sites into the bacterial expression
vector
pET-27b(+) (Novagen, EMD Biosciences, La Jolla, CA). This vector places six
histidine residues (which together comprise a "His-tag") (SEQ ID NO: 313) at
the
carboxy terminus of the expressed peptide (followed by a stop codon). A "His-
tag"
permits use of rapid, simple purification of recombinant proteins using Ni
affinity
chromatography, methods which are well known to those with skill in the art.
Alternatively, the synthetic genes could be expressed in native form without
the His-tag
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and purified using size exclusion chromatography, anion-exchange
chromatography, or
other such methods, which are also well known to a person of ordinary skill in
the art.
[0227] BL21(DE3) competent cells (Novagen) were transformed with each
plasmid and induced with 0.5 mM isopropyl-P-D-thio-galactoside (IPTG) for 4.5
hours
at 25 C. For all clones, 5 mL of IPTG induced culture was treated with
Bugbuster
Protein Extraction Reagent and Benzonase Nuclease (Novagen) to release
soluble
proteins and degrade nucleic acids according to the manufacturer's
instructions. The
recovered protein was passed over a Ni affinity column (Novagen) and eluted
with
buffer containing 1M imidazole according to protocols provided by the
manufacturer.
[0228] Both "total" and "soluble" fractions of the bacterial lysate were
examined
using SDS 10% polyaerylamide gel electrophoresis. Proteins were visualized
with
Coomassie Blue staining. Following IPTG induction, large amounts of
recombinant
proteins were produced from all 5 Archaeal RNase H2 synthetic genes. Using
this
method of purification, protein was recovered in the soluble fraction for 4
enzymes,
Pyrococcus kodakaraensis, Pyrococcus furiosus, Methanocaldococcus jannaschii,
and
Pyrococcus abyssi. No soluble protein was recovered for Sulfolobus
solfataricus
RNase H2 using this lysis procedure. Examples of induced RNase H2 proteins are
shown in Figs. 4A and 4B.
[0229] Improved methods to produce and purify the recombinant proteins were
developed to produce small scale amounts of the proteins for characterization
as
follows. To maximize the amount of soluble protein obtained for each clone, an
induction temperature of 37 C is used for 6 hours. For Pyrococcus
kodakaraensis,
Methanocaldococcus jannaschii, and Sulfolobus solfataricus, CelLyticTM B 10x
lysis
reagent (Sigma-Aldrich, St. Louis, MO) is used for lysis. A 10 fold dilution
in 500 mM
NaC1, 20 mM TrisHC1, 5 mM imidazole, pH 7.9 is made and 10 mL is used per 0.5
g of
pelleted bacterial paste from induced cultures. For Pyrococcus furiosus and
Pyrococcus abyssi, 5 mL of Bugbuster Protein Extraction Reagent (Novagen) per
100
mL of induced culture is used for cell lysis. In addition, per 100 mL induced
culture for
all clones, 5KU rLysozymeTM (Novagen) and 250U DNase I (Roche Diagnostics,
Indianapolis, IN) is used to enhance bacterial cell lysis and decrease the
viscosity of the
solution. Following centrifugation to remove cell debris, the lysates are
heated for 15
minutes at 75 C to inactive the DNase I and any other cellular nucleases
present. The
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lysates are then spun at 16,000 x g for 20 minutes to sediment denatured
protein
following heat treatment. The centrifugation step alone provides a large
degree of
functional purification of the recombinant thermostable enzymes.
[0230] The resulting soluble supernatant is passed over a Ni affinity
column
containing HisBind Resin (Novagen) and eluted with an elution buffer
containing
200 mM imidazole. The purified protein is then precipitated in the presence of
70%
ammonium sulfate and resuspended in storage buffer (10mM Tris pH 8.0, 1mM
EDTA,
100mM NaC1, 0.1% Triton X-100, 50% Glycerol) to concentrate and stabilize the
protein for long term storage. The concentrated protein is dialyzed 2 x 2
hours (x250
volumes each) against the same storage buffer to remove residual salts. The
final
purified protein is stored at -20 C. Using these protocols, for Pyrococcus
abysii, 200
mL of IPTG induced culture yields ¨2 mg of soluble protein. After passing over
a Ni
column, ¨0.7mg of pure protein is recovered. For functional use, the
concentrated
enzyme stocks were diluted in storage buffer and added 1:10 in all enzymatic
reactions
studied. Therefore all reaction buffers contain 0.01 % Triton X-100 and 5%
Glycerol.
[0231] Recombinant protein was made and purified for each of the cloned
RNase
H2 enzymes as outlined above. Samples from Pyrococcus kodakaraensis,
Pyrococcus
furiosus, Pyrococcus abyssi, and Sulfolobus solfataricus were examined using
SDS
10% polyacrylamide gel electrophoresis. Proteins were visualized with
Coomassie
Blue staining. Results are shown in Figure 5. If the expression and
purification method
functioned as predicted, these proteins should all contain a 6x Histidine tag
(SEQ ID
NO: 313), which can be detected using an anti-His antibody by Western blot.
The gel
shown in Figure 5 was electroblot transferred to a nylon membrane and a
Western blot
was performed using an anti-His antibody. Results are shown in Figure 6. All
of the
recombinant proteins were recognized by the anti-His antibody, indicating that
the
desired recombinant protein species were produced and purified.
[0232] Large scale preparations of the recombinant proteins can be better
expressed
using bacterial fermentation procedures well known to those with skill in the
art. Heat
treatment followed by centrifugation to sediment denatured proteins will
provide
substantial purification and final purification can be accomplished using size
exclusion
or anion exchange chromatography without the need for a His-tag or use of Ni-
affinity
chromatography.
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EXAMPLE 3 ¨ RNase H2 activity for the recombinant peptides
[0233] The following example demonstrates RNase H2 activity for the
recombinant peptides.
[0234] RNase H enzymes cleave RNA residues in an RNA/DNA heteroduplex. All
RNase H enzymes can cleave substrates of this kind when at least 4 sequential
RNA
residues are present. RNase H1 enzymes rapidly lose activity as the RNA
"window" of
a chimeric RNA/DNA species is shortened to less than 4 residues. RNase H2
enzymes,
on the other hand, are capable of cleaving an RNA/DNA heteroduplex containing
only
a single RNA residue. In all cases, the cleavage products contain a 3'-
hydroxyl and a
5'-phosphate (see FIG. 1). When multiple RNA residues are present, cleavage
occurs
between RNA bases, cleaving an RNA-phosphate linkage. When only a single RNA
residue is present, cleavage occurs only with Type II RNase H enzymes. In this
case
cleavage occurs on the 5'-side of the RNA base at a DNA-phosphate linkage (see
FIG.
3). RNase H enzymes require the presence of a divalent metal ion cofactor.
Typically,
RNase H1 enzymes require the presence of Mg" ions while RNase H2 enzymes can
function with any of a number of divalent cations, including but not limited
to Mg",
Mn", Co" and Ni".
[0235] The recombinant RNase H2 proteins described in Example 2 were tested
for
both types of RNase H activity and were examined for the characteristics
listed above.
[0236] Cleavage of a substrate with multiple RNA bases. The following
synthetic
30 bp substrate was used to test the activity of these enzymes for cleavage of
a long
RNA domain. The substrate is an "11-8-11" design, having 11 DNA bases, 8 RNA
bases, and 11 DNA bases on one strand and a perfect match DNA complement as
the
other strand. The oligonucleotides employed are indicated below, where upper
case
letters represent DNA bases and lower case letters represent RNA bases.
[0237] SEQ ID NO: 6
5f-CTCGTGAGGTGaugcaggaGATGGGAGGCG-3'
[0238] SEQ ID NO: 7
5f-CGCCTCCCATCTCCTGCATCACCTCACGAG-3'
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[0239] When annealed, these single-stranded (ss) oligonucleotides form the
following "11-8-11" double-stranded (ds) substrate:
[0240] SEQ ID NOS 6 and 7, respectively, in order of appearance
5f-CTCGTGAGGTGaugcaggaGATGGGAGGCG-3'
3f-GAGCACTCCACTACGTCCTCTACCCTCCGC-5'
[0241] Aliquots of each of the recombinant protein products were incubated
with
single-stranded or double-stranded oligonucleotide substrates in an 80 1,t1
reaction
volume in buffer 50 mM NaC1, 10 mM MgC12, and 10 mM Tris pH 8.0 for 20 minutes
at 45 C or 70 C. Reactions were stopped with the addition of gel loading
buffer
(formamide/EDTA) and separated on a denaturing 7M urea, 15% polyacrylamide
gel.
Gels were stained using GelStarTM (Lonza, Rockland, ME) and visualized with UV
excitation. All 5 recombinant peptides showed the ability to cleave an 8 base
RNA
sequence in an RNA/DNA heteroduplex (11-8-11) substrate. Importantly, the
recombinant proteins did not degrade the single stranded RNA-containing
oligonucleotide (SEQ ID No. 6), indicating that a double-stranded substrate
was
required. Further, a dsDNA substrate was not cleaved.
[0242] Cleavage was not observed in the absence of a divalent cation (e.g.,
no
activity was observed if Mg was absent from the reaction buffer). A Mg'
titration
was performed and high enzyme activity was observed between 2-8 mM MgC12.
Optimal activity was observed between 3-6 mM MgC12. Cleavage activity was also
detected using other divalent cations including Mn" and Co". In MnC12, good
activity
was seen from 0.3 mM to 10 mM divalent cation concentration. Enzyme activity
was
optimal in the range of 300 nM to 1 mM. For C0C12, activity was seen in the
range of
0.3 mM to 2 mM, with optimal activity in the range of 0.5 ¨ 1 mM. The isolated
enzymes therefore show RNase H activity, and divalent cation requirements that
are
characteristic of the RNase H2 class.
[0243] Digestion of the 11-8-11 substrate by recombinant RNase H2 enzymes
from
Pyrococcus kodakaraensis, Pyrococcus furiosus, and Pyrococcus abyssi is shown
in
Figure 7.
[0244] Substrate cleavage by RNase H enzymes is expected to result in
products
with a 3' -OH and 5' -phosphate. The identity of the reaction products from
the new
recombinant RNase H2 proteins was examined by mass spectrometry. Electrospray
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ionization mass spectrometry (ESI-MS) has near single Dalton resolution for
nucleic
acid fragments of this size (accuracy of +/- 0.02%). The oligonucleotide 11-8-
11
substrate (SEQ ID NOS 6 and 7) was examined by ESI-MS both before and after
digestion with the three Pyrococcus sp. RNase H enzymes. The primary masses
observed are reported in Table 7 along with identification of nucleic acid
species
consistent with the observed masses.
Table 7. Mass of species observed after RNase H2 digestion of SEQ ID NOS 6 and
7
RNase 112 Predicted
Observed
Sequence
Treatment MolWt
MolWt
None 5'
CTCGTGAGGTGaugcaggaGATGGGAGGCG (SEQ ID NO: 6) 9547 9548
(control) 3'
GAGCACTCCACTACGTCCTCTACCCTCCGC (SEQ ID NO: 7) 8984 8984
Pyrococcus 5' CTCGTGAGGTGa (SEQ ID NO: 8) 3717
3719
kodakaraen 5 P-aGATGGGAGGCG (SEQ ID
NO: 9) 3871 3871
sis 3'
GAGCACTCCACTACGTCCTCTACCCTCCGC (SEQ ID NO: 7) 8984 8984
5' CTCGTGAGGTGa (SEQ ID NO: 8) 3717
3719
Pyrococcus
P-aGATGGGAGGCG (SEQ ID NO: 9) 3871 3872
furiosus
3' GAGCACTCCACTACGTCCTCTACCCTCCGC (SEQ ID NO: 7) 8984 8984
5' CTCGTGAGGTGa (SEQ ID NO: 8) 3717
3719
Pyrococcus
5' P-aGATGGGAGGCG (SEQ ID
NO: 9) 3871 3872
abyssi
3' GAGCACTCCACTACGTCCTCTACCCTCCGC (SEQ ID NO: 7) 8984 8984
[0245] Major species identified are shown. DNA bases are indicated with
upper
case letters, RNA bases are indicated with lower case letters, and phosphate =
"P".
Molecular weights are rounded to the nearest Dalton. In the absence of other
notation,
the nucleic acids strands end in a 5' -hydroxyl or 3' -hydroxyl.
[0246] In all cases, the DNA complement strand was observed intact
(non-degraded). The RNA-containing strands were efficiently cleaved and the
observed masses of the reaction products are consistent with the following
species
being the primary fragments produced: 1) a species which contained undigested
DNA
residues and a single 3 '-RNA residue with a 3'-hydroxyl groups (SEQ ID No.
8), and 2)
a species with a 5' -phosphate, a single 5' -RNA residue, and undigested DNA
residues
(SEQ ID No. 9). The observed reaction products are consistent with the known
cleavage properties of both RNase H1 and RNase H2 enzymes.
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[0247] SEQ ID NO: 8 - 5' CTCGTGAGGTGa 3'
[0248] SEQ ID NO: 9 - 5 f P-aGATGGGAGGCG 3'
[0249] Cleavage of a substrate with a single RNA base. RNase H2 enzymes
characteristically cleave a substrate that contains a single RNA residue while
RNase H1
enzymes cannot. The following synthetic 30 bp substrates were used to test the
activity
of these enzymes for cleavage at a single RNA residue. The substrates are a
"14-1-15"
design, having 14 DNA bases, 1 RNA base, and 15 DNA bases on one strand and a
perfect match DNA complement as the other strand. Four different substrates
were
made from 8 component single-stranded oligonucleotides comprising each of the
4
RNA bases: C, G, A, and U. The oligonucleotides employed are indicated below,
where upper case letters represent DNA bases and lower case letters represent
RNA
bases.
For rC:
[0250] SEQ ID NO: 10
5f-CTCGTGAGGTGATGcAGGAGATGGGAGGCG-3'
[0251] SEQ ID NO: 11
5f-CGCCTCCCATCTCCTGCATCACCTCACGAG-3'
[0252] When annealed, these single-stranded (ss) oligonucleotides form the
following "14-1-15 rC" double-stranded (ds) substrate:
[0253] SEQ ID NOS 10 and 11, respectively, in order of appearance
5f-CTCGTGAGGTGATGcAGGAGATGGGAGGCG-3'
3f-GAGCACTCCACTACGTCCTCTACCCTCCGC-5'
For rG:
[0254] SEQ ID NO: 12 - 5' -CTCGTGAGGTGATGgAGGAGATGGGAGGCG-3'
[0255] SEQ ID NO: 13 - 5'-CGCCTCCCATCTCCTCCATCACCTCACGAG-3'
[0256] When annealed, these single-stranded (ss) oligonucleotides form the
following "14-1-15 rG" double-stranded (ds) substrate:
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[0257] SEQ ID NOS 12 and 13, respectively, in order of appearance
5f-CTCGTGAGGTGATGgAGGAGATGGGAGGCG-3'
3f-GAGCACTCCACTACCTCCTCTACCCTCCGC-5'
For TA:
[0258] SEQ ID NO: 14- 5'-CTCGTGAGGTGATGaAGGAGATGGGAGGCG-3'
[0259] SEQ ID NO: 15- 5'-CGCCTCCCATCTCCTTCATCACCTCACGAG-3'
[0260] When annealed, these single-stranded (ss) oligonucleotides form the
following "14-1-15 TA" double-stranded (ds) substrate:
[0261] SEQ ID NOS 14 and 15, respectively, in order of appearance
5f-CTCGTGAGGTGATGaAGGAGATGGGAGGCG-3'
3f-GAGCACTCCACTACTTCCTCTACCCTCCGC-5'
For rU:
[0262] SEQ ID NO: 16
5f-CTCGTGAGGTGATGuAGGAGATGGGAGGCG-3'
[0263] SEQ ID NO: 17
5f-CGCCTCCCATCTCCTACATCACCTCACGAG-3'
[0264] When annealed, these single-stranded (ss) oligonucleotides form the
following "14-1-15 rU" double-stranded (ds) substrate:
[0265] SEQ ID NOS 16 and 17, respectively, in order of appearance
5f-CTCGTGAGGTGATGuAGGAGATGGGAGGCG-3'
3f-GAGCACTCCACTACATCCTCTACCCTCCGC-5'
[0266] Aliquots of each of the recombinant protein products were incubated
with
the single-stranded and double-stranded oligonucleotide substrates indicated
above in
an 80 IA reaction volume in buffer 50 mM NaC1, 10 mM MgC12, and 10 mM Tris pH
8.0
for 20 minutes at 70 C. Reactions were stopped with the addition of gel
loading buffer
(formamide/EDTA) and separated on a denaturing 7M urea, 15% polyacrylamide
gel.
Gels were stained using GelStarTM (Lonza, Rockland, ME) and visualized with UV
excitation. All 5 recombinant peptides showed the ability to cleave a single
RNA base
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in an RNA/DNA heteroduplex (14-1-15). Each of the 4 RNA bases functioned as a
cleavable substrate with these enzymes. Importantly, the recombinant proteins
did not
degrade the single stranded RNA-containing oligonucleotides (SEQ ID Nos. 10,
12, 14,
16), indicating that a double-stranded substrate was required. The isolated
enzymes
therefore show RNase H2 activity. Titration of divalent cations was tested and
results
were identical to those obtained previously using the 8-11-8 substrate.
[0267] Digestion of the four 14-1-15 substrates (SEQ ID NOS 10-11, 12-
13, 14-15
and 16-17) and the 11-8-11 substrate (SEQ ID NOS 6 and 7) by recombinant RNase
H2
enzymes from Pyrococcus abyssi, Pyrococcus furiosus, and Methanocaldococcus
jannaschii is shown in Figure 8A and from Pyrococcus kodakaraensis in Figure
8B.
[0268] Substrate cleavage by RNase H enzymes is expected to result in
products
with a 3'-OH and 5'-phosphate. Further, cleavage of a substrate containing a
single
ribonucleotide by RNase H2 enzymes characteristically occurs at the DNA
linkage
5'-to the RNA residue. The identity of the reaction products using a single
ribonucleotide substrate from the new recombinant RNase H2 proteins was
examined
by mass spectrometry. The oligonucleotide 14-1-15 rC substrate (SEQ ID NOS 10
and
11) was examined by ESI-MS both before and after digestion with the three
Pyrococcus
sp. RNase H2 enzymes and the Methanocaldococcus jannaschii enzyme. The primary
masses observed are reported in Table 8 along with identification of nucleic
acid
species consistent with the observed masses.
Table 8. Mass of species observed after RNase H2 digestion of SEQ ID NOS 10
and 11
RNase 112 Predicted
Observed
Sequence
Treatment Mol Wt Mol Wt
5' CTCGTGAGGTGATGcAGGAGATGGGAGGCG (SEQ ID NO: 10) 9449
9450
None (control)
3' GAGCACTCCACTACGTCCTCTACCCTCCGC (SEQ ID NO: 11) 8984
8984
5' CTCGTGAGGTGATG (SEQ ID NO: 18)
4334 4335
Pyrococcus
5' P-cAGGAGATGGGAGGCG
(SEQ ID NO: 19) 5132 5133
kodakaraensis
3' GAGCACTCCACTACGTCCTCTACCCTCCGC (SEQ ID NO: 11) 8984
8984
5' CTCGTGAGGTGATG (SEQ ID NO: 18)
4334 4335
Pyrococcus
5' P-cAGGAGATGGGAGGCG
(SEQ ID NO: 19) 5132 5132
furiosus
3' GAGCACTCCACTACGTCCTCTACCCTCCGC (SEQ ID NO: 11) 8984
8984
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Table 8. Mass of species observed after RNase H2 digestion of SEQ ID NOS 10
and 11
RNase 112 Predicted
Observed
Sequence
Treatment Mol Wt
Mol Wt
5' CTCGTGAGGTGATG (SEQ ID NO: 18)
4334 4335
Pyrococcus
5' P-cAGGAGATGGGAGGCG
(SEQ ID NO: 19) 5132 5133
abyssi
3' GAGCACTCCACTACGTCCTCTACCCTCCGC (SEQ ID NO: 11) 8984 8984
Methanocaldoco 5' CTCGTGAGGTGATG (SEQ ID NO: 18)
4334 4335
ccus 5' P-cAGGAGATGGGAGGCG (
SEQ ID NO: 1 9 ) 5132 5133
jannaschii 3
GAGCACTCCACTACGTCCTCTACCCTCCGC ( SEQ ID NO: 11) 8984 8984
[0269] Major species identified are shown. DNA bases are indicated
with upper
case letters, RNA bases are indicated with lower case letters, and phosphate =
"P".
Molecular weights are rounded to the nearest Dalton. In the absence of other
notation,
the nucleic acids strands end in a 5' -hydroxyl or 3' -hydroxyl.
[0270] In all cases, the DNA complement strand was observed intact
(non-degraded). The RNA-containing strands were efficiently cleaved and the
observed masses of the reaction products are consistent with the following
species
being the primary fragments produced: 1) a species which contained undigested
DNA
residues with a 3'-hydroxyl (SEQ ID No. 18), and 2) a species with a 5' -
phosphate, a
single 5'-RNA residue, and undigested DNA residues (SEQ ID No. 19). The
observed
reaction products are consistent with the known cleavage properties of RNase
H2 class
enzymes.
[0271] SEQ ID NO: 18
5' CTCGTGAGGTGATG 3'
[0272] SEQ ID NO: 19
5' P-cAGGAGATGGGAGGCG 3'
[0273] In summary, the cloned, codon-optimized rnhb genes predicted to
encode
RNase H2 enzymes from 5 Archaeal species all produced recombinant protein
products
which displayed enzyme activities consistent with that expected for members of
the
RNase H2 family. 1) The enzymes required divalent cation to function (the
experiments presented here were done using Mg"). Activity is also present
using Mn"
or Co" ions; 2) Single-stranded nucleic acids are not degraded; 3) Double-
stranded
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heteroduplex nucleic acids are substrates where one strand contains one or
more RNA
bases; 4) For substrates containing 2 or more consecutive RNA bases, cleavage
occurs
in a DNA-RNA-DNA chimera between RNA linkages; for substrates containing a
single RNA base, cleavage occurs immediately 5'-to the RNA base in a
DNA-RNA-DNA at a DNA linkage; and 6) Reaction products have a 3-hydroxyl and
5' -phosphate.
EXAMPLE 4 ¨ Reaction temperature optimization and thermal stability of
Pyrococcus abyssi RNase H2
[0274] For this example and all subsequent work, the amount of the enzyme
employed was standardized based upon the following unit definition, where:
1 unit is defined as the amount of enzyme that results in the cleavage of 1
nmole of a heteroduplex substrate containing a single rC residue per
minute at 70 C in a buffer containing 4 mM Mg' at pH 8Ø
[0275] Substrate SEQ ID NOS 10 and 11 were employed for characterizing
RNase
H2 enzyme preparation for the purpose of normalizing unit concentration. The
following standardized buffer was employed unless otherwise noted. "Mg
Cleavage
Buffer": 4 mM MgC12, 10 mM Tris pH 8.0, 50 mM NaC1, 10 ug/m1 BSA (bovine serum
albumin), and 300 nM oligo-dT (20mer poly-dT oligonucleotide). The BSA and
oligo-dT serve to saturate non-specific binding sites on plastic tubes and
improve the
quantitative nature of assays performed.
[0276] Purified recombinant Pyrococcus abyssi RNase H2 enzyme was studied
for
thermal stability. Aliquots of enzyme were incubated at 95 C for various
periods of
time and then used to cleave the single rC containing substrate SEQ ID NOS 10
and 11.
The RNA strand of the substrate was radiolabeled with 32P using 6000 Ci/mmol
7-32P-ATP and the enzyme T4 Polynucleotide Kinase (Optikinase, US
Biochemical).
Trace label was added to reaction mixtures (1:50). Reactions were performed
using
100 nM substrate with 100 microunits ( U) of enzyme in Mg Cleavage Buffer.
Reactions were incubated at 70 C for 20 minutes. Reaction products were
separated
using denaturing 7M urea, 15% polyacrylamide gel electrophoresis (PAGE) and
visualized using a Packard CycloneTM Storage Phosphor System (phosphorimager).
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The relative intensity of each band was quantified using the manufacturer's
image
analysis software and results plotted as a fraction of total substrate
cleaved. Results are
shown in Figure 9. The enzyme retained full activity for over 30 minutes at 95
C.
Activity was reduced to 50% after 45 minutes incubation and to 10% after 85
minutes
incubation.
[0277] These results demonstrate that the Pyrococcus abyssi RNase H2 enzyme
is
sufficiently thermostable to survive prolonged incubation at 95 C and would
therefore
survive conditions typically employed in PCR reactions.
[0278] The temperature dependence of the activity of the Pyrococcus abyssi
RNase
H2 enzyme was next characterized. The activity was studied over a 40 C
temperature
range from 30 C to 70 C. The RNA strand of the rC substrate SEQ ID NOS 10 and
11
was radiolabeled as described above. Reactions were performed using 100 nM
substrate with 200 microunits ( U) of enzyme in Mg Cleavage Buffer. Reactions
were
incubated at 30 C, 40 C, 50 C, 60 C, or 70 C for 10 minutes. Reactions were
stopped
with the addition of cold EDTA containing formamide gel loading buffer.
Reaction
products were then separated using denaturing 7M urea, 15% polyacrylamide gel
electrophoresis (PAGE) and visualized using a Packard CycloneTM Storage
Phosphor
System (phosphorimager). The resulting gel image is shown in Figure 10. The
relative
intensity of each band was quantified using the manufacturer's image analysis
software
and results plotted as a fraction of total substrate cleaved (see FIG. 11).
The enzyme
shows only ¨0.1% activity at 30 C and does not attain appreciable activity
until about
50 to 60 C.
[0279] Therefore, for practical purposes the enzyme is functionally
inactive at
room temperature. Reactions employing this enzyme can therefore be set up on
ice or
even at room temperature and the reactions will not proceed until temperature
is
elevated. If Pyrococcus abyssi RNase H2 cleavage were linked to a PCR
reaction, the
temperature dependent activity demonstrated herein would effectively function
to
provide for a "hot start" reaction format in the absence of a modified DNA
polymerase.
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EXAMPLE 5¨ Cleavage at non-standard bases by RNase H2
[0280] The natural biological substrates for RNase H1 and RNase H2 are
duplex
DNA sequences containing one or more RNA residues. Modified bases containing
substitutions at the 2'-position other than hydroxyl (RNA) have not been
observed to be
substrates for these enzymes. The following example demonstrates that the
Pyrococcus
abyssi RNase H2 enzyme has activity against modified RNA-containing
substrates.
[0281] The following 14-1-15 substrates containing modified bases were
tested to
determine if RNase H2 could recognize single non-RNA 2'-modified bases as
sites for
cleavage. The modifications are located on the 2' position of the base and
include
locked nucleic acid (LNA), 2'-0-methyl (2'0Me), and 2'-fluoro (2'F); the
single
ribo-C containing substrate was employed as positive control. Hereafter, LNA
bases
will be designated with a "+" prefix (+N), 2' OMe bases will be designated
with a "m"
prefix (mN), 2'F bases will be designated with a "f' prefix (fN), and 2'-amino
bases
with an "a" prefix (aN).
Ribo-C Substrate
[0282] SEQ ID NOS 10 and 11, respectively, in order of appearance
5'-CTCGTGAGGTGATGcAGGAGATGGGAGGCG-3'
3f-GAGCACTCCACTACGTCCTCTACCCTCCGC-5'
LNA-C Substrate
[0283] SEQ ID NOS 20 and 274, respectively, in order of appearance
5f-CTCGTGAGGTGATG(+C)AGGAGATGGGAGGCG-3'
3f-GAGCACTCCACTAC G TCCTCTACCCTCCGC-5'
2'0Me-C Substrate
[0284] SEQ ID NOS 21 and 274, respectively, in order of appearance
5f-CTCGTGAGGTGATG(mC)AGGAGATGGGAGGCG-3'
3f-GAGCACTCCACTAC G TCCTCTACCCTCCGC-5'
2'F-C Substrate
[0285] SEQ ID NOS 22 and 274, respectively, in order of appearance
5f-CTCGTGAGGTGATG(fC)AGGAGATGGGAGGCG-3'
3f-GAGCACTCCACTAC G TCCTCTACCCTCCGC-5'
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[0286] The above 4 substrates were incubated in an 80 11,1 reaction volume
in
various buffers for 20 minutes at 70 C with the recombinant Pyrococcus abyssi
RNase
H2 enzyme. Buffers tested included 50 mM NaC1, 10 mM Tris pH 8.0 with either
10
mM MgC12, 10 mM C0C12, or 10 mM MnC12. Reactions were stopped with the
addition of gel loading buffer (formamide/EDTA) and separated on a denaturing
7M
urea, 15% polyacrylamide gel. Gels were stained using GelStarTM (Lonza,
Rockland,
ME) and visualized with UV excitation. Results are shown in Figure 12. The
control
substrate with a single ribo-C residue was 100% cleaved. The substrates
containing a
single LNA-C or a single 2'0Me-C residue were not cleaved. However, the
substrate
containing a single 2'-F-C residue was cleaved to a small extent. This
cleavage
occurred only in the manganese containing buffer and was not seen in either
cobalt or
magnesium buffers.
[0287] Cleavage at a 2'-F-C base was unexpected. Cleavage of 2'-fluoro
bases was
investigated further using the following substrates.
Ribo-C Substrate
[0288] SEQ ID NOS 10 and 11, respectively, in order of appearance
5'-CTCGTGAGGTGATGcAGGAGATGGGAGGCG-3'
3f-GAGCACTCCACTACGTCCTCTACCCTCCGC-5'
2'F-C Substrate
[0289] SEQ ID NOS 22 and 274, respectively, in order of appearance
5f-CTCGTGAGGTGATG(fC)AGGAGATGGGAGGCG-3'
3f-GAGCACTCCACTAC G TCCTCTACCCTCCGC-5'
2'F-U Substrate
[0290] SEQ ID NOS 23 and 275, respectively, in order of appearance
5f-CTCGTGAGGTGATG(fU)AGGAGATGGGAGGCG-3'
3f-GAGCACTCCACTAC A TCCTCTACCCTCCGC-5'
2'F-C + 2'FU (fCfU) Substrate
[0291] SEQ ID NOS 24 and 276, respectively, in order of appearance
5f-CTCGTGAGGTGATG(fCfU)GGAGATGGGAGGCG-3'
3f-GAGCACTCCACTAC G A CCTCTACCCTCCGC-5'
[0292] The above 4 substrates were incubated in an 80 11,1 reaction volume
in a
buffer containing 50 mM NaC1, 10 mM Tris pH 8.0 and 10 mM MnC12 for 20 minutes
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at 70 C with either the recombinant Pyrococcus abyssi RNase H2 enzyme or the
recombinant Pyrococcus furiosus RNase H2 enzyme. Reactions were stopped with
the
addition of gel loading buffer (formamide/EDTA) and separated on a denaturing
7M
urea, 15% polyacrylamide gel. Gels were stained using GelStarTM (Lonza,
Rockland,
ME) and visualized with UV excitation. Results are shown in Figure 13. The
control
substrate with a single ribo-C residue was 100% cleaved. The substrates
containing a
single 2'-F-C or single 2'-F-U residue were cleaved to a small extent. The di-
fluoro
substrate containing adjacent 2'-F-C and 2'-F-U residues (fCfU) was cleaved
nearly
100%. Further, both the Pyrococcus abyssi and Pyrococcus furiosus RNase H2
enzymes cleaved the modified substrate in an identical fashion. This example
demonstrates that the unexpected cleavage of the fC group was not restricted
to fC but
also occurred with fU. More importantly, a combination of 2 sequential 2'-
fluoro
modified bases was a far better substrate for RNase H2.. This novel cleavage
property
was seen for both the P. abyssi and P. furiosus enzymes. Cleavage of such
atypical
substrates may be a property common to all Archaeal RNase H2 enzymes.
[0293] The
identity of the cleavage products of the di-fluoro fCfU substrate was
studied using mass spectrometry using the methods described in Example 3.
Using
traditional ribonucleotide substrates, cleavage by RNase H enzymes results in
products
with a 3'-OH and 5'-phosphate. The fCfU substrate (SEQ ID NOS 24 and 276) were
examined by ESI-MS both before and after digestion by the recombinant
Pyrococcus
abyssi RNase H2 enzyme. The primary masses observed are reported in Table 9
along
with identification of nucleic acid species consistent with the observed
masses.
Table 9. Mass of species observed after RNase H2 digestion of SEQ ID NOS 24
and 276
RNase 112 Predicted
Observed
Sequence
Treatment Mol Wt Mol Wt
None 5f-CTCGTGAGGTGATG(fCfU)GGAGATGGGAGGCG-3' (SEQ ID NO: 24) 9446
9446
(control) 3f-
GAGCACTCCACTAC G A CCTCTACCCTCCGC-5' (SEQ ID NO: 276) 8993 8994
5' CTCGTGAGGTGATG(fC) (SEQ ID NO: 277) 4642
4643
Pyrococcus
5' P-(fU)GGAGATGGGAGGCG (SEQ ID NO: 278) 4822
4823
abyssi
3' GAGCACTCCACTAC G A CCTCTACCCTCCGC (SEQ ID NO: 276) 8993
8994
Major species identified are shown. DNA bases are indicated with upper case
letters,
2'-F bases are indicated as fC or fU, and phosphate = "P". Molecular weights
are
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rounded to the nearest Dalton. In the absence of other notation, the nucleic
acids
strands end in a 5'-hydroxyl or 3' -hydroxyl.
[0294] The mass spectrometry data indicates that digestion of a di-fluoro
substrate
such as the fCfU duplex studied above by RNase H2 results in cleavage between
the
two fluoro bases. Further, the reaction products contain a 3' -hydroxyl and
5' -phosphate, similar to the products resulting from digestion of RNA
containing
substrates.
[0295] Cleavage of the modified bases was not observed in the absence of
a
divalent cation. A titration was performed and enzyme activity was observed
between
0.25-10 mM MnC12 and 0.25-1.5 mM C0C12. Enzyme activity was optimal in the
range
of 0.5 mM to 1 mM for both MnC12 and C0C12. Hereafter 0.6 mM MnC12 was
employed in reactions or 0.5 mM C0C12. Reduced activity for cleavage of the
modified
substrate was observed using Mg buffers. Overall, optimum activity was
observed
using Mn buffers for cleavage of the di-fluoro (fNfN) substrates whereas Mg
buffers
were superior for cleavage of ribonucleotide (rN) substrates.
[0296] The ability of the RNase H2 enzymes to cleave at single or double
2'-F
bases was unexpected. The Pyrococcus abyssi RNase H2 enzyme was next tested
for
the ability to cleave a greater variety of modified substrates using the same
methods
described above in this example. The modified strand of the substrate was
radiolabeled
as described above. Reactions were performed using 100 nM substrate and 480-
1000
mU of recombinant enzyme in Mn Cleavage Buffer (10 mM Tris pH 8.0, 50 mM NaC1,
0.6 mM MnC12, 10 .tg/m1 BSA). Reactions were incubated at 70 C for 20 minutes.
Reaction products were separated using denaturing 7M urea, 15% polyacrylamide
gel
electrophoresis (PAGE) and visualized using a Packard CycloneTM Storage
Phosphor
System (phosphorimager). The relative intensity of each band was quantified
using the
manufacturer's image analysis software and results plotted as a fraction of
total
substrate cleaved are shown in Table 10.
Table 10: Cleavage of substrates containing 2'-modification by Pyrococcus
abyssi RNase H2 using increased amounts
of enzyme
2'-Mod Oligo Sequence SEQ ID NOS Cleavage
fN-fN 5' -CTCGTGAGGTGAT ( fNfN) AGGAGATGGGAGGCG- 3 ' 25 +++++
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Table 10: Cleavage of substrates containing 2'-modification by Pyrococcus
abyssi RNase H2 using increased amounts
of enzyme
2'-Mod Oligo Sequence SEQ ID NOS Cleavage
3'-GAGCACTCCACTA N N TCCTCTACCCTCCGC-5' 279
5'-CTCGTGAGGTGAT(fU+C)AGGAGATGGGAGGCG-3' 26
fU-LNA-C ++++
3'-GAGCACTCCACTA A G TCCTCTACCCTCCGC-5' 280
5' -CTCGTGAGGTGAT (mUfC) AGGAGATGGGAGGCG-3 ' 27
mU-fC +++
3'-GAGCACTCCACTA A G TCCTCTACCCTCCGC-5' 280
5' -CTCGTGAGGTGAT (mU+C) AGGAGATGGGAGGCG-3 ' 28
mU-LNA-C ++
3'-GAGCACTCCACTA A G TCCTCTACCCTCCGC-5' 279
5' -CTCGTGAGGTGAT (mUmN) AGGAGATGGGAGGCG-3 ' 29
mU-mN ++
3'-GAGCACTCCACTA A N TCCTCTACCCTCCGC-5' 281
Amino-U-LNA- 5' -CTCGTGAGGTGAT (aU+C) AGGAGATGGGAGGCG-3 ' 30
3'-GAGCACTCCACTA A G TCCTCTACCCTCCGC-5' 280
' - CTCGTGAGGTGATG ( fN) AGGAGATGGGAGGCG-3 31
fN
3 ' - GAGCACTCCACTAC N TCCTCTACCCTCCGC -5 ' 282
5' -CTCGTGAGGTGAT (mUaC) AGGAGATGGGAGGCG-3 ' 32
mU-Amino-C
3'-GAGCACTCCACTA A G TCCTCTACCCTCCGC-5' 280
5'-CTCGTGAGGTGAT(+TfC)AGGAGATGGGAGGCG-3' 33
LNA-T-fC
3'-GAGCACTCCACTA A G TCCTCTACCCTCCGC-5' 280
5 ' - CTCGTGAGGTGATG (aU) AGGAGATGGGAGGCG-3 34
Amino-U
3 ' - GAGCACTCCACTAC A TCCTCTACCCTCCGC -5 ' 275
LNA-T- 5' -CTCGTGAGGTGAT (+T+C)
AGGAGATGGGAGGCG-3 ' 35
LNA-C 3'-GAGCACTCCACTA A G TCCTCTACCCTCCGC-5' 280
5'-CTCGTGAGGTGAT(fUmC)AGGAGATGGGAGGCG-3' 36
fU-mC
3'-GAGCACTCCACTA A G TCCTCTACCCTCCGC-5' 280
5'-CTCGTGAGGTGAT(+TmC)AGGAGATGGGAGGCG-3' 37
LNA-T-mC
3'-GAGCACTCCACTA A G TCCTCTACCCTCCGC-5' 280
Uppercase letters = DNA; fN = 2'-F bases, +N = LNA bases, mN = 2'0Me bases, aN
= 2'-amino bases
Use of "N" base indicates that every possible base (A, G, C, U/T) was tested
with the appropriate perfect match
complement. Efficiency of cleavage was rated from "+++++" (100% cleavage) to "-
" (no cleavage). The mUmN
substrates did not cleave equally well and the "++" rating applies to the best
cleaving dinucleotide pair, mUmU. The
rank order of cleavage for this substrate design was mUmU > mUmA > mUmC >
mUmG.
[0297] It is clear from the above results that many different 2'-
modifications can be
cleaved by RNase H2 enzymes that were not heretofore appreciated. Of the
2'-modified substrates, the di-fluoro compounds (those with 2 sequential 2'-
fluoro
bases) were most active. Additional substrates were tested, including some
with 3 or 4
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sequential 2'-fluoro bases. No icrease in activity was seen when increasing
the
2'-fluoro content above 2 residues.
[0298] A similar series of experiments was performed using lower amounts of
enzyme. The experiment below was conducted using an identical protocol except
that
148 !AU of recombinant Pyrococcus abyssi RNase H2 was employed instead of the
480
mU previously employed (3000-fold less enzyme) and the buffer contained a
mixture
of divalent cations (3 mM MgC12 + 0.6 mM MnC12). Under these conditions, a
substrate containing a single ribonucleotide residue is completely cleaved
whereas
modified substrates are not. Results are shown in Table 11. RNase H2 is more
active in
cleaving substrates containing an RNA base than in cleaving the 2'-modified
bases.
Table 11. Cleavage of substrates containing 2'-modification by Pyrococcus
abyssi RNase H2 using small
amounts of enzyme
2'-Mod Oligo Sequence SEQ ID NOS
Cleavage
5' -CTCGTGAGGTGATGnAGGAGATGGGAGGCG- 3 ' 38
rN +++++
3'-GAGCACTCCACTACNTCCTCTACCCTCCGC-5' 282
5'-CTCGTGAGGTGAT(fUrC)AGGAGATGGGAGGCG-3' 39
fil-rC +++++
3'-GAGCACTCCACTA A G TCCTCTACCCTCCGC-5' 275
5'-CTCGTGAGGTGAT(rUfC)AGGAGATGGGAGGCG-3' 40
ril-fC ++++
3'-GAGCACTCCACTA A G TCCTCTACCCTCCGC-5' 275
5'-CTCGTGAGGTGAT(fNfN)AGGAGATGGGAGGCG-3' 25
fN-fN
3'-GAGCACTCCACTA N N TCCTCTACCCTCCGC-5' 279
5'- CTCGTGAGGTGATG(fN)AGGAGATGGGAGGCG-3' 31
fN
3'- GAGCACTCCACTAC N TCCTCTACCCTCCGC-5' 282
Uppercase letters = DNA; fN = 2'-F bases. Use of "N" base indicates that every
possible base (A, G, C, U/T) was
tested with the appropriate perfect match complement. Efficiency of cleavage
was rated from "+++++" (100%
cleavage) to "-" (no cleavage).
[0299] Thus, Pyrococcus abyssi RNase H2 can be used to cleave substrates
which
do not contain any RNA bases but instead contain 2'-modified bases. Of the
compounds studied, di-fluoro (fNfN) containing substrates performed best. Use
of the
modified substrates generally requires increased amounts of enzyme, however
the
enzyme is catalytically very potent and it presents no difficulty to employ
sufficient
enzyme to achieve 100% cleavage of a di-fluoro substrate.
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[0300] The 2'-modified substrate described in this example are not
susceptible to
cleavage by typical RNase enzymes. As such they can be employed in novel assay
formats where cleavage events are mediated by RNase H2 using substrates that
are
completely resistant to cleavage by other RNase enzymes, particularly single
stranded
ribonucleases.
EXAMPLE 6 ¨ Base preferences for cleavage of the di-fluoro MIN substrate
[0301] The following example demonstrates that all 16 possible 2'-fluoro
dinucleotides can be cleaved by RNase H2. Distinct base preferences are
observed.
[0302] The modified strand of each substrate was radiolabeled as described
above.
Reactions were performed using 100 nM substrate with 25 mU of recombinant
enzyme
in Mn Cleavage Buffer (10 mM Tris pH 8.0, 50 mM NaC1, 0.6 mM MnC12, 10 ng/m1
BSA). Reactions were incubated at 70 C for 20 minutes. Reaction products were
separated using denaturing 7M urea, 15% polyacrylamide gel electrophoresis
(PAGE)
and visualized using a Packard CycloneTM Storage Phosphor System
(phosphorimager).
The relative intensity of each band was quantified, and results plotted as a
fraction of
total substrate cleaved are shown in Figure 14. The enzyme amount was titrated
so that
the most active substrate cleaved at 90-95% without having excess enzyme
present so
that accurate assessment could be made of relative cleavage efficiency for
less active
substrates.
[0303] All 16 dinucleotide fNfN pairs were cleaved by RNase H2, however
clear
substrate preferences were observed. In general, substrates having the
sequence fNfU
performed worse, indicating that placement of a fU base at the 3'-position of
the
dinucleotide pair was unfavorable. The least active substrate was fUfU, which
showed
10% cleavage under conditions that resulted in >90% cleavage of fAfC or fAfG
substrates.
[0304] Using greater amounts of enzyme, the relative differences of
cleavage
efficiency between substrates is minor and 100% cleavage can readily be
achieved for
all substrates studied here.
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EXAMPLE 7 ¨ Optimization of 3'- and 5'- base lengths for cleavage of rN and
fNfIN substrates
[0305] The following example shows the optimization of the placement of the
cleavable domain relative to the 3' and 5' ends of a primer or probe sequence.
In the
prior examples, the substrates all had 14 or 15 DNA bases on both the 5'- and
3'-sides
flanking the cleavable domain. For use in designing cleavable probes and
primers, it
may at times be beneficial to make these flanking sequences as short as
possible, in
order to control Tm (hybridization temperature) or to improve specificity of
priming
reactions. It is therefore important to define the minimum length of duplex
needed to
obtain efficient enzymatic cleavage.
[0306] In this experiment, the synthetic substrate duplexes shown in Table
13 were
made having a single rC cleavable base, a fixed domain of 25 DNA bases 5'-
flanking
the ribonucleotide and a variable number of bases on the 3'-side.
Table 13:
3'-End Sequence (rC) SEQ ID NOS
5f-CTGAGCTTCATGCCTTTACTGTCCTcT-3' 43
3' -D1
3f-GACTCGAAGTACGGAAATGACAGGACA-5' 283
5f-CTGAGCTTCATGCCTTTACTGTCCTcTC-3' 44
3'-D2
3f-GACTCGAAGTACGGAAATGACAGGACAG-5' 284
5f-CTGAGCTTCATGCCTTTACTGTCCTcTCC-3' 45
3'-D3
3f-GACTCGAAGTACGGAAATGACAGGACAGG-5' 285
5f-CTGAGCTTCATGCCTTTACTGTCCTcTCCTT-3' 46
3'-D5
3f-GACTCGAAGTACGGAAATGACAGGACAGGAA-5' 286
5f-CTGAGCTTCATGCCTTTACTGTCCTcTCCTTC-3' 47
3'-D6
3f-GACTCGAAGTACGGAAATGACAGGACAGGAAG-5' 287
[0307] The modified strand of each substrate was radiolabeled as described
above
Reactions were performed using 100 nM substrate with 100 nU of recombinant
enzyme
in Mg Cleavage Buffer (10 mM Tris pH 8.0, 50 mM NaC1, 4 mM MgC12, 10 ng/m1
BSA). Reactions were incubated at 70 C for 20 minutes. Reaction products were
separated using denaturing 7M urea, 15% polyacrylamide gel electrophoresis
(PAGE)
and visualized using a Packard CycloneTM Storage Phosphor System
(phosphorimager).
The relative intensity of each band was quantified, and results plotted as a
fraction of
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total substrate cleaved are shown in Figure 15. Maximal cleavage occurred with
4-5
DNA bases flanking the ribonucleotide on the 3 '-side.
[0308] In the next experiment, the synthetic substrate duplexes shown in
Table 14
were made having a single rU cleavable base with a fixed domain of 25 base-
pairs
flanking the ribonucleotide on the 3'side and 2-14 base-pairs on the 5'-side.
A
minimum of 5 unpaired bases (dangling ends) were left on the unmodified
complement
to simulate hybridization to a long nucleic acid sample.
Table 14:
5'-End Sequence (rU) SEQ ID NOS
5' -CuCCTGAGCTTCATGCCTTTACTGTCC-3 ' 48
5'-D1
3'-ACGTAGAATGGACAGAAGGAGGACTCGAAGTACGGAAATGACAGGACGTA-5' 288
5'-CCuCCTGAGCTTCATGCCTTTACTGTCC-3' 49
5'-D2
3'-ACGTAGAATGGACAGAAGGAGGACTCGAAGTACGGAAATGACAGGACGTA-5' 288
5'-TCCuCCTGAGCTTCATGCCTTTACTGTCC-3' 50
5'-D3
3'-ACGTAGAATGGACAGAAGGAGGACTCGAAGTACGGAAATGACAGGACGTA-5' 288
5'-TTCCuCCTGAGCTTCATGCCTTTACTGTCC-3' 51
5'-D4
3'-ACGTAGAATGGACAGAAGGAGGACTCGAAGTACGGAAATGACAGGACGTA-5' 288
5'-CTTCCuCCTGAGCTTCATGCCTTTACTGTCC-3' 52
5'-D5
3'-ACGTAGAATGGACAGAAGGAGGACTCGAAGTACGGAAATGACAGGACGTA-5' 288
5'-TCTTCCuCCTGAGCTTCATGCCTTTACTGTCC-3' 53
5'-D6
3'-ACGTAGAATGGACAGAAGGAGGACTCGAAGTACGGAAATGACAGGACGTA-5' 288
5'-TGTCTTCCuCCTGAGCTTCATGCCTTTACTGTCC-3' 54
5'-D8
3'-ACGTAGAATGGACAGAAGGAGGACTCGAAGTACGGAAATGACAGGACGTA-5' 288
5'-CCTGTCTTCCuCCTGAGCTTCATGCCTTTACTGTCC-3' 55
5'-D10
3'-ACGTAGAATGGACAGAAGGAGGACTCGAAGTACGGAAATGACAGGACGTA-5' 288
5'-TACCTGTCTTCCuCCTGAGCTTCATGCCTTTACTGTCC-3' 56
5'-D12
3'-ACGTAGAATGGACAGAAGGAGGACTCGAAGTACGGAAATGACAGGACGTA-5' 288
5'-CTTACCTGTCTTCCuCCTGAGCTTCATGCCTTTACTGTCC-3' 57
5'-D14
3'-ACGTAGAATGGACAGAAGGAGGACTCGAAGTACGGAAATGACAGGACGTA-5' 288
[0309] The modified strand of each substrate was radiolabeled as previously
described. Reactions were performed using 100 nM substrate with 123 nU of
recombinant enzyme in a mixed buffer containing both Mg and Mn cations (10 mM
Tris pH 8.0, 50 mM NaC1, 0.6 mM MnC12, 3 mM MgC12, 10 ng/m1 BSA). Reactions
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were incubated at 70 C for 20 minutes. Reaction products were separated using
denaturing 7M urea, 15% polyacrylamide gel electrophoresis (PAGE) and
visualized
using a Packard CycloneTM Storage Phosphor System (phosphorimager). The
relative
intensity of each band was quantified and the results plotted as a fraction of
total
substrate cleaved in Figure 16. Little cleavage was seen with the short
substrates.
Activity increased with length of the 5'-DNA domain until maximum cleavage was
obtained at around 10-12 bases of duplex flanking the rU base on the 5'-side.
[0310] Similar experiments were done to determine the optimal length of the
3'- DNA domain needed for cleavage of di-fluoro (fNfN) substrates. The
duplexes
shown in Table 15 were synthesized and tested to functionally define the
length of
DNA bases needed at the 3'-end of a fUfC di-fluoro substrate. A fixed domain
of 22
base pairs was positioned at the 5'-end and the 3'-domain was varied from 2-14
bases.
Table 15:
3'-End Sequence (MC) SEQ ID NOS
5' -CTGAGCTTCATGCCTTTACTGT (fUfC) CC-SpC3-3' 58
3'-D2
3f-GACTCGAAGTACGGAAATGACA A G GGGCTGTGTGTCGAG-5' 289
5f-CTGAGCTTCATGCCTTTACTGT(fUfC)CCCG-SpC3-3' 59
3'-D4
3f-GACTCGAAGTACGGAAATGACA A G GGGCTGTGTGTCGAG-5' 289
5f-CTGAGCTTCATGCCTTTACTGT(fUfC)CCCGA-SpC3-3' 60
3'-D5
3f-GACTCGAAGTACGGAAATGACA A G GGGCTGTGTGTCGAG-5' 289
5f-CTGAGCTTCATGCCTTTACTGT(fUfC)CCCGAC-SpC3-3' 61
3'-D6
3f-GACTCGAAGTACGGAAATGACA A G GGGCTGTGTGTCGAG-5' 289
5f-CTGAGCTTCATGCCTTTACTGT(fUfC)CCCGACAC-SpC3-3' 62
3'-D8
3f-GACTCGAAGTACGGAAATGACA A G GGGCTGTGTGTCGAG-5' 289
5f-CTGAGCTTCATGCCTTTACTGT(fUfC)CCCGACACAC-SpC3-3' 63
3'-D10
3f-GACTCGAAGTACGGAAATGACA A G GGGCTGTGTGTCGAG-5' 289
5f-CTGAGCTTCATGCCTTTACTGT(fUfC)CCCGACACACAG-SpC3-3' 64
3'-D12
3f-GACTCGAAGTACGGAAATGACA A G GGGCTGTGTGTCGAG-5' 289
5f-CTGAGCTTCATGCCTTTACTGT(fUfC)CCCGACACACAGCT-SpC3-3' 65
3'-D14
3f-GACTCGAAGTACGGAAATGACA A G GGGCTGTGTGTCGAG-5' 289
[0311] The modified strand of each substrate was radiolabeled as above.
Reactions
were performed using 100 nM substrate with 37 mU of recombinant enzyme in a
mixed
buffer containing both Mg and Mn cations (10 mM Tris pH 8.0, 50 mM NaC1, 0.6
mM
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MnC12, 3 mM MgC12, 10 ug/m1 BSA). Reactions were incubated at 70 C for 20
minutes. Reaction products were separated using denaturing 7M urea, 15%
polyacrylamide gel electrophoresis (PAGE) and visualized using a Packard
CycloneTM
Storage Phosphor System (phosphorimager). The relative intensity of each band
was
quantified and the results plotted as a fraction of total substrate cleaved in
Figure 17.
No cleavage was seen with the substrate having 2 DNA bases on the 3'-side of
the
cleavable domain. Cleavage was seen with 4 DNA bases and steadily increased
until
maximal cleavage was obtained when 8-10 DNA bases were present on the 3'-side
of
the fUfC cleavage domain. Interestingly, the optimal length of DNA bases on
the
3'-side of the cleavage domain is longer for the di-fluoro substrates (8-10
bases)
compared with the single ribonucleotide substrates (4-5 bases).
[0312] In summary, for ribonucleotide containing substrates, maximal
cleavage
activity is seen when at least 4-5 DNA residues are positioned on the 3'-side
and 10-12
DNA residues are positioned on the 5'-side of the cleavable domain. For di-
fluoro
substrates, maximal cleavage activity is seen when at least 8-10 DNA residues
are
positioned on the 3'-side of the cleavable domain; from prior examples it is
clear that
activity is high when 14-15 DNA residues are positioned on the 5'-side of the
cleavable
domain.
EXAMPLE 8 ¨ Application to DNA primers: primer extension assay format and
potential utility in DNA sequencing
[0313] The examples above characterized the ability of a thermostable RNase
H2
enzyme to cleave a duplex nucleic acid at a single internal ribonucleotide or
at a
2'-fluoro dinucleotide. Example 7 establishes parameters for designing short
oligonucleotides which will be effective substrates in this cleavage reaction.
These
features can be combined to make cleavable primers that function in primer
extension
assays, such as DNA sequencing, or PCR. A single stranded oligonucleotide is
not a
substrate for the cleavage reaction, so a modified oligonucleotide primer will
be
functionally "inert" until it hybridizes to a target sequence. If a cleavable
domain is
incorporated into an otherwise unmodified oligonucleotide, this
oligonucleotide could
function to prime PCR and will result in an end product wherein a sizable
portion of the
primer domain could be cleaved from the final PCR product, resulting in
sterilization of
the reaction (lacking the priming site, the product will no longer be a
template for PCR
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using the original primer set). If the cleavable domain is incorporated into
an
oligonucleotide which is blocked at the 3'-end, then this primer will not be
active in
PCR until cleavage has occurred. Cleavage will "activate" the blocked primer.
As
such, this format can confer a "hot start" to a PCR reaction, as no DNA
synthesis can
occur prior to the cleavage event. Example 4 showed that this cleavage event
is very
inefficient with Pyrococcus abysii RNase H2 until elevated temperatures are
attained.
Additionally, the linkage between the cleavage reaction and primer extension
confer
added specificity to the assay, since both steps requireenzymatic recognition
of the
duplex formed when the primer hybridizes to the template. A schematic of this
reaction
is shown in Figure 18. Note that this schema applies to both simple primer
extension
reactions as well as PCR. It can also be exploited in other kinds of enzymatic
assays
such as ligation reactions.
[0314] The following example demonstrates the use of an RNase H2 cleavable
primer for DNA sequencing. The most common method of DNA sequencing in use
today involves sequential DNA synthesis reactions (primer extension reactions)
done in
the presence of dideoxy terminator nucleotides. The reaction is done in a
thermal
cycling format where multiple cycles of primer extension are performed and
product
accumulates in a linear fashion.
[0315] DNA sequencing was done using the Big DyeTM Terminator V3.1 Cycle
Sequencing Kit (Applied Biosystems, Foster City, CA). The following primers
were
used:
M13(-27)
[0316] SEQ ID No. 66
5f-CAGGAAACAGCTATGAC-3'
M13(-27)-rC
[0317] SEQ ID No. 67
5f-CAGGAAACAGCTATGACcATGA-SpC3-3'
[0318] As before, DNA bases are indicated in upper case, RNA bases are
indicated
in lower case, and SpC3 is a spacer C3 blocking group placed at the 3'-end of
the
oligonucleotide. The blocked cleavable primer contains 17 DNA bases on the 5'-
side
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of the ribonucleotide and 4 DNA bases on the 3'-side of the ribonucleotide (17-
1-4
design) and so conforms to the optimized design rules established in Example
7.
[0319] Sequencing reactions were set up in 20 1.1,1 volume comprising 0.75X
ABI
Reaction buffer, 160 nM primer, 0.5X Big Dye Terminators and 230 ng plasmid
DNA
template. Optionally, 4 mM additional MgC12 was supplemented into the
reaction, with
or without 14, 1.4, or 0.14 mU of recombinant Pyrococcus abyssi RNase H2. The
following cycle sequencing program was employed: 96 C for 30 seconds followed
by
25 cycles of [96 C for 5 seconds, 50 C for 10 seconds, 55 C for 4 minutes].
The DNA
sequencing reactions were run on an Applied Biosystems model 3130x1 Genetic
Analyzer. The resulting sequencing traces were examined for quality and read
length.
Results are summarized in Table 16 below.
Table 16. Results of cycle sequencing using a rC blocked cleavable primer
Read length in ABI Read length in ABI
Primer RNase H2
Buffer Buffer +4 mM MgC12
0 >800 ¨500
M13(-27) 0.14 mU >800 >800
SEQ ID No. 66 1.4 mU >800 >800
14 mU >800 >800
0 0 0
M13(-27)-rC 0.14 mU 0 0
SEQ ID No. 67 1.4 mU 0 ¨300
14 mU ¨300 >800
[0320] Control reactions using an unmodified primer resulted in high
quality DNA
sequence traces with usable read lengths slightly exceeding 800 bases. The
addition of
RNase H2 enzyme to these reactions did not compromise reaction quality. The
manufacturer (Applied Biosystems) does not disclose the cation content of the
buffer
provided in the sequencing kits, so actual reaction conditions are not
certain.
Supplementation of the reactions with an additional 4 mM MgC12 had no effect.
The rC
blocked cleavable primer did not support DNA sequencing without the addition
of
RNase H2. With the addition of RNase H2, high quality sequencing reactions
were
obtained using 14 mU of enzyme in the 20 1.1,1 reaction. Use of lower amounts
enzyme
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resulted in lower quality reactions or no functional reaction at all.
Supplementing
magnesium content of the reaction buffer was necessary to obtain cleavage and
primer
extension reactions using the blocked primers. The amount of enzyme employed
here
is 100-fold higher than is needed to achieve 100% cleavage of a rN substrate
under
optimal conditions (70 C, 20 minute incubation). In the cycle sequencing
reactions
performed herein, primer annealing was run at 50 C and extension reactions
were run at
55 C for 10 seconds and 4 minutes, respectively. These lower temperatures are
suboptimal for Pyrococcus abyssi RNase H2 (see Example 4 above). Performing
the
cycle sequencing reaction at higher temperatures will require less enzyme but
is not
necessary.
[0321] This example demonstrates that blocked primers containing an
internal
cleavage site for RNase H2 can be used with primer-extension based sequencing
methods, such as dideoxy (Sanger) sequencing, and are compatible with use of
existing
high throughput fluorescent sequencing protocols. Use of blocked primers and
the
method of the present invention can confer added specificity to the sequencing
reaction,
thus permitting sequencing to be performed for more cycles and on highly
complex
nucleic acid samples that work poorly with unmodified primers.
EXAMPLE 9 ¨ Application to DNA primers: rN primers in PCR and quantitative
real-time PCR
[0322] Example 8 demonstrated that RNase H2 could be used to cleave a
blocked
primer and that this system could be linked to DNA synthesis and primer
extension
reactions, including DNA sequencing. The following example demonstrates the
utility
of this method in PCR. The first system demonstrates use in an end point PCR
format
and the second system demonstrates use in a quantitative real-time PCR format.
[0323] The primers shown in Table 17, were made for use in a synthetic end-
point
PCR assay. The Syn-For and Syn-Rev primers are unmodified control primers
specific
for an artificial amplicon (a synthetic oligonucleotide template). The Syn-For
primer is
paired with the unmodified control Syn-Rev primer or the different modified
Syn-Rev
primers. A set of modified Syn-Rev primers were made which contain a single rU
(cleavable) base followed by 2-6 DNA bases, all ending with a dideoxy-C
residue
(ddC). The ddC residue functions as a blocking group that prevents primer
function.
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The ddC blocking group is removed with cleavage of the primer at the rU base
by the
action of RNase H2 (the unblocking step, shown in Figure 18). The synthetic
template
is a 103-base long oligonucleotide, shown below (SEQ ID No. 75). Primer
binding
sites are underlined.
Table 17:
Name Sequence SEQ ID No.
Syn-F or 5f-AGCTCTGCCCAAAGATTACCCTG-3' SEQ ID No. 68
Syn-Rev 5f-CTGAGCTTCATGCCTTTACTGT-3' SEQ ID No. 69
Syn-Rev-rU-2D 5' -CTGAGCT TCATGCCT TTACTGTuCC -ddC- 3 ' SEQ ID No. 70
Syn-Rev-rU-3D 5' -CTGAGCT TCATGCCT TTACTGTuCCC-ddC - 3 ' SEQ ID No. 71
Syn-Rev-rU-4D 5' -CTGAGCT TCATGCCT TTACTGTuCCCC -ddC- 3 ' SEQ ID No. 72
Syn-Rev-rU-5D 5' -CTGAGCT TCATGCCT TTACTGTuCCCCG-ddC - 3 ' SEQ ID No. 73
Syn-Rev-rU-6D 5' -CTGAGCT TCATGCCT TTACTGTuCCCCGA-ddC- 3 ' SEQ ID No. 74
DNA bases are shown in uppercase. RNA bases are shown in lowercase. ddC
indicates a
dideoxy-C residue which functions as a blocking group.
Synthetic template
[0324] SEQ ID No. 75
AGCTCTGCCCAAAGATTACCCTGACAGCTAAGTGGCAGTGGAAGTTGGCCTCAGAAGTAGTGGCCAGCT
GTGTGTCGGGGAACAGTAAAGGCATGAAGCTCAG
[0325] PCR reactions were performed in 20 ul volume using 200 nM primers, 2
ng
template, 200 uM of each dNTP (800 uM total), 1 unit of Immolase (a
thermostable
DNA polymerase, Bioline), 50 mM Tris pH 8.3, 50 mM KC1, and 3 mM MgC12.
Reactions were run either with or without 100 uU of Pyrococcus abyssi RNase
H2.
Reactions were started with a soak at 95 C for 5 minutes followed by 35 cycles
of [95 C
for 10 seconds, 60 C for 30 seconds, and 72 C for 1 second]. Reaction products
were
separated on a 10% non-denaturing polyacrylamide gel and visualized using
GelStar
staining. Results are shown in Figure 19. Unmodified control primers produced
a
strong band of the correct size. 3' -end blocked rU primers did not produce
any
products in the absence of RNase H2. In the presence of RNase H2, blocked
primers
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produced a strong band of the correct size using the D4, D5, and D6 primers.
No signal
was seen using the D2 or D3 primers. This example demonstrates that blocked
primers
can be used in PCR reactions using the method of the present invention.
Further, this
example is consistent with results obtained using cleavage of preformed duplex
substrates in Example 7, where the presence of 4-5 3' -DNA bases were found to
be
optimal for cleavage of rN containing primers.
[0326] The same synthetic PCR amplicon assay system described above was
next
tested in a quantitative real-time PCR assay using SYBR Green detection.
Reactions
were done in 384 well format using a Roche Lightcycler 480 platform.
Reactions
comprised lx BIO-RAD iQTM SYBR Green Supermix (BIO-RAD, Hercules, CA),
200 nM of each primer (for + rev), 2 x 106 copies of synthetic template
oligonucleotide
(SEQ ID No. 75), and 5 mU of Pyrococcus abyssi RNase H2 in 101.1,1 volume.
Thermal
cycling parameters included an initial 5 minutes soak at 95 C and then 45
cycles were
performed of [95 C for 10 seconds + 60 C for 20 seconds + 72 C for 30
seconds]. All
reactions were run in triplicate and reactions employed the same unmodified
For primer
(SEQ ID No. 68). The Rev primer was varied between the unmodified and 2-6D
modified primers (SEQ ID Nos. 69-74). Cp values, the PCR cycle number where a
positive reaction is first detected, in these experiments are shown in Table
18 below.
The Cps were essentially identical for control reactions done using unmodified
For +
Rev primers and the coupled cleavage PCR reactions performed using the D4, D5,
or
D6 blocked primers in the presence of RNase H2. In the absence of RNase H2, no
positive signal was detected using the blocked primers. As was seen in the end
point
assay, performance was reduced for the primers having shorter 3 '-DNA domains
(D2
or D3).
Table 18. Cp values for SYBR Green qPCR reactions using
cleavable blocked primers in a synthetic amplicon system with
RNase H2 present
Reverse Primer SEQ ID No. Cp Value
Syn-Rev (Control) SEQ ID No. 69 17.7
Syn-Rev-rU-2D SEQ ID No. 70 23.4
Syn-Rev-rU-3D SEQ ID No. 71 23.0
Syn-Rev-rU-4D SEQ ID No. 72 16.8
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Table 18. Cp values for SYBR Green qPCR reactions using
cleavable blocked primers in a synthetic amplicon system with
RNase H2 present
Reverse Primer SEQ ID No. Cp Value
Syn-Rev-rU-5D SEQ ID No. 73 16.6
Syn-Rev-rU-6D SEQ ID No. 74 16.9
All reactions used the same unmodified For primer, SEQ ID No.
68
[0327] The following example demonstrates use of RNase H2 cleavage using rN
blocked primers (both For and Rev) in a quantitative real-time PCR assay
format using
an endogenous human gene target and HeLa cell cDNA as template. The primers
shown in Table 19 specific for the human HRAS gene (NM_176795) were designed
and synthesized. In this case a C3 spacer was use das the blocking group.
Table 19:
SEQ ID
Name Sequence
No.
HRAS-618-For 5f-ACCTCGGCCAAGACCC-3' SEQ ID No.
76
HS-9 16-Rev 5f-CCTTCCTTCCTTCCTTGCTTCC-3' SEQ ID No.
77
HRAS-618-For-rG- SEQ ID No.
5f-ACCTCGGCCAAGACCCgGCAG-SpC3-3'
D4 78
HRAS-916-Rev-rG- SEQ ID No.
5f-CCTTCCTTCCTTCCTTGCTTCCgTCCT-SpC3-3'
D4 79
Uppercase represents DNA bases, lowercase represents RNA bases. SpC3 is a
spacer
C3 placed as a blocking group on the 3 '-end.
[0328] These primers define a 340 bp amplicon within the HRAS gene as shown
below. Primer binding sites are underlined.
HRAS assay amplicon
[0329] SEQ ID No. 80
ACCTCGGCCAAGACCCGGCAGGGCAGCCGCTCTGGCTCTAGCTCCAGCTCCGGGACCCTCTGGGACCCC
CCGGGACCCATGTGACCCAGCGGCCCCTCGCGCTGGAGTGGAGGATGCCTTCTACACGTTGGTGCGTGA
GATCCGGCAGCACAAGCTGCGGAAGCTGAACCCTCCTGATGAGAGTGGCCCCGGCTGCATGAGCTGCAA
GTGTGTGCTCTCCTGACGCAGCACAAGCTCAGGACATGGAGGTGCCGGATGCAGGAAGGAGGTGCAGAC
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GGAAGGAGGAGGAAGGAAGGACGGAAGCAAGGAAGGAAGGAAGG
[0330] Reactions were performed in 10 1 volume in 384 well format using a
Roche
Lightcycler 480 platform. Reactions comprised lx BIO-RAD iQTM SYBR Green
Supermix (BIO-RAD, Hercules, CA) using the iTAQ DNA polymerase at 25U/ml, 3
mM MgC12, 200 nM of each primer (for + rev), 2 ng cDNA (made from HeLa cell
total
RNA), with or without 5 mU of Pyrococcus abyssi RNase H2. Thermal cycling
parameters included an initial 5 minutes soak at 95 C and then 50 cycles were
performed of [95 C for 10 seconds + 60 C for 20 seconds + 72 C for 30
seconds]. All
reactions were run in triplicate. Using unmodified primers, the crossing point
(Cp)
occurred at cycle 27. In the absence of RNase H2, reactions done with blocked
primers
did not support PCR and no fluorescence signal was detected during the 50
cycle
reaction. In the presence of RNase H2, reactions done with blocked primers
produced
detectable signal at cycle 27.4, essentially identical to the control
unblocked primers.
Real time PCR fluorescence plots are shown in Figure 20.
[0331] The following example demonstrates use of RNase H2 cleavage using rN
blocked primers in a quantitative real-time PCR assay format using another
endogenous human gene target and HeLa cell cDNA as the template. The primers
specific for the human ETS2 gene (NM_005239) shown in Table 20 were designed
and
synthesized.
Table 20:
Name Sequence SEQ ID No.
ETS2-300-For 5f-CCCTGTTTGCTGTTTTTCCTTCTC-3' SEQ ID No. 81
ETS2-463-Rey 5f-CGCCGCTGTTCCTTTTTGAAG-3' SEQ ID No. 82
ETS2-300-For-rU-D4 5' -CCCTGTTTGCTGTTTTTCCTTCTCuAAAT-SpC3-3' SEQ ID No. 83
ETS2-463-Rev-rC-D4 5' -CGCCGCTGTTCCTTTTTGAAGcCACT-SpC3-3' SEQ ID No. 84
Uppercase represents DNA bases, lowercase represents RNA bases. SpC3 is a
spacer C3 placed
as a blocking group on the 3 '-end
[0332] These primers define a 184 bp amplicon within the ETS2 gene as shown
below. Primer binding sites are underlined.
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ETS2 assay amplicon
[0333] SEQ ID No. 85
CCCTGTTTGCTGTTTTTCCTTCTCTAAATGAAGAGCAAACACTGCAAGAAGTGCCAACAGGCTTGGATT
CCATTTCTCATGACTCCGCCAACTGTGAATTGCCTTTGTTAACCCCGTGCAGCAAGGCTGTGATGAGTC
AAGCCTTAAAAGCTACCTTCAGTGGCTTCAAAAAGGAACAGCGGCG
[0334] Reactions were performed in 10 n1 volume in 384 well format using a
Roche
Lightcycler 480 platform. Reactions comprised lx BIO-RAD iQTM SYBR Green
Supermix (BIO-RAD, Hercules, CA) using the iTAQ DNA polymerase at 25U/ml, 3
mM MgC12, 200 nM of each primer (for + rev), 2 ng cDNA (made from HeLa cell
total
RNA), with or without 5 mU of Pyrococcus abyssi RNase H2. Thermal cycling
parameters included an initial 5 minutes soak at 95 C and then 50 cycles were
performed of [95 C for 10 seconds + 60 C for 20 seconds + 72 C for 30
seconds]. All
reactions were run in triplicate. Using unblocked primers, the Cp occurred at
cycle 25.7.
In the absence of RNase H2, reactions done with blocked primers did not
support PCR
and no fluorescence signal was detected out to 50 cycles. In the presence of
RNase H2,
reactions done with blocked primers produced detectable signal at cycle 31.7,
a delay of
6 cycles from the unmodified control primers. Reactions done using one blocked
primer (unmodified For + blocked Rev or blocked For + unmodified Rev) showed
intermediate Cp values. Real time PCR fluorescence plots are shown in Figure
21.
[0335] Using the present reaction conditions, the HRAS assay performed
identically using unmodified vs. blocked primers. However, the ETS2 assay
showed a
delay between unmodified vs. blocked primers. In the setting of a PCR reaction
where
rapid thermal cycling occurs, primer hybridization and cleavage kinetics play
a
significant role in the efficiency of the overall reaction for reactions which
employ the
blocked primers. DNA synthesis is linked to the unblocking event, and
unblocking
requires hybridization, binding of RNase H2, and substrate cleavage before
primers
become activated and are capable of priming DNA synthesis. It should be
possible to
increase the amount of cleaved primer produced each cycle by either increasing
the
amount of RNase H2 enzyme present or by increasing the anneal time of the
reaction.
DNA synthesis occurs at the anneal temperature (60 C) nearly as well as at the
extension temperature (72 C) used in the above examples. However, unblocking
can
only take place during the duration of the anneal step (60 C) and not during
the extend
step (72 C) due to the Tm of the primers employed which only permit formation
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double-stranded substrate for RNase H2 during the anneal step but not at 72 C
(where
the primers only exist in single-stranded form).
[0336] PCR cycle parameters were changed to a 2 step reaction with
anneal/extend
as a single event done at 60 C and the duration of the anneal/extend step was
varied to
see if changing these reaction parameters could allow the blocked ETS2 primers
to
perform with similar efficiency as the unmodified control primers. Reactions
were
done in 10 IA volume in 384 well format using a Roche Lightcycler 480
platform.
Reactions comprised lx BIO-RAD iQTM SYBR Green Supermix (BIO-RAD,
Hercules, CA) using the iTAQ DNA polymerase at 25U/ml, 3 mM MgC12, 200 nM of
each primer (for + rev), 2 ng cDNA (made from HeLa cell total RNA), with or
without
rnU of Pyrococcus abyssi RNase H2. Thermal cycling parameters included an
initial
5 minutes soak at 95 C and then 45 cycles were performed of [95 C for 10
seconds +
60 C for 20-120 seconds]. All reactions were run in triplicate. The
differences
between the Cp values obtained for the blocked primers and the unmodified
control
primers (ACp) are summarized in Table 21 below.
Table 21. ACp values for SYBR Green qPCR ETS2 reactions
comparing unmodified and cleavable blocked primers
Combined time ACp Value
at 60 C (anneal/extend)
20 seconds 6.1
60 seconds 1.2
90 seconds 0.6
120 seconds 0.4
[0337] Minor adjustment of the cycling parameters and increasing the
duration of
the 60 C anneal step from 20 seconds to 1-2 minutes led to uniform performance
between the blocked-cleavable primers and the control unmodified primers.
Similar
experiments were performed keeping the cycling parameters fixed and increasing
enzyme. As predicted, it was possible to improve performance of the blocked
primers
using higher amounts of enzyme. Doubling the amount of enzyme employed to 10
mU
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RNase H resulted in minimal difference between control unblocked and blocked
cleaveable primers when using a 30 second anneal step at 60 C.
[0338] The above example demonstrates that blocked primers containing a
single
ribonucleotide residue of the optimized design taught in Example 7 can be used
with
RNase H2 in quantitative real-time PCR assays.
EXAMPLE 10¨ Application to DNA primers: MIN primers in PCR and
quantitative real-time PCR
[0339] Example 9 above demonstrated utility of RNase H2 mediated cleavage
for
use of rN blocked primers in end point and quantitative real time PCR assays.
The
present example demonstrates utility using fNfN blocked primers in
quantitative real
time PCR assays.
[0340] Since cleavage of the di-fluoro substrate by RNase H2 results in a
species
having a 3' -OH end, this product should also be able to support PCR reactions
using the
same reaction format as described in Example 9, assuming that primers bearing
a single
2'-F base (fN) are capable of priming DNA synthesis. Cleavage of a di-fluoro
substrate
proceeds best in the presence of manganese cations, whereas PCR reactions
generally
are performed in the presence of magnesium cations. PCR reactions using
unmodified
primers were tested using standard qPCR buffer containing 3 mM MgC12 and a
modified buffer containing 3 mM MgC12 + 0.6 mM MnC12. Reaction performance was
identical and the presence of this low amount of manganese did not adversely
affect the
quantitative nature of the reaction.
[0341] The ability of a terminal 3' -fN primer to function in PCR was
investigated
using the synthetic PCR amplicon system described in example 9. The following
primers shown in Table 22 were tested:
Table 22:
Name Sequence SEQ ID No.
Syn-F or 5f-AGCTCTGCCCAAAGATTACCCTG-3' SEQ ID No. 68
Syn-Rev 5f-CTGAGCTTCATGCCTTTACTGT-3' SEQ ID No. 69
Syn-Rev-fU 5f-CTGAGCTTCATGCCTTTACTGT(fU)-3' SEQ ID No. 86
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Table 22:
Name Sequence SEQ ID No.
DNA bases are shown in uppercase. 2'-fluoro bases are indicated as fN.
Synthetic template
[0342] SEQ ID No. 75
AGCTCTGCCCAAAGATTACCCTGACAGCTAAGTGGCAGTGGAAGTTGGCCTCAGAAGTAGTGGCCAGCT
GTGTGTCGGGGAACAGTAAAGGCATGAAGCTCAG
[0343] Reactions were done in 10 1.1,1 volume in 384 well format using a
Roche
Lightcycler 480 platform. Reactions comprised lx BIO-RAD iQTM SYBR Green
Supermix (BIO-RAD, Hercules, CA) using the iTAQ DNA polymerase at 25U/ml, 3
mM MgC12, 0.6 mM MnC12, 200 nM of each primer (for + rev), 2 x 106 copies of
synthetic oligonucleotide target, with or without 1.75 U of Pyrococcus abyssi
RNase
H2. Thermal cycling parameters included an initial 5 minutes soak at 95 C and
then 30
cycles were performed of [95 C for 10 seconds + 60 C for 120 seconds + 72 C
for 120
seconds]. All reactions were run in triplicate. Results are shown in Figure
22. In the
absence of RNase H2, the primer haying a 2'-F base at the 3' -end supported
PCR with
identical efficiency compared with the unmodified primer. However, in the
presence of
RNase H2, the 2'-F modified primer showed a 3.5 Cp delay compared with the
unmodified primer. This results not from the inhibition of DNA synthesis by
RNase H2,
but from a low level of cleavage of the primer from the amplification product
by RNase
H2. Following DNA synthesis, incorporation of a fN-containing primer into the
newly
formed DNA product creates a potential substrate for RNase H2 (see example 5
above).
Cleavage at the 2'-F base will remove the priming site from this strand of the
amplicon,
effectively sterilizing this product so that any products made from it will be
incapable
of further priming events. It is this reaction sequence which occurs in
polynomial
amplification. Cleavage of substrates containing a single 2'-F residue is
relatively
inefficient, however, so only a modest decrease in PCR reaction efficiency is
seen.
Extending incubation at 72 C following PCR should result in total cleavage of
the
primer from the amplification product, completely blocking the ability of
further
amplification to occur and thereby sterilizing the product. This should be
useful in
cross-contamination control of PCR reactions.
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[0344] Given that the cleavage of a single 2'-F residue is inefficient, use
of lower
amounts of enzyme, or eliminating the 72 C elongation step permits cleavage
of a
difluoro blocked primer by RNase H2 without significantly cleaving the primer
extension reaction product containing a single 2' fluoro residue.
Alternatively, it
should be possible to block this cleavage event by selective placement of a
phosphorothioate modification between the terminal 2'-F residue and the
adjacent
DNA base.
[0345] The ability of a di-fluoro blocked primer to support qPCR was
demonstrated
using the primers shown in Table 23, in the synthetic oligonucleotide amplicon
system,
described in Example 9 above.
Table 23:
Name Sequence SEQ ID No.
Syn-For 5'-AGCTCTGCCCAAAGATTACCCTG-3' SEQ ID No. 68
Syn-Rev-fU 5f-CTGAGCTTCATGCCTTTACTGT(fU)-3' SEQ ID No. 86
Syn-Rev-fUfC-D 5' -CTGAGCTTCATGCCTTTACTGT (fUfC) CCCGACACA
SEQ ID No. 87
C-SpC3-3'
DNA bases are shown in uppercase. 2'-F bases are indicated as fN. SpC3
indicates a spacer
C3 group employed to block the 3' -end
Synthetic template
[0346] SEQ ID No. 75
AGCTCTGCCCAAAGATTACCCTGACAGCTAAGTGGCAGTGGAAGTTGGCCTCAGAAGTAGTGGCCAGCT
GTGTGTCGGGGAACAGTAAAGGCATGAAGCTCAG
[0347] Reactions were done in 10 ial volume in 384 well format using a
Roche
Lightcycler 480 platform. Reactions comprised lx BIO-RAD iQTM SYBR Green
Supermix (BIO-RAD, Hercules, CA) using the iTAQ DNA polymerase at 25U/ml, 3
mM MgC12, 0.6 mM MnC12, 200 nM of each primer (for + rev), 2 x 106 copies of
synthetic oligonucleotide target, with or without 1.75 U of Pyrococcus abyssi
RNase
H2. Thermal cycling parameters included an initial 5 minutes soak at 95 C and
then 45
cycles were performed of [95 C for 10 seconds + 60 C for 120 seconds + 72 C
for 120
seconds]. All reactions were run in triplicate. The reactions run with the
control primer
having a single 2' -fluoro base at the 3'-end (which mimics the cleavage
product of the
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fNfN blocked primer) had a Cp of 20. Reactions run with the blocked fUfC
primer also
had a Cp of 20.
[0348] The amount of RNase H2 enzyme needed in the di-fluoro primer
cleavage
assay was next studied in more detail. Reactions were done in 101.1,1 volume
in 384 well
format using a Roche Lightcycler 480 platform. Reactions comprised lx BIO-RAD
iQTM SYBR Green Supermix (BIO-RAD, Hercules, CA) using the iTAQ DNA
polymerase at 25U/ml, 3 mM MgC12, 0.6 mM MnC12, 200 nM of each primer (for +
rev), 2 x 106 copies of synthetic oligonucleotide target. The same unmodified
Syn-For
primer was used in all reactions. Recombinant Pyrococcus abyssi RNase H2 was
added from 0 to 600 mU per reaction. Thermal cycling parameters included an
initial 5
minutes soak at 95 C and then 45 cycles were performed of [95 C for 10 seconds
+
60 C for 120 seconds + 72 C for 120 seconds]. All reactions were run in
triplicate. Cp
values corresponding to the varying amounts of RNase H2 for each primer are
shown in
Table 24.
Table 24. Optimization of the amount of RNase H2 for qPCR reactions using a
fUfC
blocked primer
Amount of RNase H2 used per reaction
Primer 600 mU 400 mU 200 mU 100 mU 0 mU
Syn-Rev 17.9 17.7 17.2 17.1 17.0
Syn-Rev-fU 25.6 23.2 19.9 18.5 17.0
Syn-Rev-fU 24.6 22.9 21.3 21.9 ND
fC-D10
ND = not detected.
[0349] The optimal amount of RNase H2 is 200 mU (Cp = 21.3 shown in bold
and
underlined). At higher concentrations of RNase H2 PCR reaction is less
efficient, and
to a similar degree, with both the 3' fluoroU primer and the blocked difluoro
primer.
Presumably this is due to a low level of cleavage at the fU set within the PCR
product as
discussed above.
[0350] Generally about 200 mU of Pyrococcus abysii RNase H2 per 10 1.1,1 is
the
optimal enzyme concentration for a coupled RNase H2-PCR with blocked primers
wherein the RNase H2 cleavage domain is two consecutive 2' -fluoronucleosides.
An
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increase in Cp compared to standard unmodified DNA primers of between 2 and 6
cycles is typically observed. This small difference has no effect on assay
performance
because results are always compared to a standard curve of Cp vs. target copy
number
generated with the same primers as used to test unknown samples.
[0351] In conclusion, this example has demonstrated that blocked fNfN
primers
can support qPCR reactions using RNase H2 cleavage with the methods of the
present
invention and defines optimal amounts of RNase H2 and cycling conditions to
employ.
EXAMPLE 11 ¨ Improved specificity using rN blocked primers in PCR reactions.
[0352] In theory, PCR has an almost unlimited potential for amplification
and a
PCR reaction should only be limited by consumption of reagents in the reaction
mix. In
actual practice, PCR reactions are typically limited to 40-45 cycles to help
preserve
specificity. The amplification power of PCR is enormous and, as cycle number
exceeds
40-45, it becomes increasingly common for mispriming events to give rise to
amplification of undesired products and false positive signals. This example
demonstrates how use of cleavable blocked primers with the methods of the
present
invention improves reaction specificity and permits use of a greater number of
PCR
cycles, thereby increasing the potential sensitivity of PCR.
[0353] In this example, we studied PCR reactions specific for 3 human genes
and
compared the specificity of each set of primer pairs in amplification using
human and
rat cDNA as the template. Traditional unmodified oligonucleotides were
compared
with the new cleavable blocked primers of the present invention. The following
primers, as shown in Table 25, were employed. DNA bases are shown in upper
case,
RNA bases in lower case, and the 3 '-blocking group employed was a C3 spacer
(SpC3).
The gene targets studied were human ETS2, NM 005239 (rat homolog
NM 001107107), human HRAS, NM 176795 (rat homolog NM 001061671), and
human ACACA, NM 198834 (rat homolog NM 022193).
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Table 25:
Gene Primer SEQ ID No. Sequence
ETS2 hETS2-For SEQ ID No. 88
CCCTGTTTGCTGTTTTTCCTTCTC
hET S2-F or-rU SEQ ID No. 89
CCCTGTTTGCTGTTTTTCCTTCTCuAAAT-SpC3
hET S2 -Rev SEQ ID No. 90 CGCCGCTGTTCCTTTTTGAAG
hET S2-Rev-rC SEQ ID No. 91
CGCCGCTGTTCCTTTTTGAAGcCACT-SpC3
HRAS hHRAS-For SEQ ID No. 92 ACCTCGGCCAAGACCC
hHRAS-For-rG SEQ ID No. 93 ACCTCGGCCAAGACCCgGCAG-
SpC3
hHRAS-Rev SEQ ID No. 94 CCTTCCTTCCTTCCTTGCTTCC
hHRAS-Rev-rG SEQ ID No. 95
CCTTCCTTCCTTCCTTGCTTCCgTCCT-SpC3
ACACA hACACA-For SEQ ID No. 96 GCATTTCTTCCATCTCCCCCTC
hACACA-For-rU SEQ ID No. 97
GCATTTCTTCCATCTCCCCCTCuGCCT-SpC3
hACACA-Rev SEQ ID No. 98 TCCGATTCTTGCTCCACTGTTG
hACACA-Rev-rG SEQ ID No. 99 TCCGATTCTTGCTCCACTGTTGgCTGA-SpC3
[0354] PCR reactions were done in 384 well format using a Roche Lightcycler
480 platform. Reactions comprised lx BIO-RAD iQTM SYBR Green Supermix
(BIO-RAD, Hercules, CA), 200 nM of each primer (For + Rev), and 1.3 mU of
Pyrococcus abyssi RNase H2 in 10 1.1,1 volume. Template DNA was either 2 ng of
human HeLa cell cDNA or 2 ng of rat spinal cord cDNA. Thermal cycling
parameters
included an initial 5 minutes soak at 95 C and then 60 cycles were performed
of [95 C
for 10 seconds + 60 C for 90 seconds]. Under these conditions, the Cp value
observed
for human cDNA represents a true positive event. If any signal was detected
using rat
cDNA, it was recorded as a false positive event. For these 3 genes, the human
and rat
sequences are divergent at the primer binding sites. Therefore detection of a
PCR
product in rat cDNA using human gene specific primers is an undesired, false
positive
result that originates from mispriming. Results are shown in Table 26 below.
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Table 26: False detection of products in rat cDNA using human gene specific
primers
in a 60 cycle qRT-PCR reaction
Observed Cp Observed Cp
Primers (For/Rev) ACp
Human cDNA Rat cDNA
ETS2 23.6 56.4 32.8
ETS2-blocked 24.9 ND > assay
HRAS 25.2 35.5 10.3
HRAS-blocked 26.1 ND > assay
ACACA 26.2 52.3 26.1
ACACA-blocked 26.3 ND > assay
ND = not detected
[0355] Using unmodified primers, detection of the human targets in human
cDNA
was successful and Cp's of 23-26 were observed. For all 3 PCR assays, the
human
gene-specific primers also detected products in rat cDNA when cycling was
continued,
and Cp's of 35-56 were observed. These represent undesired false positive
signals
which limit the ability of the PCR assays to detect low levels of true
positive signal.
[0356] Using modified primers, detection of the desired product in human
cDNA
was successful and Cp's were all within 1 of the values obtained for
unmodified
primers. However, no false positive signals were seen using rat cDNA with the
modified primers, even at 60 cycles. Use of the RNase H2 blocked-cleavable
primers
resulted in improved specificity, permitting use of longer, more sensitive PCR
reactions
(in this case up to 60 cycles) without detection of false priming events. This
allows for
a much greater ability to detect variant alleles in the presence of a larger
excess of the
wild type sequence.
EXAMPLE 12 ¨ Mismatch discrimination for a rC substrate under steady state
conditions
[0357] Example 11 demonstrated the ability of the methods of the invention
to
improve specificity of a qPCR reaction in the face of background mispriming
events.
The present example demonstrates the specificity of the RNase H2 cleavage
reaction
with respect to single-base differences (SNPs). The ability of the Pyrococcus
abyssi
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RNase H2 enzyme to distinguish base mismatches in a duplex substrate
containing a
single rC base was tested under steady state conditions. The following
substrates were
32P-end labeled and incubated in "Mg Cleavage Buffer" as described in Example
4
above. Reactions comprised 100 nM substrate with 100 i.tU of enzyme in 20 [IL
volume
and were incubated at 70 C for 20 minutes. Reaction products were separated
using
denaturing 7M urea, 15% polyacrylamide gel electrophoresis (PAGE) and
visualized
using a Packard CycloneTM Storage Phosphor System (phosphorimager). The
relative
intensity of each band was quantified and results plotted as a fraction of
total substrate
cleaved.
[0358] Ten duplexes were studied, including the perfect match (rC:G, SEQ ID
NOS 10 and 11) as well as each possible base mismatch at the rC base (3
duplexes, SEQ
ID Nos. 10 and 100-102), at position +1 relative to the rC (3 duplexes, SEQ ID
Nos. 10
and 103-105), and at position -1 relative to the rC (3 duplexes, SEQ ID Nos.
10 and
106-108). Results were normalized for perfect match = 100% and are shown in
Table
27 below.
Table 27: Cleavage of rC substrates with and without mismatches under steady
state
conditions
Duplex Identity
Substrate Sequence Cleavage
SEQ ID NOS
5' CTCGTGAGGTGATGcAGGAGATGGGAGGCG 3'
11 100%
3' GAGCACTCCACTACGTCCTCTACCCTCCGC 5'
10 5' CTCGTGAGGTGATGcAGGAGATGGGAGGCG 3'
46%
100 3' GAGCACTCCACTACATCCTCTACCCTCCGC 5'
10 5' CTCGTGAGGTGATGcAGGAGATGGGAGGCG 3'
35%
101 3' GAGCACTCCACTACTTCCTCTACCCTCCGC 5'
10 5' CTCGTGAGGTGATGcAGGAGATGGGAGGCG 3'
23%
102 3' GAGCACTCCACTACCTCCTCTACCCTCCGC 5'
10 5' CTCGTGAGGTGATGcAGGAGATGGGAGGCG 3'
19%
103 3' GAGCACTCCACTAAGTCCTCTACCCTCCGC 5'
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Table 27: Cleavage of rC substrates with and without mismatches under steady
state
conditions
Duplex Identity
Substrate Sequence Cleavage
SEQ ID NOS
5' CTCGTGAGGTGATGcAGGAGATGGGAGGCG 3'
65%
104 3' GAGCACTCCACTATGTCCTCTACCCTCCGC 5'
10 5' CTCGTGAGGTGATGcAGGAGATGGGAGGCG 3'
22%
105 3' GAGCACTCCACTAGGTCCTCTACCCTCCGC 5'
10 5' CTCGTGAGGTGATGcAGGAGATGGGAGGCG 3'
61%
106 3' GAGCACTCCACTACGACCTCTACCCTCCGC 5'
10 5' CTCGTGAGGTGATGcAGGAGATGGGAGGCG 3'
91%
107 3' GAGCACTCCACTACGCCCTCTACCCTCCGC 5'
10 5' CTCGTGAGGTGATGcAGGAGATGGGAGGCG 3'
46%
108 3' GAGCACTCCACTACGGCCTCTACCCTCCGC 5'
_
DNA bases are shown as uppercase. RNA bases are shown as lowercase. Mismatches
are shown in bold font and are underlined.
[0359] Pyrococcus RNase H2 was able to discriminate between single base
mismatches under these conditions. The precise degree of discrimination varied
with
which bases were paired in the mismatch. Interestingly, mismatches at position
-1 (one
base 5' to the rC base) showed relatively good mismatch discrimination while
mismatches at position +1 (one base 3' to the rC base) were in general less
effective.
Although the selectivity appears relatively modest, it becomes greatly
amplified with
repeated cycles of PCR.
EXAMPLE 13 ¨ Mismatch discrimination for rN substrates during thermal
cycling
[0360] The ability of the Pyrococcus abyssi RNase H2 enzyme to distinguish
base
mismatches for a rC substrate under steady state conditions was described in
Example
12. The ability of this enzyme to distinguish base mismatches for all rN
containing
substrates under conditions of thermal cycling was examined in the present
example.
In these conditions, the cleavable substrate is only available for processing
by the
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enzyme for a short period of time before temperature elevation disrupts the
duplex.
Mismatch discrimination was assessed in the setting of a fluorescent
quantitative
real-time PCR assay. We found that base mismatch discrimination was greatly
improved under these kinetically limited conditions than were observed under
steady-state conditions.
[0361] The following nucleic acids were employed in this example.
Oligonucleotides were synthesized to provide coverage for all nearest neighbor
pairs
and mismatches.
Unmodified For primer:
[0362] SEQ ID No. 68
5' AGCTCTGCCCAAAGATTACCCTG 3'
[0363] Blocked rN substrate rev primers (C3 spacer blocking group at the 3
'-end)
are shown below. DNA bases are uppercase and RNA bases are lower case. Regions
of
variation are indicated by bold and underlined. At total of 28 blocked primers
containing a single RNA residue were synthesized.
TA series:
[0364] SEQ ID No. 109
5' CTGAGCTTCATGCCTTTACTGTaCCCC-SpC3 3'
[0365] SEQ ID No. 110
5' CTGAGCTTCATGCCTTTACTGAaCCCC-SpC3 3'
[0366] SEQ ID No. 111
5' CTGAGCTTCATGCCTTTACTGCaCCCC-SpC3 3'
[0367] SEQ ID No. 112
5' CTGAGCTTCATGCCTTTACTGGaCCCC-SpC3 3'
[0368] SEQ ID No. 113
5' CTGAGCTTCATGCCTTTACTGTaTCCC-SpC3 3'
[0369] SEQ ID No. 114
5' CTGAGCTTCATGCCTTTACTGTaGCCC-SpC3 3'
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[0370] SEQ ID No. 115
5' CTGAGCTTCATGCCTTTACTGTaACCC-SpC3 3'
rU series:
[0371] SEQ ID No. 116
5' CTGAGCTTCATGCCTTTACTGTuCCCC-SpC3 3'
[0372] SEQ ID No. 117
5' CTGAGCTTCATGCCTTTACTGAuCCCC-SpC3 3'
[0373] SEQ ID No. 118
5' CTGAGCTTCATGCCTTTACTGCuCCCC-SpC3 3'
[0374] SEQ ID No. 119
5' CTGAGCTTCATGCCTTTACTGGuCCCC-SpC3 3'
[0375] SEQ ID No. 120
5' CTGAGCTTCATGCCTTTACTGTuTCCC-SpC3 3'
[0376] SEQ ID No. 121
5' CTGAGCTTCATGCCTTTACTGTuGCCC-SpC3 3'
[0377] SEQ ID No. 122
5' CTGAGCTTCATGCCTTTACTGTuACCC-SpC3 3'
rC series:
[0378] SEQ ID No. 123
5' CTGAGCTTCATGCCTTTACTGTcCCCC-SpC3 3'
[0379] SEQ ID No. 124
5' CTGAGCTTCATGCCTTTACTGAcCCCC-SpC3 3'
[0380] SEQ ID No. 125
5' CTGAGCTTCATGCCTTTACTGCcCCCC-SpC3 3'
[0381] SEQ ID No. 126
5' CTGAGCTTCATGCCTTTACTGGcCCCC-SpC3 3'
[0382] SEQ ID No. 127
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5' CTGAGCTTCATGCCTTTACTGTcTCCC-SpC3 3'
[0383] SEQ ID No. 128
5' CTGAGCTTCATGCCTTTACTGTcGCCC-SpC3 3'
[0384] SEQ ID No. 129
5' CTGAGCTTCATGCCTTTACTGTcACCC-SpC3 3'
rG series:
[0385] SEQ ID No. 130
5' CTGAGCTTCATGCCTTTACTGTgCCCC-SpC3 3'
[0386] SEQ ID No. 131
5' CTGAGCTTCATGCCTTTACTGAgCCCC-SpC3 3'
[0387] SEQ ID No. 132
5' CTGAGCTTCATGCCTTTACTGCgCCCC-SpC3 3'
[0388] SEQ ID No. 133
5' CTGAGCTTCATGCCTTTACTGGgCCCC-SpC3 3'
[0389] SEQ ID No. 134
5' CTGAGCTTCATGCCTTTACTGTgTCCC-SpC3 3'
[0390] SEQ ID No. 135
5' CTGAGCTTCATGCCTTTACTGTgGCCC-SpC3 3'
[0391] SEQ ID No. 136
5' CTGAGCTTCATGCCTTTACTGTgACCC-SpC3 3'
[0392] The unblocked control Rev primer (mimicing reaction product of
blocked
primers after cleavage by RNase H2) employed was:
[0393] SEQ ID NO: 290
5' CTGAGCTTCATGCCTTTACTG 3'
[0394] The following perfect-matched and mismatched synthetic templates
were
employed. The locations of varying bases are indicated in bold font with
underline.
Unique templates were made for each possible base variation at the
ribonucleotide or
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one base 5' or one base 3' of the ribonucleotide. In total, 28 templates were
synthesized
and tested.
TA templates:
[0395] SEQ ID No. 137
5'AGCTCTGCCCAAAGATTACCCTGACAGCTAAGTGGCAGTGGAAGTTGGCCTCAGAAGTAGTGGCCAG
CTGTGTGTCGGGGTACAGTAAAGGCATGAAGCTCAG-3'
[0396] SEQ ID No. 138
5'AGCTCTGCCCAAAGATTACCCTGACAGCTAAGTGGCAGTGGAAGTTGGCCTCAGAAGTAGTGGCCAG
CTGTGTGTCGGGGTCCAGTAAAGGCATGAAGCTCAG-3'
[0397] SEQ ID No. 139
5'AGCTCTGCCCAAAGATTACCCTGACAGCTAAGTGGCAGTGGAAGTTGGCCTCAGAAGTAGTGGCCAG
CTGTGTGTCGGGGTTCAGTAAAGGCATGAAGCTCAG-3'
[0398] SEQ ID No. 140
5'AGCTCTGCCCAAAGATTACCCTGACAGCTAAGTGGCAGTGGAAGTTGGCCTCAGAAGTAGTGGCCAG
CTGTGTGTCGGGGTGCAGTAAAGGCATGAAGCTCAG-3'
[0399] SEQ ID No. 141
5'AGCTCTGCCCAAAGATTACCCTGACAGCTAAGTGGCAGTGGAAGTTGGCCTCAGAAGTAGTGGCCAG
CTGTGTGTCGGGCTACAGTAAAGGCATGAAGCTCAG-3'
[0400] SEQ ID No. 142
5'AGCTCTGCCCAAAGATTACCCTGACAGCTAAGTGGCAGTGGAAGTTGGCCTCAGAAGTAGTGGCCAG
CTGTGTGTCGGGATACAGTAAAGGCATGAAGCTCAG-3'
[0401] SEQ ID No. 143
5'AGCTCTGCCCAAAGATTACCCTGACAGCTAAGTGGCAGTGGAAGTTGGCCTCAGAAGTAGTGGCCAG
CTGTGTGTCGGGTTACAGTAAAGGCATGAAGCTCAG-3'
rU templates:
[0402] SEQ ID No. 144
5'AGCTCTGCCCAAAGATTACCCTGACAGCTAAGTGGCAGTGGAAGTTGGCCTCAGAAGTAGTGGCCAG
CTGTGTGTCGGGGAACAGTAAAGGCATGAAGCTCAG-3'
[0403] SEQ ID No. 145
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5'AGCTCTGCCCAAAGATTACCCTGACAGCTAAGTGGCAGTGGAAGTTGGCCTCAGAAGTAGTGGCCAG
CTGTGTGTCGGGGATCAGTAAAGGCATGAAGCTCAG-3'
[0404] SEQ ID No. 146
5'AGCTCTGCCCAAAGATTACCCTGACAGCTAAGTGGCAGTGGAAGTTGGCCTCAGAAGTAGTGGCCAG
CTGTGTGTCGGGGACCAGTAAAGGCATGAAGCTCAG-3'
[0405] SEQ ID No. 147
5'AGCTCTGCCCAAAGATTACCCTGACAGCTAAGTGGCAGTGGAAGTTGGCCTCAGAAGTAGTGGCCAG
CTGTGTGTCGGGGAGCAGTAAAGGCATGAAGCTCAG-3'
[0406] SEQ ID No. 148
5'AGCTCTGCCCAAAGATTACCCTGACAGCTAAGTGGCAGTGGAAGTTGGCCTCAGAAGTAGTGGCCAG
CTGTGTGTCGGGCAACAGTAAAGGCATGAAGCTCAG-3'
[0407] SEQ ID No. 149
5'AGCTCTGCCCAAAGATTACCCTGACAGCTAAGTGGCAGTGGAAGTTGGCCTCAGAAGTAGTGGCCAG
CTGTGTGTCGGGAAACAGTAAAGGCATGAAGCTCAG-3'
[0408] SEQ ID No. 150
5'AGCTCTGCCCAAAGATTACCCTGACAGCTAAGTGGCAGTGGAAGTTGGCCTCAGAAGTAGTGGCCAG
CTGTGTGTCGGGTAACAGTAAAGGCATGAAGCTCAG-3'
rG templates:
[0409] SEQ ID No. 151
5'AGCTCTGCCCAAAGATTACCCTGACAGCTAAGTGGCAGTGGAAGTTGGCCTCAGAAGTAGTGGCCAG
CTGTGTGTCGGGGCACAGTAAAGGCATGAAGCTCAG-3'
[0410] SEQ ID No. 152
5'AGCTCTGCCCAAAGATTACCCTGACAGCTAAGTGGCAGTGGAAGTTGGCCTCAGAAGTAGTGGCCAG
CTGTGTGTCGGGGCTCAGTAAAGGCATGAAGCTCAG-3'
[0411] SEQ ID No. 153
5'AGCTCTGCCCAAAGATTACCCTGACAGCTAAGTGGCAGTGGAAGTTGGCCTCAGAAGTAGTGGCCAG
CTGTGTGTCGGGGCCCAGTAAAGGCATGAAGCTCAG-3'
[0412] SEQ ID No. 154
5'AGCTCTGCCCAAAGATTACCCTGACAGCTAAGTGGCAGTGGAAGTTGGCCTCAGAAGTAGTGGCCAG
CTGTGTGTCGGGGCGCAGTAAAGGCATGAAGCTCAG-3'
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[0413] SEQ ID No. 155
5'AGCTCTGCCCAAAGATTACCCTGACAGCTAAGTGGCAGTGGAAGTTGGCCTCAGAAGTAGTGGCCAG
CTGTGTGTCGGGACACAGTAAAGGCATGAAGCTCAG-3'
[0414] SEQ ID No. 156
5'AGCTCTGCCCAAAGATTACCCTGACAGCTAAGTGGCAGTGGAAGTTGGCCTCAGAAGTAGTGGCCAG
CTGTGTGTCGGGTCACAGTAAAGGCATGAAGCTCAG-3'
[0415] SEQ ID No. 157
5'AGCTCTGCCCAAAGATTACCCTGACAGCTAAGTGGCAGTGGAAGTTGGCCTCAGAAGTAGTGGCCAG
CTGTGTGTCGGGCCACAGTAAAGGCATGAAGCTCAG-3'
rC templates
[0416] SEQ ID No. 158
5'AGCTCTGCCCAAAGATTACCCTGACAGCTAAGTGGCAGTGGAAGTTGGCCTCAGAAGTAGTGGCCAG
CTGTGTGTCGGGGGACAGTAAAGGCATGAAGCTCAG-3'
[0417] SEQ ID No. 159
5'AGCTCTGCCCAAAGATTACCCTGACAGCTAAGTGGCAGTGGAAGTTGGCCTCAGAAGTAGTGGCCAG
CTGTGTGTCGGGGGTCAGTAAAGGCATGAAGCTCAG-3'
[0418] SEQ ID No. 160
5'AGCTCTGCCCAAAGATTACCCTGACAGCTAAGTGGCAGTGGAAGTTGGCCTCAGAAGTAGTGGCCAG
CTGTGTGTCGGGGGCCAGTAAAGGCATGAAGCTCAG-3'
[0419] SEQ ID No. 161
5'AGCTCTGCCCAAAGATTACCCTGACAGCTAAGTGGCAGTGGAAGTTGGCCTCAGAAGTAGTGGCCAG
CTGTGTGTCGGGGGGCAGTAAAGGCATGAAGCTCAG-3'
[0420] SEQ ID No. 162
5'AGCTCTGCCCAAAGATTACCCTGACAGCTAAGTGGCAGTGGAAGTTGGCCTCAGAAGTAGTGGCCAG
CTGTGTGTCGGGAGACAGTAAAGGCATGAAGCTCAG-3'
[0421] SEQ ID No. 163
5'AGCTCTGCCCAAAGATTACCCTGACAGCTAAGTGGCAGTGGAAGTTGGCCTCAGAAGTAGTGGCCAG
CTGTGTGTCGGGTGACAGTAAAGGCATGAAGCTCAG-3'
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[0422] SEQ ID No. 164
5'AGCTCTGCCCAAAGATTACCCTGACAGCTAAGTGGCAGTGGAAGTTGGCCTCAGAAGTAGTGGCCAG
CTGTGTGTCGGGCGACAGTAAAGGCATGAAGCTCAG-3'
[0423] Together, these nucleic acids (SEQ ID NOS 68, 291, 116 and 69,
respectively, in order of appearance) comprise PCR assays set up as indicated:
5'AGCTCTGCCCAAAGATTACCCTG 4
5'AGCTCTGCCCAAAGATTACCCTGACAGCTAAGTGGCAGTGGAAGTTGGCCTCAGAAGTAGTGGCCAG
CTGTGTGTGTCGGGGAACAGTAAAGGCATGAAGCTCAG 3'
X-CCCCuTGTCATTTCCGTACTTCGAGTC 5'
TGTCATTTCCGTACTTCGAGTC 5'
[0424] The terminal C3 spacer group (indicated by "x") blocks the rU
containing
oligonucleotide to serve as a primer. When hybridized to the template, the
duplex
becomes a substrate for RNase H2 and cleavage occurs immediately 5'- to the rU
residue, resulting in a functional primer as shown ().
[0425] Quantitative real time PCR reactions were performed using unmodified
primer SEQ ID No. 68 and pairwise combinations of rN containing primers SEQ ID
Nos. 109-136 and templates SEQ ID Nos. 137-164. Reactions were done in 384
well
format using a Roche Lightcycler 480 platform. Reactions comprised lx BIO-RAD
iQTM SYBR Green Supermix (BIO-RAD, Hercules, CA), 200 nM of each primer (for
+ rev), and 1.3 mU of Pyrococcus abyssi RNase H2 in 10 1 volume. Thermal
cycling
parameters included an initial 5 minutes soak at 95 C and then 45 cycles were
performed of [95 C for 10 seconds + 60 C for 20 seconds + 72 C for 30
seconds].
Under these conditions, the Cp value was identical for control reactions done
using For
+ Rev (unmodified) primers and control coupled RNase H2 cleavage-PCR reactions
done using the perfect match For (unmodified) + rN Rev (blocked) primers. Thus
the
reaction conditions employed had sufficient incubation time and RNase H2
concentration to cleave the perfect match species within the kinetic
constraints of the
real time thermal cycling and any deviations from this point will represent a
change in
reaction efficiency imparted by base mismatches present between the blocked
primer
and the various templates.
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[0426] Pairwise combinations of primers and templates were run as described
above and results are summarized below showing ACp, which is the difference of
cycle
threshold observed between control and mismatch reactions. Since each Cp
represents
a cycle in PCR (which is an exponential reaction under these conditions), a
ACp of 10
represents a real differential of 210, or a 1024 fold change in sensitivity. A
ACp of 4 to
cycles is generally sufficient to discriminate between SNPs in allele specific
PCR
assays.
[0427] Results for tests done varying bases at the central position over
the rN base
are shown below in Table 28 (SEQ ID NOS 292 and 293, respectively, in order of
appearance):
5' CTGAGCTTCATGCCTTTACTGTaCCCC-SpC3 3' blocked primers
3' GACTCGAAGTACGGAAATGACATGGGG... 5' templates
A
Table 28. ACp for all possible base mismatches at the
rN position
Template
A
rA 14.9 9.4 13.6 0
rC 7.4 9.2 0 6.6
rG 13.9 0 12.7 14.5
rU 0 12.2 10.9 5.3
[0428] Very large differences in reactive efficiency are seen in RNase H2
cleavage
of a rN substrate under thermal cycling conditions, ranging from a difference
of around
40-fold (ACp 5.3) to over a 30,000 fold difference (ACp 14.9). None of the
assays
showed a ACp less than 5 cycles. Thus the RNase H2 rN cleavage reaction shows
far
greater specificity in the setting of a kinetic assay (qPCR) than under steady
state
conditions and much greater selectivity than allele specific PCR with standard
DNA
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primers. Added specificity may be conferred by the design of the primers as
described
in the detailed description of the invention and demonstrated in the examples
below.
[0429] Results for tests done varying bases at the -1 position relative to
the rN base
are shown below in Table 29 (SEQ ID NOS 294 and 295, respectively, in order of
appearance):
A
5' CTGAGCTTCATGCCTTTACTGTuCCCC-SpC3 3' blocked primers
3' GACTCGAAGTACGGAAATGACAAGGGG... 5' templates
Table 29: ACp for all possible base mismatches at
position -1 relative to a rU base
Template
A
A(rU) 16.1 8.7 12.6 0
C(rU) 7.6 3.9 0 12.0
G(rU) 13.8 0 12.4 5.9
T(rU) 0 5.2 2.4 6.2
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[0430] Results for tests done varying bases at the +1 position relative to
the rN base
are shown below in Table 30 (SEQ ID NOS 296 and 297, respectively, in order of
appearance):
A
5' CTGAGCTTCATGCCTTTACTGTuCCCC-SpC3 3' blocked primers
3' GACTCGAAGTACGGAAATGACAAGGGG... 5' templates
A
Table 30. ACp for all possible base mismatches at
position +1 relative to a rU base
Template
A
(rU)A 11.4 2.5 12.2 0
(rU)C 6.4 10.4 0 9.0
(rU)G 13.8 0 4.5 3.0
(rU)T 0 11.1 11.9 2.9
[0431] Pairwise combinations were similarly tested for all sequence
variants listed
above for the -1 and +1 positions relative to the rN base, including the rA,
rC, and rG
probes. Results are shown in Tables 31-36 below.
Table 31: ACp for all possible base mismatches at position -1 relative to a rA
base
Template
A
A(rA) 14.2 8.6 11.8 0
C(rA) 6.9 12.6 0 6.8
G(rA) 12.8 0 12.6 8.9
T(rA) 0 5.1 1.4 8.6
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Table 32: ACp for all possible base mismatches at position +1 relative to a rA
base
Template
A C G T
(rA)A 3.1 1.0 6.12 0
(rA)C 9.3 10.2 0 8.3
(rA)G 13.2 0 2.5 5.9
(rA)T 0 5.0 7.1 4.0
Table 33: ACp for all possible base mismatches at position -1 relative to a rC
base
Template
A C G T
A(rC) 13.0 8.2 10.5 0
C(rC) 5.0 3.3 0 3.5
G(rC) 8.3 0 7.0 0.8
T(rC) 0 5.4 2.1 4.6
Table 34: ACp for all possible base mismatches at position +1 relative to a rC
base
Template
A C G T
(rC)A 5.6 1.8 10.2 0
(rC)C 8.8 9.6 0 8.6
(rC)G 9.8 0 3.2 0.3
(rC)T 0 2.1 0.2 0.0
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Table 35: ACp for all possible base mismatches at position -1 relative to a rG
base
Template
A C G T
A(rG) 12.4 4.8 10.4 0
C(rG) 4.5 11.1 0 2.5
G(rG) 10.3 0 10.1 3.8
T(rG) 0 3.5 2.2 5.3
Table 36: ACp for all possible base mismatches at position +1 relative to a rG
base
Template
A C G T
(rG)A 6.2 3.0 11.4 0
(rG)C 9.5 7.3 0 4.7
(rG)G 13.1 0 6.0 3.2
(rG)T 0 4.5 11.5 0.3
[0432] The relative change in reaction efficiency of cleavage of a rN
substrate by
Pyrococcus abyssi RNase H2 in the setting of a single base mismatch varies
with the
identity of the paired bases, the relative position of the mismatch to the
cleavage site,
and the neighboring bases. The mismatch charts defined in this example can be
used to
design optimal mismatch detection assays which maximize the expected
differential
(ACp)between mismatch and matched loci, and can be built into an algorithm to
automate optimization of new assay designs.
Example 14 ¨ Mismatch discrimination for fUfU substrate under steady state
conditions
[0433] The ability of the Pyrococcus abyssi RNase H2 enzyme to distinguish
base
mismatches in a duplex substrate containing a fUfU dinucleotide pair was
tested under
steady state conditions. The following substrates were 32P-end labeled and
incubated in
"Mn Cleavage Buffer" as described in Examples 5 and 6 above. Reactions
comprised
100 nM substrate with 1 U of enzyme in 20 L volume and were incubated at 70 C
for
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20 minutes. Reaction products were separated using denaturing 7M urea, 15%
polyacrylamide gel electrophoresis (PAGE) and visualized using a Packard
CycloneTM
Storage Phosphor System (phosphorimager). The relative intensity of each band
was
quantified and results plotted as a fraction of total substrate cleaved.
[0434] Fourteen duplexes shown in Table 37, were studied, including the
perfect
match (SEQ ID NOS 42 and 183), mismatches within the 2'-fluoro dinucleotide
pair
(SEQ ID Nos. 42 and 165-171), and mismatches adjacent to the 2'-fluoro
dinucleotide
pair (SEQ ID Nos. 42 and 172-177). Results were normalized for a perfect match
=
100%.
Table 37: Cleavage of fUfU substrates with and without mismatches under steady
state conditions
Duplex Identity
Substrate Sequence Cleavage
SEQ ID NOS
42
5' CTCGTGAGGTGAT (fUfU) AGGAGATGGGAGGCG 3'
183 100%
3' GAGCACTCCACTA A A TCCTCTACCCTCCGC 5'
42 5' CTCGTGAGGTGAT ( fUfU) AGGAGATGGGAGGCG 3'
5%
165 3' GAGCACTCCACTA A G TCCTCTACCCTCCGC 5'
42 5' CTCGTGAGGTGAT ( fUfU) AGGAGATGGGAGGCG 3'
14%
166 3' GAGCACTCCACTA A C TCCTCTACCCTCCGC 5'
42 5' CTCGTGAGGTGAT ( fUfU) AGGAGATGGGAGGCG 3'
1%
167 3' GAGCACTCCACTA A T TCCTCTACCCTCCGC 5'
_
42 5' CTCGTGAGGTGAT ( fUfU) AGGAGATGGGAGGCG 3'
2%
168 3' GAGCACTCCACTA C T TCCTCTACCCTCCGC 5'
42 5' CTCGTGAGGTGAT ( fUfU) AGGAGATGGGAGGCG 3'
0%
169 3' GAGCACTCCACTA G G TCCTCTACCCTCCGC 5'
42 5' CTCGTGAGGTGAT ( fUfU) AGGAGATGGGAGGCG 3'
0%
170 3' GAGCACTCCACTA C C TCCTCTACCCTCCGC 5'
42 5' CTCGTGAGGTGAT ( fUfU) AGGAGATGGGAGGCG 3'
0%
171 3' GAGCACTCCACTA T T TCCTCTACCCTCCGC 5'
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Table 37: Cleavage of fUfU substrates with and without mismatches under steady
state conditions
Duplex Identity
Substrate Sequence Cleavage
SEQ ID NOS
42 5' CTCGTGAGGTGAT ( fUfU) AGGAGATGGGAGGCG 3'
8%
172 3' GAGCACTCCACTC A A TCCTCTACCCTCCGC 5'
42 5' CTCGTGAGGTGAT ( fUfU) AGGAGATGGGAGGCG 3'
4%
173 3' GAGCACTCCACTG A A TCCTCTACCCTCCGC 5'
42 5' CTCGTGAGGTGAT ( fUfU) AGGAGATGGGAGGCG 3'
4%
174 3' GAGCACTCCACTT A A TCCTCTACCCTCCGC 5'
42 5' CTCGTGAGGTGAT ( fUfU) AGGAGATGGGAGGCG 3'
2%
175 3' GAGCACTCCACTA A A GCCTCTACCCTCCGC 5
42 5' CTCGTGAGGTGAT ( fUfU) AGGAGATGGGAGGCG 3'
8%
176 3' GAGCACTCCACTA A A CCCTCTACCCTCCGC 5'
42 5' CTCGTGAGGTGAT ( fUfU) AGGAGATGGGAGGCG 3'
2%
177 3' GAGCACTCCACTA A A ACCTCTACCCTCCGC 5
DNA bases are shown as uppercase. 2'-F bases are shown as fU.
Mismatches are shown in bold font and are underlined.
[0435] Pyrococcus RNase H2 was able to discriminate very efficiently
between
single base mismatches under these conditions. The precise degree of
discrimination
varied with which bases were paired in the mismatch. Interestingly, mismatches
at both
positions -1 and +1 (relative to the fUfU domain) were effective. Specificity
for
cleavage using the fUfU substrate was significantly higher under steady state
assay
conditions than was the rC substrate (Example 12 above).
[0436] The study above employed the fUfU dinucleotide pair, which was
previously shown in Example 6 to be the least efficient di-fluoro substrate
for cleavage
of the 16 possible dinucleotide pairs. This may impact the mismatch results.
Similar
experiments were conducted using the same complement strands, substituting a
fUfC
di-fluoro substrate strand. RNase H2 was reduced to 20 mU due to the increased
activity of cleavage seen for fUfC compared to fUfU substrates. Results are
shown in
Table 38 below.
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Table 38. Cleavage of fUfC substrates with and without mismatches under steady
state conditions
Duplex Identity
Substrate Sequence Cleavage
SEQ ID NOS
41 5' CTCGTGAGGTGAT ( fUfC) AGGAGATGGGAGGCG 3'
100%
298 3' GAGCACTCCACTA A G TCCTCTACCCTCCGC 5'
41 5' CTCGTGAGGTGAT ( fUfC) AGGAGATGGGAGGCG 3'
0%
178 3' GAGCACTCCACTA T G TCCTCTACCCTCCGC 5'
41 5' CTCGTGAGGTGAT ( fUfC) AGGAGATGGGAGGCG 3'
7%
179 3' GAGCACTCCACTA C G TCCTCTACCCTCCGC 5'
41 5' CTCGTGAGGTGAT ( fUfC) AGGAGATGGGAGGCG 3'
0%
180 3' GAGCACTCCACTA G G TCCTCTACCCTCCGC 5'
41 5' CTCGTGAGGTGAT ( fUfC) AGGAGATGGGAGGCG 3'
1%
181 3' GAGCACTCCACTA A T TCCTCTACCCTCCGC 5'
41 5' CTCGTGAGGTGAT ( fUfC) AGGAGATGGGAGGCG 3'
0%
182 3' GAGCACTCCACTA A C TCCTCTACCCTCCGC 5'
_
41 5' CTCGTGAGGTGAT ( fUfC) AGGAGATGGGAGGCG 3'
2%
183 3' GAGCACTCCACTA A A TCCTCTACCCTCCGC 5'
41 5' CTCGTGAGGTGAT ( fUfC) AGGAGATGGGAGGCG 3'
0%
184 3' GAGCACTCCACTA T C TCCTCTACCCTCCGC 5'
41 5' CTCGTGAGGTGAT ( fUfC) AGGAGATGGGAGGCG 3'
0%
185 3' GAGCACTCCACTT A G TCCTCTACCCTCCGC 5'
41 5' CTCGTGAGGTGAT ( fUfC) AGGAGATGGGAGGCG 3'
4%
186 3' GAGCACTCCACTC A G TCCTCTACCCTCCGC 5'
41 5' CTCGTGAGGTGAT ( fUfC) AGGAGATGGGAGGCG 3'
0%
187 3' GAGCACTCCACTG A G TCCTCTACCCTCCGC 5'
_
41 5' CTCGTGAGGTGAT ( fUfC) AGGAGATGGGAGGCG 3'
2%
188 3' GAGCACTCCACTA A G ACCTCTACCCTCCGC 5'
_
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Table 38. Cleavage of fUfC substrates with and without mismatches under steady
state conditions
Duplex Identity
Substrate Sequence Cleavage
SEQ ID NOS
41 5' CTCGTGAGGTGAT ( fUfC) AGGAGATGGGAGGCG 3'
4%
189 3' GAGCACTCCACTA A G CCCTCTACCCTCCGC 5
41 5' CTCGTGAGGTGAT ( fUfC) AGGAGATGGGAGGCG 3'
2%
190 3' GAGCACTCCACTA A G GCCTCTACCCTCCGC 5
DNA bases are shown as uppercase. 2'-F bases are shown as fU.
Mismatches are shown in bold font and are underlined.
[0437] Again, Pyrococcus abyssi RNase H2 was able to discriminate very
efficiently between single base mismatches. The precise degree of
discrimination
varied with which bases were paired in the mismatch. As before, mismatches at
both
positions -1 and +1 (relative to the fUfC domain) were effective. Specificity
for
cleavage using the fUfC substrate was significantly higher under steady state
assay
conditions than was the rC substrate (Example 12 above) and also showed
slightly
greater specificity than the fUfU substrate. Under kinetic assay conditions
during
thermal cycling, mismatch assays using di-fluoro substrates may show even
greater
selectivity.
Example 15 ¨ Selective placement of phosphorothioate internucleotide
modifications in the substrate
[0438] The effect of incorporation of a phosphorothioate internucleoside
linkage
was tested for several different substrates. Phosphorothioate (PS) bonds are
typically
considered relatively nuclease resistant and are commonly used to increase the
stability
of oligonucleotides in nuclease containing solutions, such as serum. PS bonds
form
two stereoisomers, Rp and Sp, which usually show different levels of
stabilization for
different nucleases.
[0439] The di-fluoro substrate was examined with a PS bond between the two
modified bases. A mixture of both diastereomers was employed for the present
study.
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Unmodified fUfC substrate:
[0440] SEQ ID NOS 41 and 298, respectively, in order of appearance
5f-CTCGTGAGGTGAT(fUfC)AGGAGATGGGAGGCG-3'
3f-GAGCACTCCACTA A G TCCTCTACCCTCCGC-5'
PS modified fU*fC substrate ("*" = PS bond):
[0441] SEQ ID NOS 191 and 299, respectively, in order of appearance
5f-CTCGTGAGGTGAT(fU*fC)AGGAGATGGGAGGCG-3'
3f-GAGCACTCCACTA A G TCCTCTACCCTCCGC-5'
(note ¨ gaps in sequence are for alignment purposes)
[0442] The above substrates were incubated for 1 hour at 70 C in "Mn
Cleavage
Buffer" using 160 pmoles of substrate in 120 n1 volume (1.3 M) and 4 units of
the
recombinant Pyrococcus RNase H2 enzyme. Reactions were stopped with the
addition
of gel loading buffer (formamide/EDTA) and separated on a denaturing 7M urea,
15%
polyacrylamide gel. Gels were stained using GelStarTM (Lonza, Rockland, ME)
and
visualized with UV excitation. The unmodified substrate was 100% cleaved under
these conditions; however the PS-modified substrate was essentially uncleaved.
The
phosphorothioate modification can effectively block cleavage of a di-fluoro
substrate.
[0443] A substrate containing a single rC residue was studied next, testing
placement of the PS modification on either side of the RNA base (5'- or 3'-
side as
indicated). A mixture of both diastereomers were employed for the present
study.
[0444] The above substrates were incubated for 1 hour at 70 C in "Mg
Cleavage
Buffer" using 160 pmoles of substrate in 120 n1 volume (1.3 M) and 4 units of
the
recombinant Pyrococcus RNase H2 enzyme. Reactions were stopped with the
addition
of gel loading buffer (formamide/EDTA) and separated on a denaturing 7M urea,
15%
polyacrylamide gel. Gels were stained using GelStarTM (Lonza, Rockland, ME)
and
visualized with UV excitation. The unmodified substrate was 100% cleaved under
these conditions. Both the 5' -*rC and 3'-rC* PS-modified substrates were
approximately 50% cleaved under these conditions. These results are most
consistent
with one stereoisomer, Rp or Sp, being more resistant to cleavage than the
other isomer.
[0445] The 3' -rC* substrate was studied in greater detail. Since RNase H2
cleaves
this substrate on the 5'-side of the ribonucleotide while other RNases (such
as RNase A,
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RNase 1, etc.) cleave this substrate on the 3'-side of the ribonucleotide, it
may be
possible to use the PS modification as a way of protecting the substrate from
unwanted
degradation by other nucleases while leaving it available as an RNase H2
substrate. It
is well known that cleavage of RNA substrates by RNase A and other single-
stranded
ribonucleases is inhibited to a greater extent by the Sp phosphorothioate
isomer than the
Rp isomer. The relative effects of the Sp vs. Rp isomer on RNase H2 cleavage
have not
been known. Therefore the two stereoisomers were purified and the relative
contributions of the Sp and Rp isomers on 3'-rC* substrate stability were
studied.
[0446] It is well known that phosphorothioate isomers can be separated by
HPLC
techniques and that this separation is readily done if only a single PS bond
exists in an
oligonucleotide. HPLC was therefore employed to purify the two PS isomers of
the
3'-rC* substrate, SEQ ID No. 192 (5'-CTCGTGAGGTGATTC(*)AGGAG
ATGGGAGGCG-3'; Figure 23). A mass of 7 nmoles of the single-stranded 3'-rC*
containing oligonucleotide was employed. Characterization showed that the test
material had a molecular weight of 9464 Daltons (calculated 9465) by ESI-MS
with a
molar purity of 95% by capillary electrophoresis. This material was injected
into a 4.6
mm x 50 mm XbridgeTM C18 column (Waters) with 2.5 micron particle size.
Starting
mobile phase (Buffer A) was 100 mM TEAA pH 7.0 with 5% acetonitrile and which
was mixed with pure acetonitrile (Buffer B) at 35 C. The HPLC method employed
clearly resolved two peaks in the sample which were collected and re-run to
demonstrate purity. HPLC traces of the mixed isomer sample and purified
specimens
are shown in Figure 23. Both the "A" and "B" peaks had an identical mass of
9464
Daltons by ESI-MS. From the original sample, 1.3 nmoles of peak "A" and 3.6
nmoles
of peak "B" were recovered.
[0447] It was not possible based upon mass or HPLC data to identify which
peak
was the Rp and which peak was the Sp isomer. Relative resistance to
degradation by
RNase A was employed to assign isomer identity to the purification fractions.
The Sp
isomer is known to confer relatively greater resistance to RNase A degradation
than the
Rp isomer. Purified products were studied in the single-stranded form. The
substrate
was radiolabeled with 32P using 6000 Ci/mmol 7-32P-ATP and the enzyme T4
Polynucleotide Kinase (Optikinase, US Biochemical). Trace label was added to
reaction mixtures (1:50). Reactions were performed using 100 nM substrate in
20 ul
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volume with 1 pg (72 attomoles) of RNase A in Mg Cleavage Buffer. Reactions
were
incubated at 70 C for 20 minutes. Reaction products were separated using
denaturing
7M urea, 15% polyacrylamide gel electrophoresis (PAGE) and visualized using a
Packard CycloneTM Storage Phosphor System (phosphorimager). The relative
intensity
of each band was quantified and results plotted as a fraction of total
substrate cleaved.
Peak "A" was more completely degraded by RNase A than peak "B"; peak "A" was
therefore assigned identity as the Rp isomer and peak "B" was assigned as the
Sp
isomer.
[0448] The relative susceptibility of each stereoisomer to RNase H2
cleavage was
studied. The RNA-containing strand of the substrate was radiolabeled with 32P
using
6000 Ci/mmol 7-32P-ATP and the enzyme T4 Polynucleotide Kinase (Optikinase, US
Biochemical). Trace label was added to reaction mixtures (1:50). Reactions
were
performed using 100 nM substrate in 20 n1 volume with 100 nU of recombinant
Pyrococcus abyssi RNase H2 in Mg Cleavage Buffer. Substrates were employed in
both single-stranded and duplex form. Reactions were incubated at 70 C for 20
minutes. Reaction products were separated using denaturing 7M urea, 15%
polyacrylamide gel electrophoresis (PAGE) and visualized using a Packard
CycloneTM
Storage Phosphor System (phosphorimager). The relative intensity of each band
was
quantified and results plotted as a fraction of total substrate cleaved. As
expected,
single-stranded substrates were not cleaved by the RNase H2 enzyme. The
control
unmodified rC duplex (SEQ ID NOS 10 and 11) were 100% cleaved under the
conditions employed. The Sp isomer 3'-rC* duplex substrate (peak "B") was
cleaved
¨30% whereas the Rp isomer (peak "A") was cleaved < 10% under these
conditions.
Therefore the relative susceptibility to cleavage of racemically pure
phosphorothioate
modified substrates at this position (3'-to the ribonucleotide) is exactly
opposite for
RNase H2 vs. RNase A. The Sp isomer is more readily cleaved by RNase H2 while
the
Rp isomer is more readily cleaved by RNase A. Therefore single ribonucleotide
containing substrates having a racemically pure Sp isomer phosphorothioate
modification on the 3 '-side of the ribonucleotide could be employed to
protect this
bond from unwanted degradation by single-stranded nucleases (such as RNase A)
while still being a functional substrate for cleavage by RNase H2. The
relationship
between enzyme cleavage and phosphorothioate stereoisomer is summarized in
Figure
24.
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EXAMPLE 16¨ Utility of rN containing dual-labeled probes in qPCR assays
[0449] The following example illustrates a real time PCR assay utilizing a
rU-containing dual labeled probe. Previously, we demonstrated in Example 9 the
feasibility for use of rN blocked primers in qPCR using a SYBR Green
detection
format. Cleavage of blocked oligonucleotides using the method of the present
invention can also be applied to the dual-labeled probe assay format. Use of
RNase H1
to cleave a dual-labeled probe containing a 4 RNA base cleavage domain in an
isothermal cycling probe assay format has been described by Harvey, J.J., et
al.
(Analytical Biochemistry, 333:246-255, 2004). Another dual-labeled probe assay
using RNase H has been described, wherein a molecular beacon containing a
single
ribonucleotide residue was employed to detect polymorphisms in an end-point
PCR
format using RNase H2 (Hou, J., et al., Oligonucleotides, 17:433-443, 2007).
In the
present example we will demonstrate use of single ribonucleotide containing
dual-labeled probes in a qPCR assay format that relies upon RNase H2 cleavage
of the
probe.
[0450] The following oligonucleotides shown in Table 39, were used as
probes and
primers in a qPCR assay with a dual-labeled fluorescence-quenched probe. The
target
was a synthetic oligonucleotide template.
Table 39:
Name Sequence SEQ ID No.
Syn-For 5f-AGCTCTGCCCAAAGATTACCCTG-3' SEQ ID No. 68
Syn-Rev 5f-CTGAGCTTCATGCCTTTACTGT-3' SEQ ID No. 69
Syn-Probe 5f-FAM-TTCTGAGGCCAACTCCACTGCCACTTA-IBFQ-3' SEQ ID No. 193
Syn-Probe-rU 5' - FAM- T TC TGAGGCCAACuCCAC TGCCAC T TA- I BFQ - 3 f SEQ ID
No. 194
DNA bases are shown in uppercase. RNA bases are shown in lowercase. FAM is
6-carboxyfluorescein and IBFQ is a dark quencher (Integrated DNA
Technologies).
[0451] Synthetic template (primer and probe binding sites are underlined).
[0452] SEQ ID No. 75
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AGCTCTGCCCAAAGATTACCCTGACAGCTAAGTGGCAGTGGAAGTTGGCCTCAGAAGTAGTGGCCAGCT
GTGTGTCGGGGAACAGTAAAGGCATGAAGCTCAG
[0453] Quantitative real time PCR reactions were performed using unmodified
primers SEQ ID Nos. 68 and 69 and probes Seq ID Nos. 193 and 194. Reactions
were
done in 384 well format using a Roche Lightcycler 480 platform. Reactions
comprised 200 nM of each primer (for + rev) and 200 nM probe, 2 x 106 copies
of the
synthetic template, and 5 mU of Pyrococcus abyssi RNase H2 in 10 n1 volume.
Thermal cycling parameters included an initial 10 minutes incubation at 95 C
and then
45 cycles were performed of [95 C for 10 seconds + 60 C for 30 seconds + 72 C
for 1
seconds]. The buffer employed varied with the polymerase used.
[0454] If PCR is performed using a thermostable DNA polymerase having
5'-exonuclease activity the polymerase will degrade the probe. Under these
conditions,
a DNA probe should perform the same as a rN modified probe. This reaction
constitutes a positive control. If a DNA polymerase is employed which is
lacking
5'-exonuclease activity, then neither probe should be degraded. This reaction
constitutes a negative control. A PCR reaction using the exo-negative
polymerase with
RNase H2, however, should degrade the rN containing probe but not the DNA
probe,
demonstrating function of the invention. For the present study, the following
two
thermostable polymerases were used: Immolase (intact 5' nuclease activity,
Bioline)
and Vent Exo- (5'-exonuclease negative mutant, New England Biolabs). Buffers
employed were the manufacturer's recommended buffers for the DNA polymerases
and were not optimized for RNase H2 activity. For Immolase, the buffer
comprised 16
mM (NH4)2504, 67 mM Tris pH 8.3, and 3 mM MgC12. For Vent Exo-, the buffer
comprised 10 mM (NH4)2504, 20 mM Tris pH 8.8, 10 mM KC1, and 3 mM Mg504.
[0455] qPCR reactions were run as described and results are shown below in
Table
40.
Table 40. Cp values of qPCR reactions comparing DNA or rU Dual-Labeled Probes
Probe Polymerase Minus RNase H2 Plus RNase H2
Syn-Probe Immolase Exo+ 21.1 21.0
Vent Exo- ND ND
Syn-Probe-rU Immolase Exo+ 21.0 20.7
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Table 40. Cp values of qPCR reactions comparing DNA or rU Dual-Labeled Probes
Probe Polymerase Minus RNase H2 Plus RNase H2
Vent Exo- ND 21.1
ND = not detectable
[0456] Using the exonuclease positive polymerase, both probes showed
similar
functional performance and gave similar Cp values, both with or without RNase
H2.
Using the exonuclease deficient mutant polymerase, however, the DNA probe did
not
produce any detectable fluorescent signal; the rU probe failed to produce
fluorescent
signal in the absence of RNase H2, but in the presence of RNase H2 was cleaved
and
resulted in signal at the expected Cp value. Similar results can be obtained
using
di-fluoro containing probes. If the RNase H2 cleavage domain is placed over a
mutation site such probes can be used to distinguish variant alleles.
[0457] RNase H-cleavable probes can also be linked with the use of blocked
primers of the present invention to additively increase the specificity of
amplification
based assay systems.
EXAMPLE 17 Utility of rN containing blocked primer to prevent primer-dimer
formation
[0458] Formation of primer-dimers or other small target independent
amplicons
can be a significant problem in both endpoint and real-time PCR. These
products can
arise even when the primers appear to be well designed. Further, it is
sometimes
necessary to employ primers which have sub-optimal design because of sequence
constraints for selection of primers which hybridize to specific regions. For
example,
PCR assays for certain viruses can be subtype or serotype specific if primers
are chosen
in areas that are variable between strains. Conversely, PCR reactions can be
designed
to broadly amplify all viral strains if primers are placed in highly conserved
regions of
the viral genome. Thus the sequence space available to choose primers may be
very
limited and "poor" primers may have to be employed that have the potential to
form
primer dimers. Use of "hot start" PCR methods may eliminate some but not all
of these
problems.
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[0459] The following example derives from one such case cited in US Patent
06001611 where primer-dimers were found to be a significant problem during
development of a PCR-based nucleic acid detection assay for the Hepatitis C
virus
(HCV) using sites in conserved domains that permit detection of a wide range
of viral
serotypes. We demonstrate herein that use of cleavable blocked primers can
prevent
unwanted primer dimer formation, specifically in the absence of a "hot-start"
DNA
polymerase.
[0460] The following oligonucleotides, as shown in Table 41, were used as
primers
in a PCR assay. The target was a cloned synthetic amplicon isolated from a
plasmid.
Table 41:
Name Sequence SEQ ID No.
5T280A-for 5f-GCAGAAAGCGTCTAGCCATGGCGTTA SEQ ID
No. 195
5T778AA-rev 5f-GCAAGCACCCTATCAGGCAGTACCACAA SEQ ID
No. 196
5T280A-for-B 5' -GCAGAAAGCGTCTAGCCATGGCGTTAgTATG-SpC3 SEQ ID
No. 197
5T778AA-rev-B 5' -GCAAGCACCCTATCAGGCAGTAccAcAAgGCCT-SpC3 SEQ ID No. 198
DNA bases are shown in uppercase. RNA bases are shown in lowercase. SpC3 is a
C3
spacer. The "B" designation indicates a blocked, cleavable primer.
Cloned synthetic target (primer binding sites are underlined).
[0461] SEQ ID No. 199
Hepatitis C virus subtype lb amplicon (242 bp):
qcagaaaqcqtctagccatggcgttagtatgagtgtcgtgcagcctccaggaccccccctcccgggaga
gccatagtggtctgcggaaccggtgagtacaccggaattgccaggacgaccgggtcctttcttggacta
aacccgctcaatgcctggagatttgggcgtgcccccgcgagactgctagccgagtagtgttgggtcgcg
aaaggccttgtggtactgcctgatagggtgcttgc
[0462] PCR reactions were done in 384 well format using a Roche Lightcycler
480 platform. Reactions comprised lx New England Biolabs (Beverly, MA) DyNAmo
reaction mix with DyNAmo DNA polymerase, 200 nM of each primer (For + Rev),
with or without 1.3 mU of Pyrococcus abyssi RNase H2 in 10 1.11 volume.
Template
DNA was either 2000 copies of the linearized HCV plasmid amplicon or no target
control. Thermal cycling parameters included an initial 2 minutes soak at 95 C
and
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then 50 cycles were performed of [95 C for 15 seconds + 60 C for 30 seconds].
Samples were separated on an 8% polyacrylamide non-denaturing gel and
visualized
using GelStar stain. Results are shown in Figure 25. The unblocked standard
primers
produced multiple products having sizes ranging from 55 bp to 90 bp in size
and no
desired full length product was seen. In the absence of RNase H2, use of the
blocked
primers did not result in any amplified product. With RNase H2, the blocked
primers
produced a single strong amplicon of the expected size and no undesired small
species
were seen.
[0463] The DyNAmo is a non hot-start DNA polymerase. Use of RNase H2
blocked primer of the present invention with a hot-start RNase H2 having
reduced
activity at lower temperatures eliminated undesired primer-dimers from the
reaction
and resulted in formation of the desired amplicon whereas standard unblocked
primers
failed and produced only small, undesired species.
EXAMPLE 18 Use of detergent in RNase H2 assay buffers
[0464] The presence of detergent was found to be beneficial to cleavage by
the
Pyrococcus abyssi RNase H2 enzyme. Different detergents were tested at
different
concentrations to optimize the reaction conditions.
[0465] Aliquots of each of the recombinant RNase H2 enzymes were incubated
with the single-stranded and double-stranded oligonucleotide substrates
indicated
above in an 80 11,1 reaction volume in buffer 50 mM NaC1, 10 mM MgC12, and 10
mM
Tris pH 8.0 for 20 minutes at 70 C. Reactions were stopped with the addition
of gel
loading buffer (formamide/EDTA) and separated on a denaturing 7M urea, 15%
polyacrylamide gel. The RNA strand of the substrate SEQ ID NOS 10 and 11 was
radiolabeled with 32P. Reactions were performed using 100 nM substrate with
100
microunits (UU) of enzyme in Mg Cleavage Buffer with different detergents at
varying
concentrations. Detergents tested included Triton-X100, Tween-20, Tween-80,
CTAB,
and N-lauryol sarcosyl. Results with Pyrococcus absii RNase H2 are shown in
Figure
26. Additional experiments were done to more finely titrate CTAB detergent
concentration. Optimum levels of detergent to obtain highest enzyme activity
were
(vol:vol): Triton-X100 0.01%, Tween-20 0.01%, and CTAB 0.0013%. The detergents
Tween-80 and N-lauryol sarcosyl did not perform as well as the other
detergents tested.
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Thus both non-ionic (Triton, Tween) and ionic (CTAB) detergents can be
employed to
stabilize thermophilic RNase H2 enzymes of the present invention.
EXAMPLE 19 Use of fluorescence-quenched (F/Q) cleavable primers in qPCR
[0466] In Example
9 above, it was demonstrated that cleavable blocked primers
function in PCR and further can be employed in real-time quantitative PCR
(qPCR)
using SYBR green detection. In this
example we demonstrate use of
fluorescence-quenched cleavage primers where the primer itself generates
detectable
signal during the course of the PCR reaction.
[0467] Figure 18
illustrates the scheme for performing PCR using blocked
cleavable primers. Figure 27 illustrates the scheme for performing PCR using
fluorescence-quenched cleavable primers. In this case one primer in the pair
is
detectably labeled with a fluorescent dye. A fluorescence quencher is
positioned at or
near the 3'-end of the primer and effectively prevents priming and DNA
synthesis when
the probe is intact. A single ribonucleotide base is positioned between the
dye and the
quencher. Cleavage at the ribonucleotide by RNase H2 separates the reporter
and
quencher, removing quenching, resulting in a detectable signal. Concomitantly,
cleavage activates the primer and PCR proceeds.
[0468] The
following synthetic oligonucleotides shown in Table 42, were
employed to demonstrate this reaction using a synthetic template. As a control
the
5' -nuclease Taqman assay was performed with unmodified primers and a
standard
fluorescence-quenched probe. Three variants of the synthetic fluorescence-
quenched
cleavable primers were compared, having 4, 5, or 6 DNA bases 3' to the RNA
base. It
was previously established that 4 DNA bases 3' to the RNA base was optimal
using
oligonucleotide substrates having a C3 spacer or ddC end group. It was
possible that
the presence of a bulky hydrophobic quencher group at or near the 3'-end might
change
the optimal number of DNA residues needed in this domain.
Table 42:
Name Sequence SEQ ID No.
Syn-F or 5'-AGCTCTGCCCAAAGATTACCCTG SEQ ID No.
68
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Table 42:
Name Sequence SEQ ID No.
Syn-Rev 5f-CTGAGCTTCATGCCTTTACTGT SEQ ID No. 69
Syn-Probe 5f-FAM-TTCTGAGGCCAACTCCACTGCCACTTA-IBFQ SEQ ID No. 200
Syn-F or
5f-FAM-CTGAGCTTCATGCCTTTACTGTuCCCC-IBFQ SEQ ID No. 201
F/Q-4D
Syn-F or
5f-FAM-CTGAGCTTCATGCCTTTACTGTuCCCCG-IBFQ SEQ ID No. 202
F/Q-5D
Syn-F or
5f-FAM-CTGAGCTTCATGCCTTTACTGTuCCCCGA-IBFQ SEQ ID No. 203
F/Q-6D
DNA bases are shown in uppercase. RNA bases are shown in lowercase. FAM is
6-carboxyfluorescein. IBFQ is Iowa Black FQ, a dark quencher.
Synthetic template
[0469] SEQ ID No. 75
AGCTCTGCCCAAAGATTACCCTGACAGCTAAGTGGCAGTGGAAGTTGGCCTCAGAAGTAGTGGCCAGCT
GTGTGTCGGGGAACAGTAAAGGCATGAAGCTCAG
[0470] PCR reactions were performed in 10 IA volume using 200 nM primers,
200
1.1,M of each dNTP (800 1.1,M total), 1 unit of iTaq (BIO-RAD), 50 mM Tris pH
8.3, 50
mM KC1, and 3 mM MgC12. Reactions were run either with or without varying
amounts of Pyrococcus abyssi RNase H2 on a Roche Lightcycler 480 platform
with 2
x 106 copies of synthetic template/target oligonucleotide (SEQ ID No. 75).
Reactions
were started with a soak at 95 C for 5 minutes followed by 45 cycles of [95 C
for 10
seconds, 60 C for 30 seconds, and 72 C for 1 second]. The For and Rev primers
(SEQ
ID Nos. 68 and 69) were used with the internally placed DLP (SEQ ED No. 200).
Alternatively, the For primer (SEQ ID No. 68) was used with the FQ primers
(individually) (SEQ ID Nos. 201-203).
[0471] Use of the F/Q cleavable primers resulted in detectable fluorescence
signal
in real time during PCR similar to that obtained using the traditional dual-
labeled probe
(DLP) (SEQ ID No. 200) in the 5'-nuclease assay format. Primer SEQ ID No. 201,
with 4 DNA residues 3' to the RNA base, showed delayed amplification relative
to the
unmodified primers. Primers SEQ ID No. 202 and 203, with 5 and 6 DNA bases 3'
to
the RNA base, were more efficient and performed equally well. It is therefore
preferable to use an oligonucleotide design with 5 DNA bases 3' to the RNA
base in
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this assay format as opposed to the 3-4 DNA base design optimal when the 3'-
blocking
group is smaller. In previous Examples using a SYBR Green assay format, 1.3 mU
of
RNase H2 resulted in priming efficiency identical to unmodified primers. In
the
present F/Q assay format, use of 1.3 mU of RNase H2 resulted in delayed
amplification
whereas use of 2.6 mU of RNase H2 resulted in identical results compared to
unmodified primers. Increasing the amount of RNase H2 for the F/Q assay format
is
therefore preferred. Both amplification and detection of signal was RNase H2
dependent.
[0472] Examples of
amplification plots for qPCR reactions run using the
5'-nuclease assay DLP (SEQ ID No. 200) and the F/Q cleavable 5D primer (SEQ ID
No. 202) are shown in Figure 28. It is evident that amplification efficiency
is similar
between both methods as the Cp values where fluorescence is first detected is
identical
(20.0). Interestingly, the ARf (the magnitude of fluorescence signal detected)
peaked at
slightly higher levels using the DLP than the FQ primer. One possible
explanation for
the difference in maximal fluorescence signal release is that the fluorescent
dye on the
FQ primer remained partially quenched at the end of the reaction. In the 5'-
nuclease
assay, the probe is degraded and the reporter dye is released into the
reaction mixture
attached to a single-stranded short nucleic acid fragment. In the FQ primer
assay
format the fluorescent reporter dye remains attached to the PCR amplicon and
is in
double-stranded format. DNA can quench fluorescein emission, so this
configuration
might lower the final signal.
[0473] We
therefore tested if changing dye/quencher configuration on the primer
would alter the fluorescence signal, comparing F/Q vs. Q/F versions of the
same primer.
In the synthetic amplicon assay used above, the preferred 5-DNA probe has a
"G"
residue present at the 3'-end. G residues tend to quenche FAM, whereas other
bases
have little effect on FAM fluorescence. The amplicon was therefore modified to
change this base. The sequences in Table 43, were synthesized and tested in a
fluorescent real-time PCR assay format.
Table 43:
Name Sequence SEQ ID No.
Syn-F or 5'-AGCTCTGCCCAAAGATTACCCTG SEQ ID No.
68
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Table 43:
Name Sequence SEQ ID No.
Syn-For(C)
5'-FAM-CTGAGCTTCATGCCTTTACTGTuCCCCC-IBFQ SEQ ID No. 204
F/Q-5D
Syn-For(C)
5f-IBFQ-CTGAGCTTCATGCCTTTACTGTuCCCCC-FAM SEQ ID No. 205
Q/F-5D
DNA bases are shown in uppercase. RNA bases are shown in lowercase. FAM is
6-carboxyfluorescein. IBFQ is Iowa Black FQ, a dark quencher.
Synthetic template
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[0474] SEQ ID No. 206
AGCTCTGCCCAAAGATTACCCTGACAGCTAAGTGGCAGTGGAAGTTGGCCTCAGAAGTAGTGGCCAGCT
GTGTGTGGGGGAACAGTAAAGGCATGAAGCTCAG
[0475] PCR reactions were performed in 10 ul volume using 200 nM primers,
200
uM of each dNTP (800 uM total), 1 unit of iTaq (BIO-RAD), 50 mM Tris pH 8.3,
50
mM KC1, and 3 mM MgC12. Reactions were run with 2.6 mU of Pyrococcus abyssi
RNase H2 on a Roche Lightcycler 480 platform with 2 x 106 copies of synthetic
template/target oligonucleotide (SEQ ID No. 206). Reactions were started with
a soak
at 95 C for 5 minutes followed by 45 cycles of [95 C for 10 seconds, 60 C for
30
seconds, and 72 C for 1 second]. The For primer (SEQ ID No. 68) was used with
either
the FQ primer (SEQ ID No. 204) or the QF primer (SEQ ID No. 205).
[0476] Use of the F/Q and Q/F cleavable primers resulted in an identical
Cp,
indicating that both primers performed with equal efficiency in the reaction.
As
predicted, the Q/F primer showed increased ARf relative to the F/Q primer.
Both
versions of the primer work equally well in the assay.
EXAMPLE 20 Use of fluorescence-quenched (F/Q) cleavable primers in multiplex
qPCR
[0477] Multiplex assays are commonly employed today to streamline
experiments
and increase throughput. It is particularly common to combine a qPCR assay
specific
for an experimental gene of interest with a second qPCR assay specific for an
internal
reference control gene for normalization purposes. One weakness of SYBR Green
detection for qPCR is that multiplex reactions are not possible. The use of
dye-labeled
fluorescence-quenched probes or primers does permit such multiplex reactions
to be
run. Real time PCR cycling and detection equipment is available today that
permits
combination of 2, 3, or 4 different fluorophores into the same reaction tube.
This
example demonstrates the utility of fluorescence-quenched (F/Q) cleavable
primers in
multiplex qPCR.
[0478] The following oligonucleotide reagents shown in Table 44, were
synthesized to perform multiplex qPCR using either a dual-labeled probe with
the
5' -nuclease assay or an F/Q cleavable primer. One assay was specific for the
human
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MYC gene (NM_002476) and the second assay was specific for the human SFRS9
gene (NM_003769), a splicing factor which is a commonly used internal
normalization
control gene.
Table 44:
Name Sequence SEQ ID No.
MYC-For 5f-TCGGATTCTCTGCTCTCCT SEQ ID No. 207
MYC-Rev 5f-CCTCATCTTCTTGTTCCTCC SEQ ID No. 208
MYC-Probe 5f-FAM-CCACCACCAGCAGCGACTCTGA-IBFQ SEQ ID No. 209
MYC-For-FQ 5' -FAM-TCGGATTCTCTGCTCTCCTcGACGG- I BFQ SEQ ID No. 210
MYC-Rev-B 5f-CCTCATCTTCTTGTTCCTCCuCAGA-SpC3 SEQ ID No. 211
SFRS9-For 5f-TGTGCAGAAGGATGGAGT SEQ ID No. 212
SFRS9-Rev 5f-CTGGTGCTTCTCTCAGGATA SEQ ID No. 213
SFRS9-Probe 5' -MAX- TGGAATATGCCCTGCGTAAACTGGA- I BFQ SEQ ID No. 214
SFRS9-For-FQ 5' -MAX- TGTGCAGAAGGATGGAGTgGGGAT- I BFQ SEQ ID No. 215
SFRS9-Rev-B 5' -CTGGTGCT TC TC TCAGGATAaAC TC -SpC 3 SEQ ID No. 216
DNA bases are shown in uppercase. RNA bases are shown in lowercase. FAM is
6-carboxyfluorescein. IBFQ is Iowa Black FQ, a dark quencher. MAX is a red
reporter
dye. SpC3 is a C3 spacer.
[0479] PCR reactions were performed in 10 ,1 volume using 200 nM primers
(and
probe where appropriate), 200 1.1,M of each dNTP (800 1.1,M total), 1 unit of
iTaq
(BIO-RAD), 50 mM Tris pH 8.3, 50 mM KC1, and 3 mM MgC12. Reactions were run
with 10 mU of Pyrococcus abyssi RNase H2 on a Roche Lightcycler 480 platform
with 2 ng of cDNA made from total HeLa cell RNA. Reactions were started with a
soak at 95 C for 5 minutes followed by 45 cycles of [95 C for 10 seconds, 60 C
for 30
seconds, and 72 C for 1 second].
[0480] The multiplex reactions for the 5'-nuclease assays included the MYC
For
and Rev primers + MYC probe (SEQ ID Nos. 207-209) and the SFRS9 For and Rev
primers + SFRS9 probe (SEQ ID Nos. 212-214). The multiplex reactions for the
FQ-cleavable primer assays included the MYC-For-FQ and MYC-Rev-B blocked
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primers (SEQ ID Nos. 210 and 211) and the SFRS9-For-FQ and SFRS9-Rev-B blocked
primers (SEQ ID Nos. 215 and 216). All assays were also run in singleplex
format for
comparison. The FAM primers and probes were detected in the fluorescein dye
channel while the MAX primers and probes were detected in the HEX dye channel.
Both the multiplexed DLP 5'-nuclease assays and the multiplexed FQ-cleavable
primer
assays worked well and resulted in very similar data, which is summarized in
Table 45
below.
Table 45. Multiplex qPCR reactions for MYC and SFRS9
Cp Value Cp Value
Reaction
FAM Channel HEX Channel
MYC FAM DLP 25.7 --
SFRS9 MAX DLP -- 24.8
MYC FAM DLP +
24.6 23.9
SFRS9 MAX DLP
MYC FAM FQ-Primer 27.2 --
SFRS9 MAX FQ Primer -- 28.0
MYC FAM FQ-Primer +
27.9 26.1
SFRS9 MAX FQ Primer
[0481] RNase H concentration was titrated and higher levels of enzyme were
needed to maintain reaction efficiency in multiplex format. For example,
blocked
primers in singleplex SYBR Green detection format required 1.3 mU of enzyme.
Blocked FQ primers in singleplex format required 2.6 mU of enzyme. Blocked FQ
primers in multiplex format required 10 mU of enzyme. It is therefore
important to
titrate the amount of RNase H2 enzyme employed when cleavable primers are used
in
different assay formats.
[0482] Another application where use of multiplex probes is common practice
is
allelic discrimination SNPs. The following assay was designed to distinguish a
SNP
pair for the SMAD7 gene at a site that is known to be relevant for development
of
colorectal carcinoma, rs4939827. FQ blocked primers were designed and
synthesized
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at this site using the standard design features taught in the above examples
without any
further optimization to discriminate between the "C" and "T" alleles in this
gene.
Sequences are shown below in Table 46.
Table 46:
Name Sequence SEQ ID No.
rs4939827 Rev 5' -CTCACTCTAAACCCCAGCATT SEQ ID No.217
rs4939827
C-FAM-FQ-For 5'-FAM-CAGCCTCATCCAAAAGAGGAAAcAGGA-IBFQ SEQ ID No.218
rs4939827
T-HEX-FQ-For 5'-HEX-CAGCCTCATCCAAAAGAGGAAAuAGGA-IBFQ SEQ ID No.219
DNA bases are shown in uppercase. RNA bases are shown in lowercase. FAM is
6-carboxyfluorescein. IBFQ is Iowa Black FQ, a dark quencher. MAX is a red
reporter
dye.
[0483] The above primers target the following 85 bp region of the SMAD7
gene
(NM 005904). Primer binding sites are underlined and the SNP location is
highlighted
as bold italic.
rs4939827 (SMAD7) C allele (SEQ ID No. 220)
CAGCCTCATCCAAAAGAGGAAACAGGACCCCAGAGCTCCCTCAGACTCCTCAGGAAACACAGACAATGC
TGGGGTTTAGAGTGAG
rs4939827 (SMAD7) T allele (SEQ ID No. 221)
CAGCCTCATCCAAAAGAGGAAATAGGACCCCAGAGCTCCCTCAGACTCCTCAGGAAACACAGACAATGC
TGGGGTTTAGAGTGAG
[0484] PCR reactions were performed in 10 ial volume using 200 nM FQ-For
and
unmodified Rev primers, 200 1.1,M of each dNTP (800 laM total), 1 unit of iTaq
(BIO-RAD), 50 mM Tris pH 8.3, 50 mM KC1, and 3 mM MgC12. Reactions were run
with 2.6 mU of Pyrococcus abyssi RNase H2 on a Roche Lightcycler 480 platform
with 2 ng of target DNA. Target DNA was genomic DNA made from cells
homozygous for the two SMAD7 alleles (Coreill 18562 and 18537). The "C" and
"T"
alleles (SEQ ID Nos. 220 and 221) were tested individually (homozygote) and
together
(heterozygote). Reactions were started with a soak at 95 C for 5 minutes
followed by
45 cycles of [95 C for 10 seconds, 60 C for 30 seconds, and 72 C for 1
second]. Data
acquisition was set for multiplex mode detecting the FAM and HEX channels.
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[0485] Results are shown in Figure 30. It is clear that the FAM-labeled "C"
probe
detected the presence of the "C" target DNA but not the "T" target DNA and
that the
HEX "T" probe detected the presence of the "T" target DNA but not the "C"
target
DNA. Thus FQ cleavable primers can be used in multiplex formats to distinguish
SNPs.
EXAMPLE 21 Use of fluorescence-quenched cleavable primers in the
primer-probe assay
[0486] We previously described a method of detecting nucleic acid samples
using
fluorescence quenched primers comprising two distinct but linked elements, a
Reporter
Domain positioned towards the 5'-end and a Primer Domain, positioned at the 3'-
end
of the nucleic acid molecule (US patent application US 2009/0068643). The
Primer
Domain is complementary to and will bind to a target nucleic acid under
conditions
employed in PCR. It is capable of priming DNA synthesis using the
complementary
target as template, such as in PCR. The Reporter Domain comprises a sequence
which
can be complementary to the target or can be unrelated to the target nucleic
acid and
does not hybridize to the target. Furthermore the Reporter Domain includes a
detectable element, such as a fluorescent reporter dye, and a quencher. The
reporter
dye and quencher are separated by a suitable number of nucleotides such that
fluorescent signal from the reporter dye is effective suppressed by the
quencher when
the Reporter Domain is in single-stranded random coil conformation. During
PCR, the
Primer Domain will prime DNA synthesis and the FQT synthetic oligonucleotide
is
thereby incorporated into a product nucleic acid, which itself can is used as
template in
the next cycle of PCR. Upon primer extension during the next cycle of PCR, the
entire
FQT probe is converted to double-stranded form, including the Reporter Domain.
Formation of a rigid double-stranded duplex physically increases the distance
between
the fluoropohore and the quencher, decreasing the suppression of fluorescence
emission (hence increasing fluorescent intensity). Thus conversion of the FQT
primer
to double-stranded form during PCR constitutes a detectable event. Further
increases
in fluorescent signal can be achieved by cleavage of the Reporter Domain at a
site
between the reporter dye and the quencher, such that the reporter dye and the
quencher
become physically separated and are no longer covalently linked on the same
nucleic
acid molecule. This cleavage event is dependent upon formation of double-
stranded
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nucleic acid sequence so that cleavage cannot occur if the FQT primer is in
its original
single-stranded state. Suitable methods to separate reporter and quencher
include, for
example, use of a restriction endonuclease to cleave at a specific sequence in
dsDNA.
Alternatively, an RNase H2 cleavage domain can be placed between the
fluorophore
and quencher. Placement of a single ribonucleotide residue between the
fluorophore
and the quencher would make the FQT primer a suitable substrate for RNase H2
during
PCR. The scheme for this reaction is shown in Figure 31. The present example
demonstrates use of a thermostable RNase H2 to mediate cleavage of a
fluorescence-quenched primer in a primer-probe real time PCR assay.
[0487] A qPCR assay was designed for the human Drosha gene including
unmodified For and Rev primers with an internally positioned dual-labeled
probe
suitable for use in the 5'-nuclease assay. The For primer was also synthesized
as an
FQT forward primer using the same Primer Domain sequence as the unmodified For
primer and adding a Reporter Domain on the 5'-end comprising a reporter dye
(Fluorescein-dT) and a dark quencher (IBFQ) separated by 11 bases including a
centrally positioned rU base (cleavage site). Sequences are shown below in
Table 47.
Table 47:
Name Sequence SEQ ID No.
Drosha-For 5f-ACCAACGACAAGACCAAGAG SEQ
ID No. 222
Drosha-Rev 5f-TCGTGGAAAGAAGCAGACA SEQ
ID No. 223
Drosha-probe 5f-FAM-ACCAAGACCTTGGCGGACCTTT-IBFQ SEQ
ID No. 224
Drosha-For-FQT 5' - I BFQ -T T TCCuGGTTT ( Fl - dT ) ACCAACGACAAGACCAAGAG SEQ
ID No. 225
DNA bases are shown in uppercase. RNA bases are shown in lowercase. Fl-dT in
an
internal Fluorescein-dT modified base. IBFQ is Iowa Black FQ, a dark quencher.
The
portion of the FQT probe that is complementary to the Drosha target is
underlined (i.e.,
the Primer Domain).
[0488] The above primers target the following 141 bp region of the human
Drosha
gene (RNASEN, NM_013235). Primer binding sites are underlined and the internal
probe binding site for the 5'-nuclease assay is in bold font.
Drosha amplicon (SEQ ID No. 226)
ACCAACGACAAGACCAAGAGGCCTGTGGCGCTTCGCACCAAGACCTTGGCGGACCTTTTGGAATCATTT
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ATTGCAGCGCTGTACATTGATAAGGATTTGGAATATGTTCATACTTTCATGAATGTCTGCTTCTTTCCA
CGA
[0489] 5'-Nuclease qPCR reactions were performed in 10 1 volume using 200
nM
unmodified For and Rev primers with 200 nM probe, 200 [tM of each dNTP (800
[tM
total), 1 unit of iTaq (BIO-RAD), 50 mM Tris pH 8.3, 50 mM KC1, and 3 mM
MgC12.
FQT qPCR reactions were performed in 10 11,1 volume using 200 nM FQT-For
primer
and 200 nM unmodified Rev primer, 200 [tM of each dNTP (800 [tM total), 1 unit
of
iTaq (hot start thermostable DNA polymerase, BIO-RAD), 50 mM Tris pH 8.3, 50
mM
KC1, and 3 mM MgC12. Reactions were run with or without 2.6 mU of Pyrococcus
abyssi RNase H2 on a Roche Lightcycler 480 platform. Reactions were run with
or
without 10 ng of cDNA made from HeLa total cellular RNA. Reactions were
started
with a soak at 95 C for 5 minutes followed by 45 cycles of [95 C for 10
seconds, 60 C
for 30 seconds, and 72 C for 1 second].
[0490] Results for the 5'-nuclease qPCR reaction are shown in Figure 32A. A
positive signal was seen at cycle 26. Results for the FQT primer qPCR
reactions are
shown in Figure 32B. A positive signal was seen at cycle 27, nearly identical
to the
5'-nuclease assay results. In this case, signal was dependent upon RNase H2
cleavage.
Thus cleavage at an internal RNA residue by RNase H2 can be used to generate
signal
from FQT primers that have a distinct fluorescence-quenched reporter domain.
EXAMPLE 22 Use of modified bases in cleavable blocked primers to improve
mismatch discrimination
[0491] We demonstrated that blocked cleavable primers can be used in qPCR
to
distinguish single base mismatches in the SYBR Green assay format in Example
13 and
in the fluorescence-quenched (FQ) assay format in Example 20. Depending upon
the
precise base mismatch and the sequence context, detectable signal for the
mismatch
target occurred from 5 to 15 cycles after detection of the perfect match
target. There
may be circumstances where greater levels of mismatch discrimination are
desired,
such as detection of a rare mutant allele in the background of predominantly
wild type
cells. We demonstrate in this example that selective placement of 2'0Me RNA
modified residue within the cleavable primer can improve mismatch
discrimination.
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[0492] Example 5 above demonstrated that modified bases could be compatible
with cleavage of a heteroduplex substrate by RNase H2 depending upon the type
of
modification employed and placement relative to the cleavage site. Here we
demonstrate in greater detail use of the 2' OMe modification in blocked
primers having
a single unmodified ribonucleotide base. The following primers, shown below in
Table
48, were synthesized and used in qPCR reactions in the SYBR Green format with
a
synthetic oligonucleotide template. Blocked cleavable primers having a single
rU
residue were synthesized either without additional modification (SEQ ID No.
116) or
with a 2' OMe base 5'- to the rU (SEQ ID No. 228) or with a 2' OMe base 3'- to
the rU
(SEQ ID No. 229). If the 2'0Me residue is positioned 5'- to the
ribonucleotide, then it
will remain in the final primer which results from cleavage by RNase H2.
Therefore a
Syn-Rev-mU primer was made specific for the synthetic template bearing a 3'-2'
OMe
U residue at the 3'-end to mimic this reaction product (SEQ ID No. 227).
Table 48:
Name Sequence SEQ ID No.
Syn-F or 5f-AGCTCTGCCCAAAGATTACCCTG SEQ ID No. 68
Syn-Rev 5f-CTGAGCTTCATGCCTTTACTGT SEQ ID No. 69
Syn-Rev-mU 5' -CTGAGCTTCATGCCTTTACTG (mU) SEQ ID No. 227
Syn-Rev-rU-C3 5f-CTGAGCTTCATGCCTTTACTGTuCCCC-SpC3 SEQ ID No. 116
Syn-Rev-mUrU-C3 5' -CTGAGCTTCATGCCTTTACTG (mU) uCCCC-SpC3 SEQ ID No. 228
Syn-Rev-rUmC-C3 5' -CTGAGCTTCATGCCTTTACTGTu (mC) CCC-SpC3 SEQ ID No. 229
DNA bases are shown in uppercase. RNA bases are shown in lowercase. 2' OMe RNA
bases are indicated as (mN).
[0493] The following synthetic oligonucleotide was used as template. Primer
binding sites are underlined.
Synthetic template, SEQ ID No. 144:
AGCTCTGCCCAAAGATTACCCTGACAGCTAAGTGGCAGTGGAAGTTGGCCTCAGAAGTAGTGGCCAGCT
GTGTGTCGGGGAACAGTAAAGGCATGAAGCTCAG
[0494] PCR reactions were performed in 10 ial volume using 200 nM
unmodified
For primer pairwise with 200 nM of each of the different Rev primers shown
above in
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Bio-Rad SYBR Green master mix. Reactions were run with or without 1.3 - 200 mU
of
Pyrococcus abyssi RNase H2 on a Roche Lightcycler 480 platformwith no target
or 2
x 106 copies of the synthetic oligonucleotide template. Reactions were started
with a
soak at 95 C for 5 minutes followed by 45 cycles of [95 C for 10 seconds, 60 C
for 20
seconds, and 72 C for 30 seconds]. Results are summarized in Table 49.
Table 49. Cp values of qPCR reactions comparing blocked primers with or
without a
2' OMe base flanking a cleavable ribonucleotide.
Syn-Rev Syn-Rev-mU Syn-Rev-rU-C3
RNase SEQ ID SEQ ID No. SEQ ID No. Syn-Rev-mUrU-C3 Syn-Rev-rUmC-C3
SEQ ID
22 116 No. 228 SEQ ID
No. 229
H2 No. 89 7
None 17.8 19.8 >40 >40 >40
17.8 19.8 17.2 21.6 >40
mU
100
17.8 19.6 17.2 19.7 >40
mU
150
17.8 19.9 17.2 19.5 >40
mU
200
17.8 19.8 17.2 19.1 >40
mU
[0495] The unblocked primer with a 3'-terminal 2'0Me base (SEQ ID No. 227)
showed a 2 cycle delay relative to the unmodified primer (SEQ ID No. 69),
indicating
that the terminal 2'0Me base slightly decreased priming efficiency but
nevertheless
was functional as a PCR primer. The blocked primer containing a single rU base
(SEQ
ID No. 116) performed as expected (see Example 13) and worked well with low
concentrations of RNase H2 (data not shown). For the 2'0Me RNA containing
primers
a higher concentration of RNase H2 was needed. The primer having a 2' OMe
residue
5'- to the ribonucleotide (SEQ ID No. 228) showed good activity at 50 mU RNase
H2
and performed identically to the unblocked 2'0Me control primer (SEQ ID No.
227)
when 100 mU or higher RNase H2 was employed. The primer having a 2' OMe
residue
3'-to the ribonucleotide (SEQ ID No. 229) did not function at any level of
RNase H2
tested. The primer having a 2' OMe residue 5'- to the ribonucleotide (SEQ ID
No. 228)
was next tested in a mismatch discrimination qPCR assay.
[0496] The standard configuration blocked RNase H2 cleavable primer (SEQ ID
No. 116) was compared with the 5'-2'0Me version of this sequence (SEQ ID No.
228).
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These two "Rev" primers were used with the unmodified "For" primer (SEQ ID No.
68)
together with 3 different synthetic oligonucleotide templates (originally used
in
defining mismatch discrimination potential in Example 13). These templates
provide a
perfect match control (Template SEQ ID No. 144), a T/U mismatch (Template SEQ
ID
No. 137), or a G/U mismatch (Template SEQ ID No. 158). The 3 templates
oligonucleotides are shown below with the cleavable blocked primer (SEQ ID No.
116)
aligned beneath to illustrate the regions of match vs. mismatch.
Synthetic template, SEQ ID No. 144 (A:U match):
AGCTCTGCCCAAAGATTACCCTGACAGCTAAGTGGCAGTGGAAGTTGGCCTCAGAAGTAGTGGCCAGCT
GTGTGTCGGGGAACAGTAAAGGCATGAAGCTCAG-3'
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
1 1 1 1 1 1 1 1 1 1 1 1
3'-C3-CCCCuTGTCATTTCCGTACTTCGAGTC-5'
Synthetic template, SEQ ID No. 137 (T:U mismatch):
AGCTCTGCCCAAAGATTACCCTGACAGCTAAGTGGCAGTGGAAGTTGGCCTCAGAAGTAGTGGCCAGCT
GTGTGTCGGGGTACAGTAAAGGCATGAAGCTCAG-3'
_
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
1 1 1 1 1 1 1 1 1 1 1
3'-C3-CCCCuTGTCATTTCCGTACTTCGAGTC-5'
Synthetic template, SEQ ID No. 158 (G:U mismatch):
AGCTCTGCCCAAAGATTACCCTGACAGCTAAGTGGCAGTGGAAGTTGGCCTCAGAAGTAGTGGCCAGCT
GTGTGTCGGGGGACAGTAAAGGCATGAAGCTCAG-3'
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
1 1 1 1 1 1 1 1 1 1 1
3'-C3-CCCCuTGTCATTTCCGTACTTCGAGTC-5'
[0497] PCR reactions were performed in 10 ial volume using 200 nM
unmodified
For primer with 200 nM of cleavable blocked Rev primer (SEQ ID No. 116) or
5'mU
containing cleavable blocked Rev primer (SEQ ID No. 228) in Bio-Rad SYBR Green
master mix. Reactions were run with 1.3 mU (primer SEQ ID No. 116) or 100 mU
(primer SEQ ID No. 228) of Pyrococcus abyssi RNase H2. Reactions were run on a
Roche Lightcycler 480 platform with 2 x 106 copies of the different synthetic
oligonucleotide templates (SEQ ID Nos. 137, 144, or 158). Reactions were
started with
a soak at 95 C for 5 minutes followed by 45 cycles of [95 C for 10 seconds, 60
C for 20
seconds, and 72 C for 30 seconds]. Results are summarized in Table 50 and are
shown
as ACp (ACp = Cp mismatch ¨ Cp match).
Table50. Cp values of qPCR reactions comparing mismatch discrimination of
blocked
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primers with or without a 2' OMe base on the 5'-side of an RNA residue.
1.3 mU RNase H2 100 mU RNase H2
SEQ ID No. 116 SEQ ID No. 228
rU primer mUrU primer
Match (A:U) 0 0
Mismatch (T:U) 5.3 12.7
Mismatch (G:U) 10.9 14.4
(ACp = Cp mismatch ¨ Cp match)
[0498] In both cases tested, addition of a 2'0Me residue directly 5' to the
cleavable
ribonucleotide significantly improved mismatch discrimination. The T/U
mismatch
improved from a ACp of 5.3 to 12.7 and the G/U mismatch improved from a ACp of
10.9 to 14.4. This new primer design required use of 100 mU of RNase H2
compared
with 1.3 mU (in a 10 ul assay), however the enzyme is inexpensive and the
boost in
reaction specificity was considerable. We conclude that the use of chemically
modified
residues in select positions within the cleavable primer can significantly
improve the
mismatch discrimination capability of the assay.
EXAMPLE 23 Use of double-mismatch design in cleavable blocked primers to
improve mismatch discrimination
[0499] Some nucleic acid probes that are complementary to a wild type (WT)
sequence will bind to both the perfect match WT target and a mutant target
bearing a
single base mismatch with sufficiently similar affinity that the two sequences
(WT and
mutant) are not easily distinguished. While a single mismatch introduced
between the
probe and target sequence may not significantly disrupt binding to the wild
type target
(which has 1 mismatch with the probe) disrupts binding to the mutant target
(which
now has 2 mismatches with the probe). This strategy has been used to improve
selectivity of hybridization based assays as well as assays dependent upon
interaction
with nucleic acid binding proteins. The present example demonstrates use of a
double-mismatch strategy to improve base discrimination with use of
cleavable-blocked primers of the present invention.
[0500] For the present study, the SMAD7 qPCR SNP discrimination assay
presented in Example 20 was employed as a model system, except that the SYBR
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Green detection format was used instead of the FQ format. Blocked-cleavable
primers
were synthesized with the base mismatch in the positioned at the cleavable
ribonucleotide. Using the present probe design, any mismatch placed 5'- to the
cleavage site (RNA base) will be retained in the primer extension product and
thus will
be replicated during PCR. In order to maintain the presence of the double-
mismatch
during PCR, the new mismatch must be positioned 3'- to the cleavable RNA
residue in
the domain that is cleaved off and is not retained in daughter products. It is
desirable
that the intentionally added second mismatch not disrupt function of the
primer with a
perfect match target. It was demonstrated in Example 13 that mismatches
present in the
"+1 position" (i.e., immediately 3'- to the RNA base) can have a significant
impact
upon cleavage and functional primer efficiency. The double mismatch was
therefore
placed at the "+2 position" 3'- to the RNA base with the expectation that this
configuration would not be disruptive as a single mismatch but would be
disruptive as a
double mismatch.
[0501] Blocked-cleavable primers were designed and synthesized at this site
using
standard design features to discriminate between the "C" and "T" alleles in
the SMAD7
gene (SNP locus rs4939827). The same unmodified Rev primer was used in all
assays
(SEQ ID No. 217). The perfect match "C" allele primer is SEQ ID No. 231 and
the
perfect match "T" allele primer is SEQ ID No. 235. Next, a series of primers
were
made bearing a mutation at position +2 relative to the ribonucleotide (2 bases
3'- to the
RNA residue). It was anticipated that the identity of the base mismatch would
alter the
relative perturbation that having a mismatch at this position would introduce
into the
assay. Therefore, perfect match (wild type) and all 3 possible base mismatches
were
synthesized and studied (SEQ ID Nos. 232-234 and 236-238). Sequences are shown
below in Table 51.
Table 51:
Name Sequence SEQ ID No.
rs4939827 Rev 5'-CTCACTCTAAACCCCAGCATT SEQ ID No. 217
rs4939827 For 5'-CAGCCTCATCCAAAAGAGGAAA SEQ ID No. 230
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Table 51:
Name Sequence SEQ ID No.
rs4939827 C-For
5f-CAGCCTCATCCAAAAGAGGAAAcAGGA-SpC3 SEQ ID No. 231
WT
rs4939827 C-For
5f-CAGCCTCATCCAAAAGAGGAAAcAAGA-SpC3 SEQ ID No. 232
CAA _
rs4939827 C-For
5f-CAGCCTCATCCAAAAGAGGAAAcACGA-SpC3 SEQ ID No. 233
CAC
rs4939827 C-For
5f-CAGCCTCATCCAAAAGAGGAAAcATGA-SpC3 SEQ ID No. 234
CAT _
rs4939827 T-For
sf-CAGCCTCATCCAAAAGAGGAAAuAGGA-SpC3 SEQ ID No. 235
WT
rs4939827 T-For
sf-CAGCCTCATCCAAAAGAGGAAAuAAGA-SpC3 SEQ ID No. 236
UAA _
rs4939827 T-For
sf-CAGCCTCATCCAAAAGAGGAAAuACGA-SpC3 SEQ ID No. 237
UAC
rs4939827 T-For
5f-CAGCCTCATCCAAAAGAGGAAAuATGA-SpC3 SEQ ID No. 238
UAT _
DNA bases are shown in uppercase. RNA bases are shown in lowercase. SpC3 is a
spacer C3 used as a 3'-blocking group. Mutations
introduced to create
double-mismatches are indicated with bold underline.
[0502] The above
primers target the following 85 bp region of the SMAD7 gene
(NM 005904). Primer binding sites are underlined and the SNP location is
highlighted
as bold italic. Primers are aligned with target in Figure 33 to help
illustrate the scheme
of the "double mutant" approach to improve SNP discrimination.
rs4939827 (S1VIAD7) C allele (SEQ ID No. 220)
CAGCCTCATCCAAAAGAGGAAACAGGACCCCAGAGCTCCCTCAGACTCCTCAGGAAACACAGACAATGC
TGGGGTTTAGAGTGAG
rs4939827 (S1VIAD7) T allele (SEQ ID No. 221)
CAGCCTCATCCAAAAGAGGAAATAGGACCCCAGAGCTCCCTCAGACTCCTCAGGAAACACAGACAATGC
TGGGGTTTAGAGTGAG
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[0503] PCR reactions were performed in 10 11,1 volume using 200 nM of the
unmodified Rev primer (SEQ ID No. 217) and the series of cleavable blocked For
primers (SEQ ID Nos. 231-238) in Bio-Rad SYBR Green master mix. Reactions were
run with 2.6 mU ofPyrococcus abyssi RNase H2 on a Roche Lightcycler 480
platform
with 2 ng of target DNA. Target DNA was genomic DNA made from cells
homozygous for the two SMAD7 alleles (Coreill 18562 and 18537). The "C" and
"T"
alleles (SEQ ID Nos. 220 and 221) were tested individually. Reactions were
started
with a soak at 95 C for 5 minutes followed by 80 cycles of [95 C for 10
seconds, 60 C
for 30 seconds, and 72 C for 1 second]. Results are shown in Table 52 below.
Table 52. Cp and ACp values of qPCR reactions comparing mismatch
discrimination of
blocked primers with or without a second mutation at position +2 relative to
the
ribonucleotide.
Cp "C" Allele Cp "T" Allele Cp
SEQ ID No. Unblocked
27.5 26.5 ----
230 control
SEQ ID No.
rCAG (WT) 29.2 39.9 10.7
231
SEQ ID No.
rCAA 29.0 47.8 18.8
232
SEQ ID No.
rCAC 31.6 45.4 13.8
233
SEQ ID No.
rCAT 30.2 42.8 12.6
234
SEQ ID No.
rUAG (WT) 42.6 29.2 13.4
235
SEQ ID No.
rUAA 49.3 40.1 9.2
236
SEQ ID No.
rUAC 74.1 49.9 24.2
237
SEQ ID No.
rUAT 62.5 45.3 17.2
238
(ACp = Cp mismatch - Cp match)
[0504] For the "C" allele, the standard design perfectly matched probe (SEQ
ID No.
231) showed amplification efficiency similar to unmodified control primers and
the
mismatch discrimination was 10.7 cycles (ACp = 10.7) against the "T" target.
The
mismatch primers showed a minor decrease in detection efficiency with the "C"
allele
target (a shift of up to 2.4 cycles was observed) but mismatch discrimination
at the SNP
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site increased significantly with a ACp of 18.8 cycles seen for the rCAA
primer (SEQ
ID No. 232).
[0505] For the "T" allele, the standard design perfect match probe (SEQ ID
No. 235)
also showed amplification efficiency similar to unmodified control primers and
the
mismatch discrimination was 13.4 cycles (ACp = 13.4) against the "C" target.
However, unlike the "C" allele, the mismatch primers for the "T" allele showed
a large
decrease in detection efficiency with the "T" allele target. Shifts as large
as 20 cycles
were observed. Nevertheless the relative SNP discrimination was improved with
a
ACp of 24.2 cycles seen for the rUAC primer (SEQ ID No. 237). For this region
of the
SMAD7 gene, the "T" allele creates an "AT-rich" stretch at the site of the
cleavable
RNA base and this sequence has low thermal stability. The presence of a
mismatch at
the +2 position must destabilize the structure in this region much more for
the "T" allele
than the higher stability "C" allele, which would account for the observed
increase Cp
for the "T" allele probes against the "T" target. However, this shift in Cp
values does
not limit utility of the assay. Given the inherent increased specificity of
the
blocked-cleavable primers (see Example 11), there should be no problem with
routinely
extending reactions to 60-80 or more cycles. In certain settings, the
increased
discrimination power of the double-mismatch format will be of sufficient value
to
accept the lower overall reaction efficiency. In "AT-rich" regions like the
SMAD7 "T"
allele, it might also be useful to position the double mismatch at the +3
position,
removing its disruptive effects further from the cleavable ribonucleotide.
EXAMPLE 24 Identity of reaction products made by PCR amplification at SNP
sites using cleavable-blocked primers
[0506] For use in PCR or any primer extension application, if a base
mismatch
(SNP site) is positioned directly at the ribonucleotide residue in blocked-
cleavable
primers, then a cleavage event that occurs 5'- to the RNA base will result in
a primer
extension product that reproduces the base variant present in the template
nucleic acid.
A cleavage event that occurs 3'- to the RNA base will result in a primer
extension
product that changes the product to the RNA base present in the primer,
creating an
error that will be replicated in subsequent PCR cycles. Cleavage on the 3' -
side of the
ribonucleotide is therefore an undesired event. Given the enormous
amplification
power of PCR, even a small amount of 3' -cleavage could lead to the
accumulation of a
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sizeable amount of products containing a sequence error. For example, cleavage
at a
rate of 0.1% would lead to 1 out of 1000 molecules having the "wrong" base at
the site
of the RNA residue which would then be detectable as "perfect match" in
subsequent
PCR cycles. This would equate to a 10 cycle shift (A.Cp = 10) in a qPCR
reaction.
Using the design parameters taught in Example 13, cycle shifts for SNP
discrimination
varied from 5-15. Thus a small amount of undesired and unsuspected 3'-cleavage
could easily account for the delayed false-positive signals seen in Example 13
during
SNP interrogation.
[0507] A false positive signal in an allele-specific SNP discrimination
reaction
could arise from two sources. First, ongoing inefficient cleavage at the
"normal"
RNase H2 cleavage site at the 5'-side of the RNA base (see Figure 3) in spite
of the
mismatch. This reaction will result in primer extension products identical to
the
starting target. Second, a false positive signal in an allele-specific SNP
discrimination
reaction could also arise from inefficient cleavage at an "abnormal" position
anywhere
on the 3 '-side of the RNA base. This reaction would produce primer extension
products identical to the primer and which would then amplify with high
efficiency
using this same primer. If the first scenario were correct, then the products
from a
reaction performed using allele "A" primer with allele "B" target should
produce
mostly allele "B" products, which would continue to amplify inefficiently with
allele
"A" primers. If the second scenario were correct, then the products from a
reaction
performed using allele "A" primer with allele "B" target should produce mostly
allele
"A" products, which would amplify efficiently with allele "A" primers.
[0508] To distinguish between these possibilities, a re-amplification
experiment
was performed wherein a first round of PCR amplification was performed using a
SMAD7 "T" allele primer with SMAD7 "T" allele target DNA or with SMAD7 "C"
allele target DNA. The reaction products were diluted 108 fold and re-
amplification
was performed using the "T" vs. "C" allele primers to determine if the
identity of the
SNP base present in the reaction products changed during the first round of
amplification. The SMAD7 rs4939827 allele system was employed using the
following primers and target DNAs, which are shown below in Table 53.
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Table 53:
Name Sequence SEQ ID No.
rs4939827 Rev 5f-CTCACTCTAAACCCCAGCATT SEQ ID No. 217
rs4939827 For 5f-CAGCCTCATCCAAAAGAGGAAA SEQ ID No. 230
rs4939827 C-For
5f-CAGCCTCATCCAAAAGAGGAAAcAGGA-SpC3 SEQ ID No. 231
WT
rs4939827 T-For
sf-CAGCCTCATCCAAAAGAGGAAAuAGGA-SpC3 SEQ ID No. 235
WT
DNA bases are shown in uppercase. RNA bases are shown in lowercase. SpC3 is a
spacer C3 used as a 3'-blocking group.
[0509] The above primers target the following 85 bp region of the SMAD7
gene
(NM 005904). Synthetic oligonucleotides were synthesized for use as pure
targets in
the SMAD7 system and are shown below. Primer binding sites are underlined and
the
SNP location is highlighted as bold italic.
rs4939827 (SMAD7) C allele (SEQ ID No. 220)
CAGCCTCATCCAAAAGAGGAAACAGGACCCCAGAGCTCCCTCAGACTCCTCAGGAAACACAGACAATGC
TGGGGTTTAGAGTGAG
rs4939827 (SMAD7) T allele (SEQ ID No. 221)
CAGCCTCATCCAAAAGAGGAAATAGGACCCCAGAGCTCCCTCAGACTCCTCAGGAAACACAGACAATGC
TGGGGTTTAGAGTGAG
[0510] PCR reactions were performed in 10 1.1,1 volume using 200 nM of the
unmodified Rev primer (SEQ ID No. 217) and the "T" allele cleavable blocked
For
primer (SEQ ID No. 235) in Bio-Rad SYBR Green master mix. Reactions were run
with 2.6 mU of Pyrococcus abyssi RNase H2 on a Roche Lightcycler 480 platform
with 6.6 x 105 copies of synthetic oligonucleotide target SMAD7 "C" allele
(SEQ ID
No. 220) or SMAD7 "T" allele (SEQ ID No. 230). Reactions were started with a
soak
at 95 C for 5 minutes followed by 80 cycles of [95 C for 10 seconds, 60 C for
30
seconds, and 72 C for 1 second]. Results of qPCR amplifications done at this
SNP site
are shown in Table 54 below.
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Table 54. Cp and ACp values of qPCR reactions showing mismatch discrimination
of
cleavable-blocked primers at a SMAD7 C/T allele.
Cp for: Cp for:
"T" Target "C" Target ACp
SEQ ID No. 221 SEQ ID No. 220
Primer
rs4939827 T-For WT 32.5 18.9 13.6
SEQ ID No. 235
[0511] The "T" allele primer performed similar to pervious results showing
a ACp
of 13.6 between reactions run using the match "T" allele target DNA and the
mismatch
"C" allele target DNA.
[0512] This experiment was repeated using a 108 dilution of the reaction
products
from the above PCR amplifications as target DNA. If cleavage at the mismatch
site
occurred at the expected position 5'- to the ribonucleotide, then the reaction
products
should remain "true" and "T" allele product would be made from input "T"
allele
template and "C" allele product would be made for input "C" allele template.
However,
if any appreciable cleavage occurred 3'- to the ribonucleotide, then the
reaction
products should be converted to the sequence of the primer at the SNP site. In
this case,
a "T" allele product would be made from a "C" allele target.
[0513] PCR reactions were performed in 10 IA volume using 200 nM of the
unmodified Rev primer (SEQ ID No. 217) and the "T" allele cleavable blocked
For
primer (SEQ ID No. 235) or the "C" allele cleavable blocked For primer (SEQ ID
No.
231) in Bio-Rad SYBR Green master mix. Reactions were run with 2.6 mU of
Pyrococcus abyssi RNase H2 on a Roche Lightcycler 480 platform. Input target
DNA
was a 108 dilution of the reaction products shown in Table 54 above. Reactions
were
started with a soak at 95 C for 5 minutes followed by 45 cycles of [95 C for
10 seconds,
60 C for 30 seconds, and 72 C for 1 second]. Results of qPCR amplifications
done at
this SNP site are shown in Table 55 below.
Table 55. Cp and ACp values of qPCR reactions showing mismatch discrimination
of
cleavable-blocked primers at a SMAD7 C/T allele.
Cp for: Cp for:
"T" Target amplified "C" Target amplified ACp
by "T" primer by "T" primer
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Cp for: Cp for:
"T" Target amplified "C" Target amplified AC p
by "T" primer by "T" primer
Primer
rs4939827 T-For WT 27.7 29.2 1.5
SEQ ID No. 235
Primer
rs4939827 C-For WT 38.6 38.5 0.1
SEQ ID No. 231
[0514] The reactions products previously made (Table 54) using the "T"
allele
primer with both the "T" allele target and the "C" allele target now show
nearly
identical amplification efficiency using the "T" allele primer whereas
previously a ACp
of 13.6 was observed between the two different starting target DNAs. This is
most
consistent with the product nucleic acids having similar sequence, i.e., both
are now
predominantly "T" allele. Consistent with this hypothesis, both of these
samples now
show similar delayed Cp using the "C" allele primer. Thus it appears that the
product
from the "T" allele primer amplification using the "C" allele target was
largely
converted to "T" allele, consistent with that product originating with a
primer cleavage
event occurring 3'- to the ribonucleotide base. The reaction products from the
original
amplification using the "T" allele primer (Table 54) were subcloned and DNA
sequence determined. All clones identified had the "T" allele present, whether
the
starting template was the "T" allele or the "C" allele, adding further support
to this
conclusion.
EXAMPLE 25 Use of phosphorothioate modified internucleotide linkages in
cleavable blocked primers to improve mismatch discrimination
[0515] The results form Example 24 indicates that PCR performed with a
mismatched primer/target combination can produce a product with sequence
matching
the primer instead of the target. The most likely scenario that would result
in this kind
of product starts with cleavage of the mismatched primer at a position 3'- to
the
ribonucleotide residue. Use of chemical modifications that prevent unwanted
cleavage
in this domain of the primer may improve performance of the cleavable-blocked
primers especially in SNP discrimination. The following primers, as shown in
below in
Table 56, were synthesized with nuclease-resistant phosphorothioate (PS)
modified
internucleotide linkages placed at positions 3'- to the ribonucleotide as
indicated. It
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was established in Example 15 that placement of a PS bond at the 3'-linkage
directly at
the RNA base can decrease cleavage efficiency. This modification survey
therefore
focused on the DNA linkages further 3'- to this site. The synthetic amplicon
system
previously used in Example 13 was employed.
Table 56:
Name Sequence SEQ ID No.
Syn-F or 5'-AGCTCTGCCCAAAGATTACCCTG SEQ ID No. 68
Syn-Rev-rU-C3 5'-CTGAGCTTCATGCCTTTACTGTuCCCC-SpC3 SEQ ID No. 116
Syn-Rev-rU-
5f-CTGAGCTTCATGCCTTTACTGTuC*CCC-SpC3 SEQ ID No. 301
C*CCC-C3
Syn-Rev-rU-
5f-CTGAGCTTCATGCCTTTACTGTuCC*CC-SpC3 SEQ ID No. 239
CC*CC-C3
Syn-Rev-rU-
5f-CTGAGCTTCATGCCTTTACTGTuCCC*C-SpC3 SEQ ID No. 240
CCC*C-C3
CSyn-Rev-rU-
C-C3 5f-CTGAGCTTCATGCCTTTACTGTuC*C*C*C-SpC3 SEQ ID No. 241
C*C**
DNA bases are shown in uppercase. RNA bases are shown in lowercase.
"*" indicates a phosphorothioate (PS) modified intemucleotide linkage.
[0516] The standard configuration blocked RNase H2 cleavable primer (SEQ ID
No. 116) was compared with PS-modified versions of this sequence (SEQ ID Nos.
301
and 239-241). This set of "Rev" primers were used with the unmodified "For"
primer
(SEQ ID No. 68) together with two different synthetic oligonucleotide
templates
(originally used in defining mismatch discrimination potential in Example 13).
These
templates provide a perfect match control (Template SEQ ID No. 144) and a T/U
mismatch (Template SEQ ID No. 137). The two templates and oligonucleotides are
shown below with the cleavable blocked primer (SEQ ID No. 116) aligned beneath
to
illustrate the regions of match vs. mismatch.
Synthetic template, SEQ ID No. 144 (A:U match):
AGCTCTGCCCAAAGATTACCCTGACAGCTAAGTGGCAGTGGAAGTTGGCCTCAGAAGTAGTGGCCAGCT
GTGTGTCGGGGAACAGTAAAGGCATGAAGCTCAG-3'
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
1 1 1 1 1 1 1 1 1 1 1 1
3f-C3-CCCCuTGTCATTTCCGTACTTCGAGTC-5'
Synthetic template, SEQ ID No. 137 (T:U mismatch):
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AGCTCTGCCCAAAGATTACCCTGACAGCTAAGTGGCAGTGGAAGTTGGCCTCAGAAGTAGTGGCCAGCT
GTGTGTCGGGGTACAGTAAAGGCATGAAGCTCAG-3'
_
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
1 1 1 1 1 1 1 1 1 1 1
3f-C3-CCCCuTGTCATTTCCGTACTTCGAGTC-5'
[0517] PCR reactions were performed in 10 11,1 volume using 200 nM of the
unmodified For primer (SEQ ID No. 68) and the different cleavable blocked Rev
primers shown above (SEQ ID Nos. 116, 301 and 239-241) in Bio-Rad SYBR Green
master mix. Reactions were run with 1.3 mU of Pyrococcus abyssi RNase H2 on a
Roche Lightcycler 480 platform. Input target DNA was 2 x 106 copies of the
synthetic
target sequences shown above (SEQ ID Nos. 137 and 144). Reactions were started
with an incubation at 95 C for 5 minutes followed by 45 cycles of [95 C for 10
seconds,
60 C for 30 seconds, and 72 C for 1 second]. Results of qPCR amplifications
done at
this SNP site are shown in Table 57 below.
Table 57. Cp values of qPCR reactions comparing mismatch discrimination of
blocked
primers with or without PS linkages 3'- to the cleavable ribonucleotide.
Match Target Mismatch Target
(A:U) (T:U) ACp
SEQ ID No. 144 SEQ ID No. 137
SEQ ID No. 116
18.5 26.2 7.7
CCCC Primer
SEQ ID No. 301
19.5 31.9 12.4
C*CCC Primer
SEQ ID No. 239
18.2 26.7 8.5
CC*CC Primer
SEQ ID No. 240
18.4 26.3 7.9
CCC*C Primer
SEQ ID No. 241
18.5 29.1 10.6
C*C*C*C Primer
(ACp = Cp mismatch ¨ Cp match
[0518] Placement of a PS modified linkage at the 3' "+1" position (rUC*CCC)
led
to almost a 5 cycle improvement in SNP discrimination in this assay (SEQ ID
No. 241
vs. 116), demonstrating that increasing nuclease stability in the domain 3'-
to the
ribonucleotide can significantly improve assay performance. Modification o the
linkages further 3' from the ribonucleotide had minimal impact. Modification
of all of
the linkages in this area (rUC*C*C*C, nucleotides 23-27 of SEQ ID No. 241)
also
showed benefit, improving relative SNP discrimination by 3 cycles, but
unexpectedly
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showed less benefit than using just a single modification at the 3'+l linkage.
This may
relate to the lowered binding affinity Tm that also results from the PS
modification.
[0519] Thus, adding nuclease resistant modifications at the linkages 3'- to
the
cleavable ribonucleotide can increase SNP discrimination for the RNase H2
mediated
cleavable-blocked primer PCR assay. Typically, only one of the two
stereoisomers at a
PS linkage (the Rp or Sp isomer) confers benefit. Improved activity might
therefore be
realized by isolation a chirally pure PS compound here, as was demonstrated in
Example 15. Other nuclease resistant modifications may be suitable in this
area, such
as the non-chiral phosphorodithioate linkage, the methyl phosphonate linkage,
the
phosphoramidate linkage, a boranophosphate linkage, and abasic residues such
as a C3
spacer to name a few.
EXAMPLE 26 Use of cleavable primers having an unblocked 3'-hydroxyl in a
qPCR assay
[0520] In the above Examples, a blocking group was placed at the 3' -end of
the
primer to prevent primer extension from occurring prior to RNase H2 cleavage.
For
certain primer designs and applications, it may not be necessary or even
desirable to
employ a 3 '-blocking group. We have previously described a method of nucleic
acid
amplification termed polynomial amplification that employs primers that are
chemically modified in ways that block template function while retaining
primer
function. A variety of groups can be used for this purpose, including internal
C3
spacers and internal 2'0Me RNA bases. Using nested primers, high specificity
is
achieved and amplification power is dependent upon the number of nested
primers
employed, with amplification occurring according to a polynomial expansion
instead of
the exponential amplification seen in PCR (see US Patent 7,112,406 and pending
US
Patent applications 2005/0255486 and 2008/0038724). Combining elements of the
polynomial amplification primers with an RNase H2 cleavable domain of the
present
invention results in a novel primer design that has an unblocked 3 '-hydroxyl
and is
capable of supporting primer extension yet cannot support PCR. Upon cleavage,
the
template blocking groups are removed and primer function for use in PCR is
restored.
The present example demonstrates use of cleavable template-blocked primers
having a
3' -hydroxyl in qPCR.
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[0521] The
following primers, as shown below in Table 58, were synthesized for
use with the artificial synthetic amplicon used in previous Examples.
Table 58:
SEQ ID
Name Sequence
No.
Syn-F or 5f-AGCTCTGCCCAAAGATTACCCTG SEQ ID
No.68
Syn-Rev 5f-CTGAGCTTCATGCCTTTACTGT SEQ ID
No.69
ID
Syn-For-rA-C3 5f-AGCTCTGCCCAAAGATTACCCTGaCAGC-SpC3 SEQ
No. 242
ID
Syn-For-rA-iC3-D1 5 -AGCTCTGCCCAAAGATTACCCTGaCAGC ( SpC3-SpC3) A SEQ
No. 243
ID
Syn-For-rA-iC3-D2 5 -AGCTCTGCCCAAAGATTACCCTGaCAGC ( SpC3-SpC3) AG SEQ
No. 244
ID
Syn-For-rA-iC3-D4 5 -AGCTCTGCCCAAAGATTACCCTGaCAGC ( SpC3-SpC3) AGTG SEQ
No. 245
ID
Syn-For-rA-iC3-D5 5' -AGCTCTGCCCAAAGATTACCCTGaCAGC ( SpC3-SpC3) AGTGG SEQ
No. 246
DNA bases are shown in uppercase. RNA bases are shown in lowercase.
SpC3 is a Spacer C3 group, positioned either internal within the primer or at
the 3'-end.
[0522] The
synthetic amplicon oligonucleotide template (SEQ ID No. 143) is
shown below with the unmodified and various modified cleavable For primers
shown
aligned above and the unmodified Rev primer aligned below. DNA bases are
uppercase, RNA bases are lowercase, and "x" indicates a Spacer-C3 group.
5'AGCTCTGCCCAAAGATTACCCTG SEQ ID No. 68 unmodified
5'AGCTCTGCCCAAAGATTACCCTGaCAGC-x SEQ ID No. 242 3'-block
5'AGCTCTGCCCAAAGATTACCCTGaCAGCxxA SEQ ID No. 243 Int D1
5'AGCTCTGCCCAAAGATTACCCTGaCAGCxxAG SEQ ID No. 244 Int D2
5'AGCTCTGCCCAAAGATTACCCTGaCAGCxxAGTG SEQ ID No. 245 Int D4
5'AGCTCTGCCCAAAGATTACCCTGaCAGCxxAGTGG SEQ ID No. 246 Int D5
11111111111 11111111111 111111 11111
5'AGCTCTGCCCAAAGATTACCCTGACAGCTAAGTGGCAGTGGAAGTTGGCCTCAGAAGTAGTGGCCAG
CTGTGTGTCGGGGAACAGTAAAGGCATGAAGCTCAG-3'
1111111111111111111111
TGTCATTTCCGTACTTCGAGTC-5' SEQ ID No. 69
[0523] PCR
reactions were performed in 10 n1 volume using 200 nM of the
individual For primers (SEQ ID Nos. 68, 242-246) and the unmodified Rev primer
(SEQ ID No. 69) in Bio-Rad SYBR Green master mix. Reactions were run with or
without 1.3 mU ofPyrococcus abyssi RNase H2 on a Roche Lightcycler 480
platform.
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Input target DNA was 2 x 106 copies of the synthetic target shown above (SEQ
ID No.
144). Reactions were started with an incubation at 95 C for 5 minutes followed
by 60
cycles of [95 C for 10 seconds, 60 C for 30 seconds, and 72 C for 1 second].
Results of
qPCR amplifications are shown in Table 59 below.
Table 59. Cp values of qPCR reactions comparing performance of cleavable
primers
having a 3 '-blocking group vs. cleavable primers having internal template
blocking
groups.
Without + 1.3 mU
RNase H2 RNase H2
Unblocked For SEQ ID No. 68
17.0 17.2
Unblocked Rev SEQ ID No. 69
3'-blocked For SEQ ID No. 242
>60 17.1
Unblocked Rev SEQ ID No. 69
It-blocked For SEQ ID No. 243 (D1)
>60 17.1
Unblocked Rev SEQ ID No. 69
It-blocked For SEQ ID No. 244 (D2)
>60 17.1
Unblocked Rev SEQ ID No. 69
It-blocked For SEQ ID No. 245 (D4)
>60 17.1
Unblocked Rev SEQ ID No. 69
It-blocked For SEQ ID No. 246 (D5)
>60 17.9
Unblocked Rev SEQ ID No. 69
[0524] The unblocked primers gave detectable signal at around cycle 17 in
this
assay system. Using the unblocked Rev primer with the 3 '-blocked For primer,
no
signal was detected within the 60 cycle PCR run without RNase H2, however with
RNase H2 a similar cycle detection time of around 17 was seen. The internally
blocked
For primers that had a free 3'-hydroxyl group behaved identically to the 3'-
modified
primer. In spite of the unblocked 3'-hydroxyl, primer cleavage with RNase H2
was
required for function in PCR, presumably due to the loss of template function
imposed
by the internal C3 spacers. C3 spacers placed near the 3 '-end may also
inhibit primer
extension to a certain degree. No signal was detected in the absence of RNase
H2; with
RNase H2, cleavage and amplification proceeded normally.
[0525] This example demonstrates that cleavable primers do not need to be
modified at the 3'-terminal residue to function in a cleavable-primer PCR
assay and
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that primers having internal modifications that disrupt template function can
perform
equally well. Given the significance of this finding to primer design, a
similar
experiment was performed using an endogenous human gene target using human
genomic DNA to ensure that these results could be generalized.
[0526] The
following primers, as shown below in Table 60, were synthesized based
upon the human SMAD7 gene used in previous Examples, using only the "C"
allele.
Table 60:
Name Sequence SEQ ID No.
rs4939827 Rev 5' -CTCACTCTAAACCCCAGCATT SEQ ID No. 217
rs4939827 For 5' -CAGCCTCATCCAAAAGAGGAAA SEQ ID No. 230
rs4939827
5f-CAGCCTCATCCAAAAGAGGAAAcAGGA-SpC3 SEQ ID No. 231
C-For-C3
rs4939827
5f-CAGCCTCATCCAAAAGAGGAAAcAGGA(SpC3-SpC3)C SEQ ID No. 247
C-F or-iC3 -D1
rs4939827
5f-CAGCCTCATCCAAAAGAGGAAAcAGGA(SpC3-SpC3)CC SEQ ID No. 248
C-For-iC3-D2
rs4939827
5f-CAGCCTCATCCAAAAGAGGAAAcAGGA(SpC3-SpC3)CCAG SEQ ID No. 249
C-For-iC3-D4
rs4939827
5f-CAGCCTCATCCAAAAGAGGAAAcAGGA(SpC3-SpC3)CCAGA SEQ ID No. 250
C-For-iC3-D5
DNA bases are shown in uppercase. RNA bases are shown in lowercase.
SpC3 is a Spacer C3 group, positioned either internal within the primer
(template block)
or at the 3'-end (primer block).
[0527] The SMAD7
amplicon sequence (SEQ ID No. 220) is shown below with the
unmodified and various modified cleavable For primers shown aligned above. DNA
bases are uppercase, RNA bases are lowercase, and "x" indicates a Spacer-C3
group.
5'CAGCCTCATCCAAAAGAGGAAA SEQ ID No. 230 unmodified
5'CAGCCTCATCCAAAAGAGGAAAcAGGA-x SEQ ID No. 231 3'-block
5'CAGCCTCATCCAAAAGAGGAAAcAGGAxxC SEQ ID No. 247 It-block
5'CAGCCTCATCCAAAAGAGGAAAcAGGAxxCC SEQ ID No. 248 It-block
5'CAGCCTCATCCAAAAGAGGAAAcAGGAxxCCAG SEQ ID No. 249 It-block
5'CAGCCTCATCCAAAAGAGGAAAcAGGAxxCCAGA SEQ ID No. 250 It-block
111111111111111111111111111 11111
5'CAGCCTCATCCAAAAGAGGAAACAGGACCCCAGAGCTCCCTCAGACTCCTCAGGAAACACAGACAAT
GCTGGGGTTTAGAGTGAG-3'
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[0528] PCR reactions were performed in 10 11,1 volume using 200 nM of the
individual For primers (SEQ ID Nos. 230-231, 247-250) and the unmodified Rev
primer (SEQ ID No. 217) in Bio-Rad SYBR Green master mix. Reactions were run
with or without 2.6 mU of Pyrococcus abyssi RNase H2 on a Roche Lightcycler
480
platform. Input target DNA was 2 ng of genomic DNA from a human cell line
(Coreill
18562, SMAD7 "C" allele). Reactions were started with an incubation at 95 C
for 5
minutes followed by 60 cycles of [95 C for 10 seconds, 60 C for 30 seconds,
and 72 C
for 1 second]. Results of qPCR amplifications are shown in Table 61 below.
Table 61. Cp values of qPCR reactions comparing performance of cleavable
primers
having a 3 '-blocking group vs. cleavable primers having internal template
blocking
groups.
Without + 2.6 mU
RNase H2 RNase H2
Unblocked For SEQ ID No. 230
25.8 25.5
Unblocked Rev SEQ ID No. 217
3'-blocked For SEQ ID No. 231
>60 26.3
Unblocked Rev SEQ ID No. 217
It-blocked For SEQ ID No. 247 (D1)
>60 26.3
Unblocked Rev SEQ ID No. 217
It-blocked For SEQ ID No. 248 (D2)
>60 26.2
Unblocked Rev SEQ ID No. 217
It-blocked For SEQ ID No. 249 (D4)
>60 26.2
Unblocked Rev SEQ ID No. 217
It-blocked For SEQ ID No. 250 (D5)
>60 26.7
Unblocked Rev SEQ ID No. 217
[0529] The unblocked primers gave detectable signal around cycle 26 in this
assay
system using human genomic DNA. Using the unblocked Rev primer with the
3'-blocked For primer, no signal was detected within the 60 cycle PCR run
without
RNase H2, however with RNase H2 a similar cycle detection time of around 26
was
seen. All of the internally blocked For primers that had a free 3'-hydroxyl
group
behaved identically to the 3 '-modified primer. No signal was detected in the
absence of
RNase H2; with RNase H2, cleavage and amplification proceeded normally with
detection occurring around 26 cycles.
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[0530] This example further demonstrates that cleavable primers do not need
to be
modified at the 3'-end to function in the cleavable primer qPCR assay. Primers
haying
internal modifications that disrupt template function still require a primer
cleavage
event to function as primers in the assay. When cleavage is done using RNase
H2 at an
internal cleavable residue, like a single RNA base, amplification efficiency
is identical
to that seen using unmodified primers. This novel version of template-blocked
cleavable primers can be employed to perform PCR in complex nucleic acid
samples
like human genomic DNA.
EXAMPLE 27 Cleavable primers with internal template blocking groups and a
3'-hydroxyl can prime DNA synthesis
[0531] The cleavable template-blocked primers disclosed in Example 26 have
an
unblocked 3'-hydroxyl group that should permit the oligonucleotides to
function as
primers in linear primer extension reactions but the internal template-
blocking groups
prevent function in PCR as most of the primer cannot be replicated.
Consequently, no
primer binding site exists in the daughter products. Cleavage of the primer by
RNase
H2 removes the domain containing the template-blocking groups and restores
normal
primer function. The present example demonstrates that these compositions can
function to prime DNA synthesis.
[0532] The following primers shown below in Table 62 were employed to
perform
linear primer extension reactions using the artificial synthetic amplicon
system used in
previous Examples.
Table 62:
SEQ ID
Name Sequence
No.
Syn-F or 5f-AGCTCTGCCCAAAGATTACCCTG SEQ ID
No. 68
SEQ ID
Syn-For-rA-C3 5f-AGCTCTGCCCAAAGATTACCCTGaCAGC-SpC3
No. 242
SEQ ID
Syn-For-rA-iC3-D1 5' -AGCTCTGCCCAAAGATTACCCTGaCAGC (SpC3-SpC3) A
No. 243
SEQ ID
Syn-For-rA-iC3-D2 5' -AGCTCTGCCCAAAGATTACCCTGaCAGC (SpC3-SpC3) AG
No. 244
SEQ ID
Syn-For-rA-iC3-D4 5' -AGCTCTGCCCAAAGATTACCCTGaCAGC (SpC3-SpC3)AGTG
No. 245
SEQ ID
Syn-For-rA-iC3-D5 5' -AGCTCTGCCCAAAGATTACCCTGaCAGC (SpC3-SpC3)AGTGG
No. 246
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DNA bases are shown in uppercase. RNA bases are shown in lowercase.
SpC3 is a Spacer C3 group, positioned either internal within the primer or at
the 3' -end.
[0533] A newly
synthesized 103mer oligonucleotide template was made which was
complementary to the Syn-For primers above (SEQ ID No. 251), which is shown
below
with the unmodified and various modified cleavable For primers aligned above.
DNA
bases are uppercase, RNA bases are lowercase, and "x" indicates a Spacer-C3
group.
5fAGCTCTGCCCAAAGATTACCCTG SEQ ID No. 68 unmodified
5fAGCTCTGCCCAAAGATTACCCTGaCAGC-x SEQ ID No. 242 3f-block
5fAGCTCTGCCCAAAGATTACCCTGaCAGCxxA SEQ ID No. 243 Int D1
5fAGCTCTGCCCAAAGATTACCCTGaCAGCxxAG SEQ ID No. 244 Int D2
5fAGCTCTGCCCAAAGATTACCCTGaCAGCxxAGTG SEQ ID No. 245 Int D4
5fAGCTCTGCCCAAAGATTACCCTGaCAGCxxAGTGG SEQ ID No. 246 Int D5
11111111111 11111111111 111111 11111
3fTCGAGACGGGTTTCTAATGGGACTGTCGATTCACCGTCACCTTCAACCGGAGTCTTCATCACCGGTC
GACACACAGCCCCTTGTCATTTCCGTACTTCGAGTC-5f
[0534] The six For
primers shown above were radiolabeled with 32P as described
above. Primer extension reactions were performed in a 20 [tL volume using 0.8
U iTaq
polymerase (Bio-Rad), 800 [tM dNTPs, 3 mM MgC12, in lx iTaq buffer (20 mM Tris
pH 8.4, 50 mM KC1) and 2 nM primer and template (40 fmole of each
oligonucleotide
in the 20 [tL reaction). Reactions were started with an incubation at 95 C for
5 minutes
followed by 35 cycles of [95 C for 10 seconds, 60 C for 30 seconds, and 72 C
for 1
second] on an MJ Research PTC-100 thermal cycler. Reactions were stopped with
the
addition of cold EDTA containing formamide gel loading buffer. Reaction
products
were separated using denaturing 7M urea, 15% polyacrylamide gel
electrophoresis
(PAGE) and visualized using a Packard CycloneTM Storage Phosphor System
(phosphorimager). The relative intensity of each band was quantified as above
and
results plotted as a fraction of total radioactive material present in the
band representing
the primer extension product. Results are shown in Figure 34.
[0535] Under these
reaction conditions, 61% of the control unblocked primer (SEQ
ID No. 68) was converted into a higher molecular weight primer extension
product. As
expected, the 3' -end blocked cleavable primer (SEQ ID No. 242) did not show
any
primer extension product. Similarly, the D1 and D2 cleavable primers with
internal C3
groups and a 3' -hydroxyl (SEQ ID Nos. 243-244) also did not support primer
extension.
The cleavable primers having a slightly longer terminal DNA domains (the D4
and D5
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sequences, SEQ ID Nos. 245-246) did support primer extension with the D4
showing
47% conversion and the D5 showing 60% conversion of the primer into an
extension
product, a reaction efficiency identical to the unmodified control primer.
Thus when
internal C3 spacers are placed very near the 3' -end both priming and template
function
are disrupted. When placed more than 4 residues from the 3' -end only template
function is blocked.
EXAMPLE 28 Use of cleavable primers with internal template blocking groups
and a 3'-hydroxyl to improve mismatch discrimination
[0536] Example 24 demonstrated that cleavage of an RNA-containing primer on
the 3' -side of the RNA base by RNase H2 is an undesired event that can
contribute to
late arising false positive signals in a qPCR SNP discrimination assay.
Example 25
demonstrated that modifications which confer nuclease resistance to this
domain can
improve SNP discrimination. The novel compositions described in examples 26
and 27
place internal C3 groups on the 3' -side of the cleavable ribonucleotide which
disrupts
template function of the primer in a domain that is removed by RNase H2
cleavage.
This example demonstrates that positioning the C3 spacer groups close to the
RNA
base improves performance of the cleavable primer in SNP discrimination using
a
format that leaves the probe "unblocked", having an unmodified 3' -hydroxyl.
[0537] The following primers, as shown below in Table 63, were synthesized
for
the human SMAD7 gene similar to previous Examples. Primers were made specific
for
the "C" allele and were tested on both "C" allele and "T" allele genomic DNA
targets.
Table 63:
Name Sequence SEQ ID No.
SEQ ID No.
rs4939827 Rev 5f-CTCACTCTAAACCCCAGCATT
217
SEQ ID No.
rs4939827 For 5f-CAGCCTCATCCAAAAGAGGAAA
230
rs4939827 SEQ ID No.
5f-CAGCCTCATCCAAAAGAGGAAAcAGGA-SpC3
C-For-C3 231
rs4939827 SEQ ID No.
5f-CAGCCTCATCCAAAAGAGGAAAcA(SpC3-SpC3)A
C-For-A(C3C3)A 252
DNA bases are shown in uppercase. RNA bases are shown in lowercase.
SpC3 is a Spacer C3 group, positioned either internal within the primer or at
the 3' -end.
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[0538] The SMAD7
amplicon sequence (SEQ ID No. 220, "C" target) is shown
below with the unmodified and two modified cleavable For primers aligned above
it.
DNA bases are uppercase, RNA bases are lowercase, and "x" indicates a Spacer-
C3
group. The site of the rs4939827 C/T SNP is indicated with bold underline.
5fCAGCCTCATCCAAAAGAGGAAA SEQ ID No. 230 unmodified
5fCAGCCTCATCCAAAAGAGGAAAcAGGA-x SEQ ID No. 231 3f-block
5fCAGCCTCATCCAAAAGAGGAAAcAxxA SEQ ID No. 252 It-block
111111111111111111111111 I
5fCAGCCTCATCCAAAAGAGGAAACAGGACCCCAGAGCTCCCTCAGACTCCTCAGGAAACACAGACAAT
GCTGGGGTTTAGAGTGAG-3f
[0539] The same
primers are aligned with the mismatch SMAD7 amplicon
sequence (SEQ ID No. 221, "T" target).
5fCAGCCTCATCCAAAAGAGGAAA SEQ ID No. 230 unmodified
5fCAGCCTCATCCAAAAGAGGAAAcAGGA-x SEQ ID No. 231 3f-block
5fCAGCCTCATCCAAAAGAGGAAAcAxxA SEQ ID No. 252 It-block
1111111111111111111111 1 1
5fCAGCCTCATCCAAAAGAGGAAATAGGACCCCAGAGCTCCCTCAGACTCCTCAGGAAACACAGACAAT
GCTGGGGTTTAGAGTGAG-3f
[0540] PCR
reactions were performed in 10 IA volume using 200 nM of the
individual For primers (SEQ ID Nos. 230-231, 247-250) and the unmodified Rev
primer (SEQ ID No. 217) in Bio-Rad SYBR Green master mix. Reactions were run
with or without 2.6 mU of Pyrococcus abyssi RNase H2 on a Roche Lightcycler
480
platform. Input target DNA was 2 ng of genomic DNA from human cell lines
homozygous for the SMAD7 "C" and "T" alleles (Coreill 18562 and 18537).
Reactions were started with an incubation at 95 C for 5 minutes followed by 75
cycles
of [95 C for 10 seconds, 60 C for 30 seconds, and 72 C for 1 second]. Results
of qPCR
amplifications are shown in Table 64 below.
Table 64. Cp values of qPCR reactions comparing performance of cleavable
primers
having a 3'-blocking group vs. cleavable primers having internal template
blocking
groups in a SNP discrimination assay.
Unmodified Control 3'-C3 Blocked Int-C3, 3'-unblocked
SEQ ID No. 230 SEQ ID No. 231 SEQ ID No. 252
õp, "c" õr, "c"
ACp
Allele Allele ACp Allele Allele AC') Allele Allele
No
27.5 25.9 >75 >75 >75 >75
RNaseH
2.6 mU
27.3 26.1 27.8 37.8 10.0 41.8 68.4 26.6
RNaseH
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Unmodified Control 3'-C3 Blocked Int-C3, 3'-unblocked
SEQ ID No. 230 SEQ ID No. 231 SEQ ID No. 252
mU
27.1 25.8 - 27.0 40.0 13.0 29.9 53.7 23.8
RNaseH
50 mU
27.1 26.0 - 27.0 28.5 1.5 27.6 53.3 25.7
RNaseH
200 mU
27.1 25.8 - 27.0 26.1 - 27.5 41.4 13.9
RNaseH
(ACp = Cp mismatch "T" - Cp match "C")
[0541] The unmodified primers are designed to be non-discriminatory and
amplified both alleles with similar efficiency, producing a detectable signal
at around
26-27 cycles. Both cleavable primers were dependent upon RNase H2 for function
and
did not produce any detectable signal for either allele in the absence of
cleaving enzyme.
Using low amounts of RNase H2 (2.6¨ 10 mU), the 3'-blocked cleavable primer
(SEQ
ID No. 231) produced detectable signal around cycle 27 for the match "C"
allele and
showed a delayed Cp of 38-40 cycles for the mismatch "T" allele (ACp of 10-
13).
Using higher amounts of RNase H2, specificity was lost and both alleles
amplified with
similar efficiency. The cleavable primer having two C3 spacers 3'- to the
ribonucleotide (SEQ ID No. 252) required higher levels of RNase H2 for
efficient
cleavage/priming and showed delayed Cp values even for the perfect match "C"
allele
using 2.6 and 10 mU of enzyme. It is not surprising that modifications of this
kind near
the RNA cleavable site require higher amounts of enzyme. Example 22
demonstrated
that placing a 2'0Me modification adjacent to the ribonucleotide required 100
mU of
RNase H2 to achieve full activity. Using higher amounts of enzyme resulted in
efficient cleavage and positive signal was detected at ¨27 cycles using 50 or
200 mU of
RNase H2. Importantly, SNP discrimination was markedly improved using this
primer
design, with the ACp for the "T" allele being around 25 cycles using RNase H2
in the
concentration range of 2.6-50 mU. Mismatch discrimination decreased when using
200
mU of the enzyme; however, SNP discrimination was still almost at a 14 cycle
ACp.
Optimal enzyme concentration was 50 mU, at which point priming efficiency was
similar to unmodified primers and SNP discrimination showed a 25.7 cycle ACp.
[0542] Therefore the present cleavable primer design with two internal C3
spacer
groups near the ribonucleotide and an unblocked 3 '-hydroxyl, "RDxxD", showed
significantly improved mismatch discrimination over the original primer
design,
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"RDDDD-x" (where R = RNA base, D = DNA base, and x = C3 spacer). Related
designs, such as "RDDxxD" or "RDxxDD", may show similarly improved function
and small optimizations in design may be beneficial depending upon the precise
sequence context of the SNP of interest. Utilizing chemical modifying groups
like the
C3 spacer that disrupt template function but leave the 3 '-hydroxyl unmodified
can
enhance the specificity of cleavage at the ribonucleotide by RNase H2 and
improve
SNP discrimination.
EXAMPLE 29 Use of RNase H2-cleavable ligation probes in DNA sequencing
methods
[0543] The previous Examples described the use of RNase H2 cleavable
oligonucleotide compositions for applications as primers where the cleavable
oligonucleotide primes a DNA synthesis reaction. Applications disclosed in the
above
Examples include both end-point and real time PCR in several different
detection
formats. Example 8 showed use of cleavable primers in a DNA sequencing
application
using the Sanger sequencing method with DNA polymerase and dideoxynucleotide
terminators; in this case the RNase H2-cleavable oligonucleotide also
functioned as a
primer. RNase H2-cleavable oligonucleotides can also be used in ligation
format
assays as well. One such application is DNA sequencing using cleavable
ligation
probes. The current Example demonstrates use of RNase H2-cleavable ligation
probes
in a format suitable for use in DNA sequencing.
[0544] The use of ligation probes to sequentially interrogate the identity
of bases in
an unknown nucleic acid sequence (i.e., DNA sequencing) has been described
(see
Patents US 5,750,341 and US 6,306,597 and US application 2008/0003571). The
basic
scheme for sequencing in the 5' to 3' direction by ligation begins with a
nucleic acid
acceptor molecule hybridized to an unknown nucleic acid sequence. A series of
base
interrogation probes are hybridized to this sequence which have a known fixed
DNA
base at the 5'-end followed by random bases or universal bases to permit
stable nucleic
acid hybridization of the probe to the target nucleic acid of unknown
sequence.
Hybridization and subsequent ligation reactions are dependent upon perfect or
near
perfect match between the ligation probe and the target; perfect match is
required at the
site of ligation. Ligation leads to a detectable event which permits
identification of the
specific base present at the ligation site. An RNase H2 cleavable site is
contained
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within the ligation probe. Following ligation the probe is cleaved by RNase
H2,
releasing the bulk of the probe but leaving the newly identified base ligated
to the
acceptor nucleic acid sequence, which has now been elongated by one residue as
a
result of the cycle of ligation and cleavage. This series of enzymatic and
chemical
events is repeated through multiple cycles of ligation, base identification,
and cleavage
and the unknown nucleic acid sequence is thereby determined.
[0545] While the patent references cited above teach methods for sequencing
by
ligation, the methods suggested therein to achieve cleavage and release of the
ligation
probe permitting multiple cycles of ligation/detection are inefficient and
difficult to
perform. RNase H2-cleavable oligonucleotides using the methods of the present
invention offer an improvement over pre-existing method and permit
construction of
less costly, easier to use cleavable ligation probes for DNA sequencing. One
scheme
for DNA sequencing using RNase H2 cleavable ligation probes is shown in Figure
35.
[0546] The RNase H2 cleavable ligation probes in this method contain a
fixed
known DNA base (or bases) at the 5'-end. The fixed known base(s) can be the
single
5' -most base or can include 2 or 3 or more bases towards the 5'-end. The
present
Example employs a system wherein only the single DNA base at the 5'-end of the
probe
is fixed. The synthetic oligonucleotide has a 5' -phosphate to permit
enzymatic ligation
using a DNA ligase. An activated adenylated form of the probe can also be
used. As
mentioned, the first base at the 5' -end is fixed (known). Thus four
independent probes
are needed to perform DNA sequencing, an "A" probe, a "C" probe, a "T" probe,
and a
"G" probe. Obviously more probes will be needed if the number of fixed bases
are
greater than one (for example, 16 ligation probes will be needed if the first
2 bases are
used as fixed known sequence, one for each possible dinucleotide pair). The
first base
following the fixed known DNA residue (in this case, the second base from the
5'-end)
is a residue which is cleavable by RNase H2. In the present Example, an RNA
base is
employed, however a 2'-F residue or other cleavable modified base (such as are
described in previous Examples) can also be used. The remaining bases in the
probe
will be random bases (heterogeneous mixes of the 4 DNA bases) and/or universal
bases
(such as inosine, 5-nitroindole, or other such groups as are well known to
those with
skill in the art). Total length of the probe will usually be around 8-9 bases,
however
longer or shorter probe length is possible depending on the particular ligase
enzyme
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employed. When using T4 DNA ligase, a length of 8 is sufficient to achieve
efficient
hybridization and enzymatic ligation. Longer probes can also be used.
[0547] Complexity
of the probe population increases according to 4N, where N =
the number of random bases employed. For example, the probe "pTn " has a
fixed "T" base at the 5'-end, a single "n" RNA base, and 6 "N" DNA bases,
totaling 7
random residues (p = phosphate, n = RNA, N = DNA). This presents a complexity
of 47
molecules (16,384) in the population. The complexity of the probe can be
decreased by
substituting universal base groups for random N bases. This is particularly
effective
towards the 3'-end. For example, using 3 inosine residues would convert the
above
probe to "pTnNNNIII" (as before, with I = inosine). This probe has a
complexity of 44
molecules (256). It will require a significantly lower mass input of ligation
probe to
achieve 100% ligation with a probe having a complexity of 256 than one having
a
complexity of 16,384. Use of one or more universal bases is generally
preferred.
Finally, the ligation probe has a dye molecule at or near the 3'-end to
provide a
detectable signal that can be resolved following ligation. The 3 '-modifying
group also
serves to block ligation at the 3'-end so that the ligation probe itself
cannot serve as an
acceptor nucleic acid.
[0548] Use of
RNase H2 cleavable ligation probes of this design in DNA
sequencing is shown schematically in Figure 35. A universal primer or acceptor
nucleic acid is hybridized to the unknown nucleic acid. Attachment of a
universal
adaptor sequence on the end of the unknown sequence may be required to permit
hybridization of the acceptor molecule, and this strategy permits use of the
same
acceptor nucleic acid for all reactions. The acceptor nucleic acid must have a
3'-hydroxyl group available for ligation. The mix of ligation probes is
introduced into
the reaction in molar excess (>256 fold excess for the 8mer inosine containing
probe
design described above) and T4 DNA ligase is used to perform enzymatic
ligation.
Free probe is removed by washing and retained fluorescent signal is measured.
The
color of the dye retained identifies which probe (A vs. G vs. C vs. T) was
attached
during the ligation reaction. RNase H2 is then used to cleave the probe,
removing the
"N" bases and universal bases but leaving the known base attached to the
acceptor
nucleic acid. In this manner the identity of the corresponding base within the
template
is determined, the acceptor nucleic acid has been extended by one base, and an
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accessible 3'-hydroxyl is once again available for ligation, permitting
cycling of the
process.
[0549] The following oligonucleotides shown below in Table 65, were made as
a
representative synthetic system to demonstrate ligation and subsequent
cleavage of
RNA-containing fluorescent ligation probes using the methods of the present
invention.
The ligation probes here have a fixed 9 base sequence (without any "N" bases
or
universal base modifications). The designation "CLP" indicates "cleavable
ligation
probe". The designation "ANA" indicates an "acceptor nucleic acid" which
provides
the 3'-hydroxyl acceptor site for a ligation reaction. "Targ-A" is a target
nucleic acid,
which directs a ligation reaction involving the complementary "T" ligation
probe
("CLP-T-Cy3"). "Targ-T" is a target nucleic acid, which directs a ligation
reaction
involving the complementary "A" ligation probe ("CLP-A-FAM").
Table 65:
CLP-C-TR 5f-pCaGCTGAAG-TR SEQ ID
No. 253
SEQ ID
CLP-G-Cy5 5,-pGaGCTGAAG-Cy5
No. 254
CLP-A-FAM 5f-pAaGCTGAAG-FAM SEQ ID
No. 255
SEQ ID
CLP-T-Cy3 5f-pTaGCTGAAG-Cy3
No. 256
ANA 5f-CCCTGTTTGCTGTTTTTCCTTCTC SEQ ID
No. 257
Targ-A 5f-
AGTGTTTGCTCTTCAGCTAGAGAAGGAAAAACAGCAAACAGGG SEQ ID
No. 258
Targ-T 5f-
AGTGTTTGCTCTTCAGCTTGAGAAGGAAAAACAGCAAACAGGG SEQ ID
No. 259
DNA bases are shown in uppercase. RNA bases are shown in lowercase. "p" is
5'-phosphate. TR is the fluorescent dye Texas Red. Cy5 is the fluorescent dye
Cyanine-5. Cy3 is the fluorescent dye Cyanine-3. FAM is the fluorescent dye
6-carboxyfluorescein. The position of base variation between Targ-A and Targ-T
is
underlined, which is complementary to the 5'-base of the corresponding
ligation probe.
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[0550] Figure 36 shows the predicted results for a ligation-cleavage
reaction cycle
using the synthetic oligonucleotide sequences shown above. "Targ-A" (SEQ ID
No.
258) will direct hybridization and ligation of the "CLP-T-Cy3" probe (SEQ ID
No. 256)
while "Targ-T" (SEQ ID No. 259) will direct hybridization and ligation of the
"CLP-A-FAM" probe (SEQ ID No. 255). Assuming that the reactions have high
specificity, the remaining two ligation probes do not have a matching target
in this
experiment and so should not participate in the ligation reaction. Following
ligation,
the newly formed fusion of the "ANA" + "CLP" product will become a substrate
for
RNase H2. Cleavage by RNase H2 will result in release of the 3'-end of the
ligation
probe (including the RNA base and the fluorescent reporter dye), leaving the
"ANA"
molecule longer by one base.
[0551] The "T" target nucleic acid (SEQ ID No. 259) or the "A" target
nucleic acid
(SEQ ID No. 258) and the "ANA" acceptor nucleic acid (SEQ ID No. 257) were
mixed
at 1.75 uM and all 4 ligation probes (SEQ ID Nos. 253-256) were added to a
concentration of 3.5 uM (each) in T4 DNA Ligase buffer (50 mM Tris-HC1 pH 7.5,
10
mM MgC12, 10 mM dithiothreitol, 1 mM ATP) in a volume of 80 uL, heated to 70 C
for
3 minutes and cooled slowly to 25 C. Ligation reactions were incubated at 37
C for 5
minutes with or without 140 units of T4 DNA Ligase. The reactions were stopped
by
heating at 65 C for 10 minutes. Reaction volumes were then adjusted to 200 uL
with
the addition of RNase H2 buffer [Tris-HC1 pH 8.0 (final concentration 10 mM),
NaC1
(final concentration 50 mM), MgC12 (final concentration 4 mM)] and 20 units of
RNase
H2 was added to each tube. Reaction mixtures were incubated at 60 C for 30
minutes,
followed by desalting over a Sephadex G25 column, and the samples were
lyophilized.
Samples were rehydrated in 70 uL of water and 10 uL aliquots were analyzed on
a 20%
acrylamide, 7M urea, denaturing gel, followed by visualization using GelStar
stain
(#50535 GelStar Nucleic Acid Gel Stain, Lonza). The remainder of the reactions
was
saved at -20 C for future testing, including mass spectrometry or other
methods as
needed.
[0552] The gel image is shown in Figure 37. Lanes 1 and 5 show the
component
oligonucleotides in the absence of enzymes to visualize migration relative to
size
markers (lane M). Lanes 2 and 3 are duplicate reactions where Targ-A (SEQ ID
No.
258) was incubated with the 4 cleavage ligation probes (SEQ ID Nos. 253-256)
in the
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presence of T4 DNA Ligase. An upward size shift of the acceptor nucleic acid
(ANA,
SEQ ID No. 257) is clearly seen which represents ligation with CLP-T-Cy3 (SEQ
ID
No. 256) and is identified as the ligation product. Specific ligation with the
correct
CLP-T-Cy3 probe and not the other 3 probes (mismatched bases) occurred, which
was
verified by visual inspection of the color of the dye (this cannot be
appreciated in the
black and white image shown in Figure 37) and was further verified by mass
spectrometry. Similarly, lanes 7 and 8 are duplicate reactions where Targ-T
(SEQ ID
No. 259) was incubated with the 4 cleavage ligation probes (SEQ ID Nos. 253-
256) in
the presence of T4 DNA Ligase. An upward size shift of the acceptor nucleic
acid
(ANA, SEQ ID No. 257) is clearly seen which represents ligation with CLP-A-FAM
(SEQ ID No. 255) and is identified as the ligation product. Specific ligation
with the
correct CLP-A-FAM probe and not the other 3 probes (mismatched bases)
occurred,
which was verified again by visual inspection of the color of the dye and
confirmed by
mass spectrometry analysis. Finally, lanes 4 and 8 demonstrate that these
ligation
products are reduced in size when treated with RNase H2, indicating that
cleavage
occurred. Note that the resulting bands show slightly reduced mobility
compared with
the original ANA band, indicating that this new species is longer than the
starting
material. Mass spectrometry confirmed that actual mass of the reaction
products in
lanes 4 and 8 were consistent with the predicted 1-base elongation of the
starting ANA
nucleic acid, that the correct base was inserted, and that the new "ANA+1"
species had
a 3'-hydroxyl. The new ANA+1 species is now prepared for a second cycle of
ligation/cleavage.
[0553] This example has therefore demonstrated that short RNA-containing
short
probes can be specifically ligated to an acceptor nucleic acid in the presence
of a
complementary target nucleic acid. Ligation is sensitive to the identify of
the template
base matching the 5'-terminal base of the ligation probe and specific ligation
of the
correct complementary probe can be detected from within a heterogeneous mix of
different probe sequences. Finally, RNase H2 can cleave the ligation probe at
the
5'-side of the RNA base, releasing the bulk of the probe, resulting in an
acceptor
nucleic acid molecule which has been extended by one base in length. The
extended
acceptor nucleic acid contains a 3'-hydroxyl and can be used in repeated
cycles of
ligation/cleavage.
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EXAMPLE 30 Use of universal bases in RNase H2-cleavable ligation probes
[0554] In Example 29 above it was proposed that universal bases, such as
5'-nitroindole or inosine, could be used in cleavable ligation probes. The
present
example demonstrates use of the universal base 5-nitroindole in a model system
where
the probe sequence is fixed (does not contain random N-bases). The
oligonucleotides,
shown below in Table 66, were synthesized based upon the synthetic
probe/template
system in Example 29. Cleavage ligation probes were designed as 8mers with a
5'-phosphate, an "A" base at the 5'-end (to direct ligation to the "T"
target), a single
ribonucleotide, and 2 or 3 additional fixed DNA bases. Three or four 5-
nitroindole
bases were positioned towards the 3'-end. A FAM fluorescent dye was attached
at the
3'-end. The same acceptor nucleic acid (ANA) and T-target nucleic acid were
employed as in Example 29. A reaction scheme showing alignment of
oligonucleotide
components for this example is shown in Figure 38.
Table 66:
CLP-A-FAM SEQ ID
5f-pAaGCTXXX-FAM
-3 x5NI No. 260
CLP-A-FAM SEQ ID
5f-pAaGCXXXX-FAM
-4x5NI No. 261
ANA 5f-CCCTGTTTGCTGTTTTTCCTTCTC SEQ ID
No. 257
Targ-T 5f-
AGTGTTTGCTCTTCAGCTTGAGAAGGAAAAACAGCAAACAGGG SEQ ID
No. 259
DNA bases are shown in uppercase. RNA bases are shown in lowercase. "p" is
5'-phosphate. "X" is the universal base 5-nitroindole. FAM is the fluorescent
dye
6-carboxyfluorescein. The position of base hybridization with the 5' -end of
the ligation
probe is underlined on the target.
[0555] The "T" target nucleic acid (SEQ ID No. 259) and the "ANA" acceptor
nucleic acid (SEQ ID No. 257) were mixed at 2 M with the 3X or 4X 5'-
nitroindole
containing CLPs (cleavable ligation probes, SEQ ID Nos. 260-261) in T4 DNA
Ligase
buffer (50 mM Tris-HC1 pH 7.5, 10 mM MgC12, 10 mM dithiothreitol, 1 mM ATP).
The reactions were heated at 70 C for 5 minutes and cooled slowly to 25 C. T4
DNA
Ligase (New England Biolabs) was added at a range of 7.5-120 units and the
ligation
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reactions were incubated at 25 or 37 C for 5 minutes. The reactions were
terminated by
the addition of EDTA to a final concentration of 50 mM. Final reaction volumes
were
50 L. An equal volume of 90% formamide, lx TBE loading buffer was added to
each
sample, which were then heat denatured at 70 C for 3 minutes and cooled on
ice.
Samples were separated on a denaturing 7M urea, 20% polyacrylamide gel. Gels
were
stained using GelStarTM stain and visualized with UV excitation. The gel image
is
shown in Figure 39.
[0556] The 8mer cleavable ligation probe with three 5-nitroindole universal
bases
(SEQ ID No. 260) worked well and showed near 100% ligation efficiency using
the
higher enzyme amounts (60-120 units T4 DNA Ligase). In contrast, the 8mer
cleavable
ligation probe with four 5-nitroindole universal bases (SEQ ID No. 261) did
not ligate
to the acceptor nucleic acid using any amount of enzyme. The same results were
seen at
25 C and at 37 C suggesting that this difference in reactivity does not relate
to
difference in Tm of the two probes. It is more likely that the differential
reactivity
relates to substrate preferences for the T4 DNA Ligase enzyme. This Example
demonstrates that three 5-nitroindole bases can be positioned at the 3' -end
of an 8mer
ligation probe and retain good function. This same experiment was repeated
using
9mer ligation probes. In this case, a probe having "six DNA + three 5-
nitroindole
bases" and a probe having "five DNA + four 5-nitroindole bases" were both
substrates
for T4 DNA Ligase but a probe with "four DNA bases + five 5-nitroindole bases"
did
not (data not shown), consistent with the idea that T4 DNA Ligase requires 5
fixed
DNA bases towards the S'-end of the ligation probe to function well and that
5' -nitroindole bases can be introduced after this requirement is met. The
precise
optimal probe design can vary with different ligase enzymes.
[0557] These findings are significant as it permits synthesis of lower
complexity
pools of ligation probes.
EXAMPLE 31 Use of random bases and universal bases in RNase H2-cleavable
ligation probes
[0558] Examples 29 and 30 demonstrated use of RNase H2-cleavable ligation
probes where some or all of the probe sequence was a perfect match to the
target. In
sequencing a nucleic acid of unknown sequence, it is necessary to use probes
that
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contain primarily random bases so that probe hybridization can occur for any
sequence
encountered. The present Example demonstrates use of 8mer cleavable ligation
probes
having a random base (Nmer) domain, a universal base (5-nitroindole) domain
and only
a single fixed DNA base at the 5'-end. The following oligonucleotides shown in
Table
67 were employed:
Table 67:
CLP-A-FAM SEQ ID
5f-pAnNNNXXX-FAM
4N+3 x5NI No. 262
CLP-T-Cy3 SEQ ID
5f-pTnNNNXXX-Cy3
4N+3 x5NI No. 263
CLP-G-Cy5 SEQ ID
5f-pGnNNNXXX-Cy5
4N+3 x5NI No. 264
ANA 5f-CCCTGTTTGCTGTTTTTCCTTCTC SEQ ID
No. 257
Targ-T 5f-
AGTGTTTGCTCTTCAGCTTGAGAAGGAAAAACAGCAAACAGGG SEQ ID
No. 259
DNA bases are shown in uppercase. RNA bases are shown in lowercase. "p" is
5'-phosphate. "N" represents a random mix of the DNA bases A, G, C, and T. "n"
represents a random mix of the RNA bases A, C, G, and U. "X" is the universal
base
5-nitroindole. FAM is the fluorescent dye 6-carboxyfluorescein. Cy5 is the
fluorescent
dye Cyanine-5. Cy3 is the fluorescent dye Cyanine-3. The position of base
hybridization with the 5'-end of the ligation probe is underlined on the
target.
[0559] The "T" target nucleic acid (SEQ ID No. 259) and the "ANA" acceptor
nucleic acid (SEQ ID No. 257) were mixed together at a final concentration of
0.4 M
each and the three cleavable ligation probes (SEQ ID Nos. 262-264) were
individually
added at a final concentration of 50 M (125-fold excess over the target and
acceptor)
in T4 DNA Ligase buffer in a final reaction volume of 50 L. Reactions were
heated to
70 C for 5 minutes and cooled slowly to 25 C. T4 DNA Ligase was added (400 U)
and
the ligation reactions were incubated at 37 C for 30 minutes. The reactions
were
stopped by heating at 65 C for 10 minutes followed by desalting over a
Sephadex G25
column, after which the samples were lyophilized and rehydrated in 10 L of
water
mixed with 10 L of 90% formamide, lx TBE loading buffer. Samples were heat
denatured at 70 C for 3 minutes and cooled on ice. Reaction products were
separated
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on a 20% acrylamide 7M urea denaturing gel, followed by visualization using
GelStar
stain with UV transillumination (50535 GelStar Nucleic Acid Gel Stain, Lonza).
Results are shown in Figure 40.
[0560] The target nucleic acid contained a "T" base at the site
complementary to
the point of ligation. This template correctly directed ligation of the "A-
FAM" ligation
probe (SEQ ID No. 262) but not the mismatch "T-Cy3" (SEQ ID No. 263) or "G-
Cy5"
(SEQ ID No. 264) ligation probes. Ligation specificity was directed by a
single fixed
DNA base at the 5'-end of the ligation probes which otherwise comprised random
"N"
bases or universal 5-nitroindole bases. The ligation probes were added to the
ligation
reactions at 125-fold molar excess over the target and the acceptor nucleic
acids. The
ligation probes contain a 4-base "N" domain, so the complexity of the nucleic
acid
mixture was 44 (256). Thus the reaction theoretically contained sufficient
perfect
matched probe to ligate with only about 50% of the input acceptor nucleic
acid. It is
evident from the relative fluorescent images in Figure 40 that approximate
half of the
acceptor was present in the longer ligation product species and half was
unreacted,
indicating that the reaction proceeded as expected. If mismatched sequences
ligated to
the acceptor with any appreciable efficiency, then the 125-fold excess of
ligation probe
would most likely have reacted with >50% of the acceptor nucleic acid
molecules,
which was not observed. Thus ligation reactions using cleavable ligation
probes of this
design were both efficient and specific.
EXAMPLE 32 Use of RNase H2-cleavable probes in an oligonucleotide ligation
assay (OLA)
[0561] Use of cleavable ligation probes in DNA sequencing represents just
one
potential format/application for this general class of assay. The sequencing
application
is unique in that the target nucleic acid is of unknown sequence. More
typically,
oligonucleotide ligation assays are employed to determine the presence or
absence of a
known nucleic acid sequence within a sample nucleic acid of interest. For
example, an
OLA can be employed to detect the presence of a nucleic acid sequence specific
for
pathogenic organisms in the background of human DNA. Another example would be
to determine the presence or absence of a known, defined polymorphism at a
specific
target nucleic acid locus (e.g., an allelic discrimination assay or SNP
assay). In all of
these applications, one ligation probe is positioned so that the 3'-most or 5'-
most base
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aligns with the SNP site and a second perfect-match nucleic acid is positioned
adjacent
so that if the probe sequence is a match for the SNP base, then a ligation
event can occur.
If the probe sequence is a mismatch for the SNP base, then ligation is
inhibited. The
ligation event results in formation of a detectable species.
[0562] An allelic discrimination (SNP) assay is shown in this Example to
demonstrate utility of the novel RNase H2 cleavable ligation oligonucleotide
probes of
the present invention. Sequence designs shown herein place the SNP site
towards the
3'-end of the acceptor ligation probe.
[0563] A traditional OLA employs three synthetic oligonucleotides to
discriminate
between two alleles (Figure 41A). If the SNP site comprises a "C" allele and
an "A"
allele, then two acceptor oligonucleotides are required, one bearing a "G"
base (match
for the "C" allele) and one bearing a "T" base (match for the "A" allele). The
acceptor
oligonucleotides have a free 3'-hydroxyl group. A third oligonucleotide (a
donor
nucleic acid) is employed that hybridizes to the target so as to place its 5'-
end adjacent
to the 3'-end of the ligation probe. The acceptor nucleic acid will have a 5'-
phosphate;
generally the 3'-end of the donor oligonucleotide is blocked so that it cannot
participate
in a ligation reaction. In this way, perfect match hybridization of both a
acceptor and
the donor probes on the target will position the two oligonucleotides in a
head-to-tail
fashion that enables ligation between the 3'-hydroxyl of the acceptor with the
5'-phosphate of the donor (Figure 41B). In contrast, a mismatch at the SNP
site
disrupts this structure and inhibits ligation. In the traditional OLA, the
identity of the
SNP base is interrogated once at the time of hybridization/ligation and
specificity is
entirely dependent upon the ability of the DNA Ligase to perform ligation on
the
perfect matched but not the mismatched species. Typically the three
oligonucleotides
(two ligation probes and the acceptor) have a similar Tm so that they can
function
together with the target nucleic acid under identical conditions.
[0564] The new RNase H2 OLA of the present invention employs four synthetic
nucleic acids to discriminate between two alleles (Figure 42A). If the SNP
site
comprises a "C" allele and an "A" allele, then two cleavable acceptor ligation
probes
are required in this embodiment, one bearing a "G" base (match for the "C"
allele) and
one bearing a "T" base (match for the "A" allele). The cleavable acceptor
ligation
probes have a single RNA base positioned towards the 3'-end of the molecule
that is
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aligned to be complementary or not (match vs. mismatch) with the base at the
target
SNP site. Additional DNA bases are positioned 3'- to the RNA base (preferably
four
DNA bases, all being complementary to the target) and a blocking group is
placed at the
3'-end to prevent ligation. The general design and function of the cleavable
ligation
probe is similar to the cleavable primers demonstrated in Example 13 in a qPCR
format
SNP discrimination assay. The cleavable ligation probes can also be designed
using
various chemically modified bases and abasic residues as outlined in the above
Examples to improve SNP discrimination at the RNase H2 cleavage site (see
Examples
22, 23, 25, and 28). Preferably the cleavable ligation probes will be designed
to have a
Tm in the range of 60-70 C (in RNase H2 cleavage buffer) to permit
hybridization of
the cleavable probe with target in the optimal temperature range for the
enzyme.
[0565] Unlike the traditional OLA format, the donor oligonucleotides in the
RNase
H2 OLA format are also SNP interrogation probes. Thus two donor probes are
required,
one bearing a "G" base (match for the "C" allele) and one bearing a "T" base
(match for
the "A" allele). Both donoror probes have a phosphate at the 5'-end to enable
ligation
and optionally are blocked at the 3 '-end (Figure 42A). The two donor ligation
probes
can have a lower Tm than the RNase H2 cleavable ligation probes so that
hybridization
of the cleavable ligation probes and the donor ligation probes with the target
nucleic
acid can be differentially regulated by control of reaction temperature. These
donor
probes in the assay format do not interact with RNase H2.
[0566] To perform an RNase H2 OLA, all four OLA probes are mixed in the
presence of the target nucleic acid in a buffer compatible with RNase H2
activity (see
above examples). Preferably this will be done around 60-70 C. The RNase H2
cleavable acceptor oligonulceotide is complementary to and will hybridize to
the target
nucleic acid under these conditions. If the RNA base of the acceptor probe and
the base
at the target SNP site match, then RNase H2 cleavage can occur (Figure 42B).
It is
preferred that the donor ligation probe (the non-cleavable probe) has a lower
Tm than
the cleavable probe. The first stage of the reaction (hybridization of the
acceptor
oligonucleotide and cleavage by RNase H2) can then be carried out at a
temperature
that is sufficiently above the Tm of the non-cleavable donor probes that they
do not
hybridize to target. Cleavage of the acceptor probe by RNase H2 removes the
RNA
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base and uncovers the SNP site, making it available to hybridize with the non-
cleavable
ligation probe (the donor oligonucleotide).
[0567] Once the RNase H2 cleavage phase of the OLA is complete, reaction
temperature is lowered to permit hybridization of the non-cleavable ligation
probe to
the target. In the presence of DNA Ligase, the 5'-end of the non-cleavable
probe will
ligate to the 3'-end of the adjacent cleaved probe (Figure 42B), if the base
at the 5'-end
of the donor probe pairs with the base at the SNP site. Thus the RNase H2 OLA
assay
interrogates the identity of the base at the SNP site twice, once during RNase
H2
mediated cleavage of the acceptor oligonucleotide probe and again at the
ligation
reaction (Figure 42C). Double interrogation of the identity of the SNP base by
two
different enzymatic events provides for greater specificity than can be
achieved using a
traditional OLA.
EXAMPLE 33 SNP discrimination using RNase H2-cleavable probes in an OLA
[0568] A variety of methods exist that enable detection of OLA products. In
the
present Example, fluorescence detection is performed in a bead capture assay
format to
perform an RNase H2 OLA allelic discrimination assay as outlined in Example
32.
Sequences were designed that were compatible for use with the Luminex xMAP
fluorescent microbead system with detection on a Luminex L100 detection system
(Luminex, Austin, TX).
[0569] The "OLA-C-antitag" and "OLA-T-antitag" sequences (SEQ ID Nos.
265-266) were made with a 5'-amino modifier to permit conjugation to
carboxylate
xMAP fluorescent beads using carbodiimide coupling chemistry. The "OLA-T-Tag"
and "OLA-C-Tag" sequences (SEQ ID Nos. 269-270) which serve as donor
oligonucleotides in the ligation reaction have a 12-base sequence towards the
5'-end
which is complementary to the target sequence and positions the SNP site (C/T
base) at
the 5'-end. Tm for these 12-base domains is estimated to be 50-53 C (in 10 mM
Mg'
containing buffer). Both sequences have a 5'-phosphate to permit ligation. The
3'-end
of these sequences is a "tag" sequence which is complementary to the "antitag"
sequence and permits capture to antitag bearing beads by hybridization. The
"OLA-C"
and "OLA-T" probes (SEQ ID Nos. 267-268) serve as the acceptor fragment and
are
complementary to the target and position the single ribonucleotide base (rC or
rU) at
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the SNP site. Tm for the cleavable ligation probes is estimated to be ¨75 C
(in 10 mM
Mg containing
buffer). Both of the oligonucleotide probes have a biotin at the 5'-end
which will enable binding of a reporter dye, Streptavidin-phycoerythrin, for
detection
by the Luminex L100 system. Synthetic 98mer oligonucleotide targets
corresponding
to the "C" allele (G base in the target, SEQ ID No. 271) and "T" allele (A
base in the
target, SEQ ID No. 272) were employed in this Example. The sequences
corresponding to SEQ ID Nos. 265-272 are shown below in Table 68. Alignment
and
interaction of the different probe, target, tag, and antitag sequences during
the various
step in this assay are shown in Figure 43..
Table 68:
OLA-C SEQ ID
5' aminoC12-GATTTGTATTGATTGAGATTAAAG
antitag No. 265
OLA-T SEQ ID
5' aminoC12-GATTGTAAGATTTGATAAAGTGTA
antitag No. 266
rs4939827 SEQ ID
5' Biotin-CACCATGCTCACAGCCTCATCCAAAAGAGGAAAcAGGA-x
OLA C No. 267
rs4939827 SEQ ID
5' Biotin-CACCATGCTCACAGCCTCATCCAAAAGAGGAAAuAGGA-x
OLA T No. 268
rs4939827
SEQ ID
OLA C 5' pCAGGACCCCAGACTTTAATCTCAATCAATACAAATC-x
No. 269
Tag
rs4939827
SEQ ID
OLA T 5' pTAGGACCCCAGATACACTTTATCAAATCTTACAATC-x
No. 270
Tag
5'CCCAGCATTGTCTGTGTTTCCTGAGGAGTCTGAGGGAGCTCTGGGGTC SEQ ID
Targ-C
CTGTTTCCTCTTTTGGATGAGGCTGTGAGCATGGTGGATTAGAGACAGCC No. 271
5'CCCAGCATTGTCTGTGTTTCCTGAGGAGTCTGAGGGAGCTCTGGGGTC SEQ ID
Targ-T
CTATTTCCTCTTTTGGATGAGGCTGTGAGCATGGTGGATTAGAGACAGCC No. 272
DNA bases are uppercase. RNA bases are lowercase. Biotin is a Biotin-TEG
group. X
represents a C3 spacer. For the OLA C/T Tag oligonucleotides, the portion of
the
sequence which is the "tag" and binds the "antitag" sequence is underline. The
site of
the SNP with the target sequence under interrogation is underlined and in
bold.
[0570] Coupling of antitag oligos to xMAP microspheres. Anti-tag
oligonucleotides containing a 5' amino group were coupled to 1.25x107 xMAP
Multi-Analyte COOH Microspheres (L100-C127-01 and L 100-C138-01, Luminex,
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Austin, TX) using 3 mg/mL N-(3-Dimethylaminopropy1)-N'-ethylcarbodiimide
hydrochloride (03449-1G, Sigma Aldritch),in 0.1 M MES, pH 4.5 buffer (M-8250
Sigma-Aldritch) at room temperature for 90 minutes in the dark (modified
manufacturer's protocol). After coupling, the microspheres were washed once
with
0.02% Tween20, and then once with 0.1% SDS. Microspheres were re-suspended in
200 L of TE pH 7.5. The concentration of microspheres was determined by
counting
with a hemocytometer under a light microscope (Nikon TMS, Freyer Company,
Carpentersville, IL). Successful coupling was determined by hybridizing 25-250
fmoles of complementary oligonucleotides containing a 5' biotin modification
and
detecting the hybrids with 2 ng/mL streptavidin R-phycoerythrin conjugate
(S866 1
mg/mL, Invitrogen, Carlsbad, CA). Mean fluorescence intensity had to increase
in a
concentration dependent manner. No cross hybridization was observed between
the
two anti-tag sequences.
[0571] OLA Assay. RNase H2 digestion mixtures (10 L) were prepared
containing rs4939827 OLA C and rs4939827 OLA T oligos (SEQ ID Nos. 267-268) at
a final concentration of 250 nM, and either C, T or C/T mix template
oligonucleotides
(SEQ ID Nos. 271-272) at 125 nM in a 20 mM Tris-HC1 (pH 7.6 at 25 C), 25 mM
KAc,
mM MgAc, 10 mM DTT, 1 mM NAD, and 0.1% Triton X-100 buffer (Taq DNA
Ligase buffer, New England Biolabs, Ipswitch, MA). Samples were incubated for
30
minutes at 65 C with or without 5 mU of Pyrococcus abyssi RNase H2. For each
RNase H2 digestion reaction, the volume was increased to 25 L by adding 2.5
pmoles
of rs4939827 OLA 12C Tag and 2.5 pmoles rs4939827 OLA 12T Tag oligonucleotides
(SEQ ID Nos. 269-270) (100 nM final concentration for each oligo), with or
without 40
U of Taq DNA Ligase (New England Biolabs, Ipswitch, MA), maintaining a final
buffer composition of 20 mM Tris-HC1 (pH 7.6 at 25 C), 25 mM KAc, 10 mM MgAc,
10 mM DTT, 1 mM NAD, and 0.1% Triton X-100. The ligation reactions were
incubated at 45 C for 30 minutes.
[0572] Capture of ligation product on fluorescent beads and detection of
signal. 10 L of each ligation mixture was combined with 15 L of H20, and 25
L of
the xMAP bead mixture (Bead sets 127 and 138) at a density of 100 beads of
each
type/nL. The samples were heated to 70 C for 90 seconds followed by 50 C for
30
minutes. The samples were transferred to a Millipore Multiscreen filtration
plate
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(MABVN1250, Millipore, Bedford, MA), and washed two times with 100 L of 50 C
0.2 M NaCl, 0.1 M Tris pH 8.0, 0.08% Triton X-100 buffer. Microspheres were
incubated at 50 C for 15 minutes with 75 L of a 2 ng/mL solution of
streptavidin-R
phycoerythrin (S866 1 mg/mL, Invitrogen, Carlsbad, CA). Mean fluorescence was
measured on a Luminex L100 detection system (Luminex, Austin, TX).
[0573] Results are shown in Figure 44. Fluorescent beads bearing the "C"
allele
antitag sequences showed positive fluorescent signal only when the reaction
was run in
the present of the "C" allele target or the "C/T" mix. Fluorescent beads
bearing the "T"
allele antitag sequences showed positive fluorescent signal only when the
reaction was
run in the present of the "T" allele target or the "C/T" mix. Signal was
dependent on
use of RNase H2 and was not observed in the absence of target DNA. Thus the
RNase
H2 cleavable oligonucleotide ligation assay of the invention was demonstrated
to be
effective at distinguishing the presence of a C/T SNP present in a target DNA
in a
highly specific fashion.
EXAMPLE 34 Double-interrogation of mismatches through the use of forward
and reverse overlapping, cleavable primers with internal template blocking
groups
[0574] Example 28 demonstrated the utility of internal template blocking
groups.
The present example demonstrates the utility of combining overlapping forward
and
reverse cleavable primers with internal template blocking groups to improve
mismatch
discrimination.
[0575] In previous examples, a single blocked-cleavable primer was employed
to
perform SNP interrogation using PCR with one blocked-cleavable primer
positioned
with the cleavable RNA residue at the SNP site paired with an unmodified
primer.
Blocked-cleavable primers can be designed complementary for either the top or
bottom
(sense or antisense) strand of a double-stranded DNA target. Thus two
different SNP
discrimination assays of this type can be made for every SNP. A schematic
outlining
the single blocked-cleavable primer approach for the "For" orientation is
shown in
Figure 45a and for the "Rev" orientation in Figure 45b. An alternative
approach is to
employ two blocked-cleavable primers which are both specific for the SNP under
interrogation, one serving as the "forward" primer and one serving as the
"reverse"
primer. In this case, the 3 '-ends of the two primers will overlap each other
when in the
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inactive blocked state but will not overlap each other when activated
following
cleavage by RNase H2. A schematic outlining the dual blocked-cleavable primer
approach is shown in Figure 45c. The use of dual allele-specific blocked-
cleavable
primers will increase specificity of the reaction by providing interrogation
for base
identity at the SNP site twice for each cycle of PCR.
[0576] The following primers, as shown below in Table 69, were synthesized
for
the human SMAD7 gene similar to previous Examples. Primers were either
non-specific and would amplify either allele with similar efficiency or were
specific for
either the "C" allele or the "T" allele. Primer sets were tested on both "C-
allele" and
"T-allele" genomic DNA targets.
Table 69:
SEQ ID
Name Sequence
No.
SEQ ID No.
rs4939827 Rev 5f-CTCACTCTAAACCCCAGCATT
217
SEQ ID No.
rs4939827 For 5f-CAGCCTCATCCAAAAGAGGAAA
230
rs4939827 SEQ ID No.
5f-CAGCCTCATCCAAAAGAGGAAAcAGGA-SpC3
C-For-AGGA(C3) 231
rs4939827 SEQ ID No.
5f-CAGCCTCATCCAAAAGAGGAAAuAGGA-SpC3
T-For-AGGA(C3) 235
rs4939827 SEQ ID No.
5f-CAGCCTCATCCAAAAGAGGAAA0A(SpC3-SpC3)A
C-For-A(C3C3)A 252
rs4939827 SEQ ID No.
5f-CAGCCTCATCCAAAAGAGGAAAuA(SpC3-SpC3)A
T-For-A(C3C3)A 273
SEQ ID No.
rs4939827 For v2 5' -GGCTGTCTCTAATCCACCAT
374
SEQ ID No.
rs4939827 Rev v2 5' -GAGGGAGCTCTGGGGTCCT
275
rs4939827 SEQ ID No.
5f-GAGGGAGCTCTGGGGTCCTgTTTC(SpC3)
C-Rev-AGGA(C3) 276
rs4939827 SEQ ID No.
5f-GAGGGAGCTCTGGGGTCCTaTTTC(SpC3)
T-Rev-AGGA(C3) 277
rs4939827 SEQ ID No.
5f-GAGGGAGCTCTGGGGTCCTgT(SpC3-SpC3)C
C-For-A(C3C3)A 278
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Table 69:
SEQ ID
Name Sequence
No.
rs4939827 SEQ ID No.
5f-GAGGGAGCTCTGGGGTCCTaT(SpC3-SpC3)C
T-For-A(C3C3)A 279
DNA bases are shown in uppercase. RNA bases are shown in lowercase.
SpC3 is a Spacer C3 group, positioned either internal within the primer or at
the 3' -end.
[0577] An 85 base
pair SMAD7 amplicon sequence (SEQ ID No. 280) is shown
below. The site of the rs4939827 C/T SNP is indicated in parenthesis.
SEQ ID No. 280
5f-GGCTGTCTCTAATCCACCATGCTCACAGCCTCATCCAAAAGAGGAAA(C/T)AGGACCCCAGAGCT
CCCTCAGACTCCTCAGGAAACACAGACAATGCTGGGGTTTAGAGTGAG-3'
The relative specificity of allelic discrimination assays using PCR with
blocked-cleavable primers was tested in the context of the SMAD7 amplicon
shown
above. Assays were tested using a single blocked-cleavable primer in the "For"
direction, a single blocked-cleavable primer in the "Rev" direction, or dual
blocked-cleavable primers in both directions. Primer
designs included the
"RDDDD-x" and "RDxxD" variants as defined in Example 28 above. As a control,
unmodified primers which were not allele-specific were also employed.
The "For" orientation primers used are aligned with the SMAD7 target below.
SEQ ID No. 273 5' CAGCCTCATCCAAAAGAGGAAA u AxxA
SEQ ID No. 252 5' CAGCCTCATCCAAAAGAGGAAA c AxxA
SEQ ID No. 235 5' CAGCCTCATCCAAAAGAGGAAA u AGGAx
SEQ ID No. 231 5' CAGCCTCATCCAAAAGAGGAAA c AGGAx
SEQ ID No. 230 5' CAGCCTCATCCAAAAGAGGAAA
5f-GGCTGTCTCTAATCCACCATGCTCACAGCCTCATCCAAAAGAGGAAA(C/T)AGGACCCCAGAGCT
CCCTCAGACTCCTCAGGAAACACAGACAATGCTGGGGTTTAGAGTGAG-3'
3'-
TTACGACCCCAAATCTCACTC-5' SEQ ID No. 217
Where DNA bases are uppercase, RNA bases are lowercase, and "x" is a C3
spacer (propanediol).
[0578] PCR
reactions were performed on a Roche Lightcycler 480 platform in 10
pi volume using 200 nM of the modified or unmodified For primers (SEQ ID Nos.
230,
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231, 235, 252, and 273) paired with the unmodified Rev primer (SEQ ID No. 217)
with
20 ng genomic DNA (Cone!! GM07048 homozygous C/C allele or GM18976
homozygous T/T allele). Reactions were run in Bio-Rad SYBR Green master mix
with
2.6 mU of Pyrococcus abyssi RNase H2 for the "RDDDD-x" primers (SEQ ID Nos.
231 and 235) or 200 mU of Pyrococcus abyssi RNase H2 for the "RDxxD" primers
(SEQ ID Nos. 252 and 273). Reactions were started with a soak at 95 C for 5
minutes
followed by 45 cycles of [95 C for 10 seconds and 60 C for 30 seconds].
Results of
qPCR amplifications done at this SNP site are shown in Table 70 below.
Table 70. Cp and ACp values of qPCR reactions showing mismatch discrimination
at a
SMAD7 C/T allele using a "For" orientation assay with a single blocked-
cleavable
primer.
C/C No Template
For Primer Employed T/T Target ACp
Target Control
SEQ ID No. Unmodified
23.0 23.1 - >75
230 control
SEQ ID No. rU-DDDD-x 33.4 23.4 10.0 >75
235
SEQ ID No' rC-DDDD-x 22.6 31.5 8.9 >75
231
SEQ ID No.
rU-DxxD 47.6 23.4 24.2 >75
273
SEQ ID No.
rC-DxxD 22.9 41.7 18.8 >75
252
DNA samples homozygous C/C or T/T were readily distinguished using either the
"RDDDD-x" or the "RDxxD" design primers, with the "RDxxD" version showing
better separation of signal between match and mismatch (larger ACp values).
The allele discrimination experiment was next performed using the "Rev"
oriented
reactions. The "Rev" orientation primers used are aligned with the SMAD7
target
below.
5f-GGCTGTCTCTAATCCACCAT SEQ ID No. 274
5f-GGCTGTCTCTAATCCACCATGCTCACAGCCTCATCCAAAAGAGGAAA(C/T)AGGACCCCAGAGCT
TCCTGGGGTCTCGA
3' xCTTT g
TCCTGGGGTCTCGA
3' xCTTT a
TCCTGGGGTCTCGA
3' CxxT g
TCCTGGGGTCTCGA
3' CxxT a
TCCTGGGGTCTCGA
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CCCTCAGACTCCTCAGGAAACACAGACAATGCTGGGGTTTAGAGTGAG-3'
GGGAG-5' SEQ ID No. 275
GGGAG-5' SEQ ID No. 276
GGGAG-5' SEQ ID No. 277
GGGAG-5' SEQ ID No. 278
GGGAG-5' SEQ ID No. 279
Where DNA bases are uppercase, RNA bases are lowercase, and "x" is a C3
spacer (propanediol).
[0579] PCR
reactions were performed on a Roche Lightcycler 480 platform in 10
pi volume using 200 nM of the modified or unmodified Rev primers (SEQ ID Nos.
275,
276, 277, 278, and 279) paired with the unmodified Rev primer (SEQ ID No. 274)
with
20 ng genomic DNA (Coriell GM07048 homozygous C/C allele or GM18976
homozygous T/T allele). Reactions were run in Bio-Rad SYBR Green master mix
with
2.6 mU of Pyrococcus abyssi RNase H2 for the "RDDDD-x" primers (SEQ ID Nos.
276 and 277) or 50 mU of Pyrococcus abyssi RNase H2 for the "RDxxD" primers
(SEQ ID Nos. 278 and 279). Reactions were started with a soak at 95 C for 5
minutes
followed by 45 cycles of [95 C for 10 seconds and 60 C for 30 seconds].
Results of
qPCR amplifications done at this SNP site are shown in Table 71 below.
Table 71. Cp and ACp values of qPCR reactions showing mismatch discrimination
at a
SMAD7 C/T allele using a "Rev" orientation assay with a single blocked-
cleavable
primer.
C/C No Template
Rev Primer Employed T/T Target ACp
Target Control
SEQ ID No. Unmodified
24.1 24.5 >75
275 control
SEQ ID No' rA-DDDD-x 38.0 24.4 13.6 >75
277
SEQ ID No. rG-DDDD-x 22.7 35.5 12.8 >75
276
SEQ ID No.
rA-DxxD 42.2 25.8 16.4 >75
279
SEQ ID No.
rG-DxxD 24.3 46.8 22.5 >75
278
DNA samples homozygous C/C or T/T were readily distinguished using either the
"RDDDD-x" or the "RDxxD" design primers, with the "RDxxD" version showing
better separation of signal between match and mismatch (larger ACp values).
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The experiment was next performed using the new dual interrogation "For + Rev"
oriented reaction method. The primers used are aligned with the SMAD7 target
below.
SEQ ID No. 273 5f-CAGCCTCATCCAAAAGAGGAAA u AxxA
SEQ ID No. 252 5f-CAGCCTCATCCAAAAGAGGAAA c AxxA
SEQ ID No. 235 5f-CAGCCTCATCCAAAAGAGGAAA u AGGAx
SEQ ID No. 231 5f-CAGCCTCATCCAAAAGAGGAAA c AGGAx
SEQ ID No. 230 5f-CAGCCTCATCCAAAAGAGGAAA
5f-TAATCCACCATGCTCACAGCCTCATCCAAAAGAGGAAA(C/T)AGGACCCCAGAGCTCCCTCA-3'
SEQ ID No. 275
TCCTGGGGTCTCGAGGGAG-5'
SEQ ID No. 276 3' xCTTT g
TCCTGGGGTCTCGAGGGAG-5'
SEQ ID No. 277 3' xCTTT a
TCCTGGGGTCTCGAGGGAG-5'
SEQ ID No. 278 3' CxxT g
TCCTGGGGTCTCGAGGGAG-5'
SEQ ID No. 279 3' CxxT a
TCCTGGGGTCTCGAGGGAG-5'
Where DNA bases are uppercase, RNA bases are lowercase, and "x" is a C3
spacer (propanediol).
[0580] PCR reactions were performed on a Roche Lightcycler 480 platform in
10
pi volume using 200 nM of the modified or unmodified For primers (SEQ ID Nos.
230,
231, 235, 252, and 273) paired with the modified or unmodified Rev primers
(SEQ ID
Nos. 294, 295, 296, 297, and 298) with 20 ng genomic DNA (Coriell GM07048
homozygous C/C allele or GM18976 homozygous T/T allele). Reactions were run in
Bio-Rad SYBR Green master mix with 2.6 mU of Pyrococcus abyssi RNase H2 for
the
"RDDDD-x" primers (SEQ ID Nos. 231, 235, 276 and 277), 50 mU of Pyrococcus
abyssi RNase H2 for the "RDxxD" "C-allele" primers (SEQ ID Nos. 252 and 276),
or
200 mU of Pyrococcus abyssi RNase H2 for the "RDxxD" "T-allele" primers (SEQ
ID
Nos. 273 and 279). Reactions were started with a soak at 95 C for 5 minutes
followed
by 45 or more cycles of [95 C for 10 seconds and 60 C for 30 seconds].
Reactions
using the "RDDDD-x" primers were run for 45 cycles. Reactions using the
"RDxxD"
primers were run for 75 cycles. Results of qPCR amplifications done at this
SNP site
are shown in Table 72 below.
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Table 72. Cp and ACp values of qPCR reactions showing mismatch discrimination
at a
SMAD7 C/T allele using a dual interrogation "For + Rev" orientation assay
format with
two blocked-cleavable primers.
C/C T/T No
Template
For Primer Employed Rev Primer Employed ACp
Target Target Control
SEQ ID Unmodified SEQ ID Unmodified
24.1 23.9 >45
No. 230 control No. 275 control
SEQ ID SEQ ID
rU-DDDD-x rA-
DDDD-x 32.0 24.2 7.8 31.1
No. 235 No. 277
SEQ ID SEQ ID
rC-DDDD-x rG-
DDDD-x 24.1 28.2 4.1 28.8
No. 231 No. 276
SEQ ID SEQ ID
No. 273 No. 279
rU-DxxD rA-DxxD 24.9 58.3 33.4 >75
SEQ ID SEQ ID
No. 252 No. 278
rC-DxxD rG-DxxD 25.9 >75 >49 >75
[0581] The "dual interrogation" assays that employed "RDDDD-x" design
blocked-cleavable primers (Table 72) showed reduced mismatch discrimination
compared with the single interrogation assay format (Tables 70 and 71).
Specificity
was limited by background and these primer pairs showed amplification in the
absence
of template. A "dual interrogation" format has been used previously to
increase the
specificity of SNP interrogation in a PCR format using the "pyrophosphorolysis
activated polymerization" (PAP) method (see Liu and Sommer, BioTechniques
36:156-166, 2004), which did not suffer from background issues. In the PAP
format,
the blocked For and Rev primers only overlapped by a single base at the 3'-
end. The
"RDDDD-x" primers overlap by 9 bases, as shown in the alignment below.
SEQ ID No. 231 5f-CAGCCTCATCCAAAAGAGGAAAcAGGAx-3'
SEQ ID No. 276 3 xCTTTgTCCTGGGGTCTCGAGGGAG-5'
Where DNA bases are uppercase, RNA bases are lowercase, and "x" is a C3
spacer (propanediol).
This amount of overlap apparently is sufficient to enable cleavage by RNase H2
of one or
both of the blocked-cleavable primers as a "primer-dimer event". Following
cleavage and
activation of one of the blocked primers, a functional primer-dimer template
is formed
which could support PCR, as shown below.
SEQ ID No. 230 5f-CAGCCTCATCCAAAAGAGGAAA 4
SEQ ID No. 276 3' xCTTTgTCCTGGGGTCTCGAGGGAG-5'
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Where DNA bases are uppercase, RNA bases are lowercase, and "x" is a C3
spacer (propanediol).
[0582] In
contrast, the "dual interrogation" assays that employed "RDxxD"
design blocked-cleavable primers (Table 72) showed significantly improved
mismatch
discrimination compared with the single interrogation assay format (Tables 70
and 71).
This format allows for the additive effect from SNP discrimination from both
the "For"
and the "Rev" primers. Using this blocked-cleavable primer design format only
5
discontinuous bases of overlap exists between the "For" and "Rev" primers,
which is
insufficient to allow "primer-dimer events" to occur.
SEQ ID No. 252 5f-CAGCCTCATCCAAAAGAGGAAAcAxxA-3'
SEQ ID No. 278 3' CxxTgTCCTGGGGTCTCGAGGGAG-5'
Where DNA bases are uppercase, RNA bases are lowercase, and "x" is a C3
spacer (propanediol).
[0583] Therefore
the present double cleavable primer design with two internal C3
spacer groups near the ribonucleotide and an unblocked 3'-hydroxyl, "RDxxD",
showed even further improvement over the single forward blocked primer design.
This
new format should have particular utility in demanding applications such as
rare allele
detection assays.
EXAMPLE 35 Improved detection of a mutant allele in a vast excess of wild-type
DNA using blocked-cleavable primers
[0584] Previous
examples demonstrated the utility of blocked-cleavable primers to
discriminate between matched versus mismatched base pairing at the cleavable
RNA
residue. The present example demonstrates the utility of using this method to
detect the
presence of a rare mutant allele in the presence of a vast excess of wild-type
DNA (rare
allele detection).
[0585] The ability
to detect rare allele(s) in the presence of a high background of
the wild-type sequence is of growing importance in both medical diagnostics
and basic
research. These species may be present at levels of 10-2 to 10-5 or lower.
With this type
of target nucleic acid, unbiased amplification of all alleles present linked
to a biased
detection probe system does not offer sufficient sensitivity, and such methods
typically
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can only detect the mutant allele at levels of 10-1 to 10-2 relative to the
wild type alelle.
Biased amplification methods, where the sequence of interest is selectively
amplified
relative to related sequences that may differ by as little as a single base,
can greatly
improve upon these results. The blocked-cleavable primers with RNase H2
cleavage as
described herein offers a version of biased amplification that is useful in
this
application and permits detection of the rare allele at levels or 10-4 or
lower, well within
the range needed for utility in medical diagnostic applications.
[0586] Reactions
were performed using a Lightcycler 480 in 10 1.1,L 384-well
format containing 0.4 U iTaq DNA polymerase, lx iTaq reaction buffer, 0.01%
Triton
X-100, 3 mM MgC12, 800 1.1,M dNTPs, and 200 nM forward and reverse primers.
P.a.
RNase H2 was added at different concentrations as indicated depending on the
design
of the primer. Detection was done using a 5'-nuclease assay with the dual-
labeled
probe (SEQ ID No. 283) at a concentration of 200 nM. Sequences of the
different
primers employed and the probe are provided in Table 73 below. The
dual-interrogation reactions were run under identical conditions except that
BIO-RAD
iQ SYBRTM Green Master Mix was employed without use of a 5'-nuclease probe
oligonucleotide. Target nucleic acids were human genomic DNAs (GM18562 or
GM18537) obtained from the Coriell Institute for Medical Research Cell
Repository.
Genomic DNA of one genotype was used as background at either 0 or 200 ng (-
66,000
copies) and was mixed with genomic DNA of the second genotype at 2 ng (-600
copies), 0.2 ng (-60 copies), 0.02 ng (-6 copies), or 0 ng per reaction.
Thermal cycling
was performed using an initial 5 minute soak at 95 C followed by 50 cycles of
10
seconds at 95 C and 30 seconds at 60 C. Cp and ACp values were computed as
described in previous example.
Table 73. Sequeces of primers and probes employed in rare allele detection
assays
Table 73:
SEQ ID
Name Sequence
No.
SEQ ID No.
rs4939827 Rev 5'-CTCACTCTAAACCCCAGCATT
217
SEQ ID No.
rs4939827 For 5'-CAGCCTCATCCAAAAGAGGAAA
230
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Table 73:
SEQ ID
Name Sequence
No.
SEQ ID No.
rs4939827 "C" For 5' -CAGCCTCATCCAAAAGAGGAAAC
281
SEQ ID No.
rs4939827 "T" For 5' -CAGCCTCATCCAAAAGAGGAAAT
282
rs4939827 SEQ ID No.
5f-CAGCCTCATCCAAAAGAGGAAAcAGGA-SpC3
C-For-AGGA(C3) 231
rs4939827 SEQ ID No.
5f-CAGCCTCATCCAAAAGAGGAAAuAGGA-SpC3
T-For-AGGA(C3) 235
rs4939827 SEQ ID No.
5f-CAGCCTCATCCAAAAGAGGAAA0A(SpC3-SpC3)A
C-For-A(C3C3)A 252
rs4939827 SEQ ID No.
5f-CAGCCTCATCCAAAAGAGGAAAuA(SpC3-SpC3)A
T-F or-A(C3C3)A 273
rs4939827 SEQ ID No.
5f-GAGGGAGCTCTGGGGTCCTgT(SpC3-SpC3)C
C-Rev-A(C3C3)A 278
rs4939827 SEQ ID No.
5f-GAGGGAGCTCTGGGGTCCTaT(SpC3-SpC3)C
T-Rev-A(C3C3)A 279
SEQ ID No.
rs4939827 probe FAM-CTCAGGAAACACAGACAATGCTGGG-IBFQ
283
DNA bases are shown in uppercase. RNA bases are shown in lowercase.
SpC3 is a Spacer C3 group, positioned either internal within the primer or at
the 3 '-end.
FAM is 6-carbocyfluorescein and IBFQ is Iowa Black FQ, a dark fluorescence
quencher.
[0587] Results are shown in Table 74. Use of the standard unmodified
allele-specific primers (For SEQ ID Nos. 281 or 282 paired with Rev SEQ ID No.
217)
resulted in Cp detection values essentially identical to non-specific control
primers (For
SEQ ID No. 230 with Rev SEQ ID No. 217). The "RDDDDx" design primers (For
SEQ ID Nos. 231 or 235 paired with Rev SEQ ID No. 217) were able to detect a
1%
level of both the "C" allele in a background of "T" allele and the "T" allele
in a
background of "C" allele with a 3 cycle detection threshold above background.
The
"RDxxD" design primers (For SEQ ID Nos. 252 or 273 paired with Rev SEQ ID No.
217) gave superior results and detected the presence of 0.1% level of both the
"C" allele
in a background of "T" allele and the "T" allele in a background of "C" allele
with a 6
cycle detection threshold above background; detection at a 0.01% level of the
rare
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allele was achieved with a 3 cycle detection threshold above background. The
bi-directional assay using "RDxxD" design primers (For SEQ ID Nos. 252 paired
with
Rev SEQ ID No. 278 and For SEQ ID Nos. 273 paired with Rev SEQ ID No. 279)
performed at a similar stringency for the "T" allele and was significantly
better for the
"C" allele. In particular, the "C" allele bidirectional assay (For SEQ ID Nos.
252 paired
with Rev SEQ ID No. 278) showed a greater than 14 cycle detection threshold
above
background, so it is likely that this assay would be effective at even lower
rare allele
levels (0.001% or lower).
Copies Mismatch / Match input human DNA
3'-Primer mU
66000 66000 66000
Sequences RNase H2 0 / 0 / 0 / 0 /
66000 /
666 66 6 0
0
666 66 6
...AA - 28.7 32.2 35.9 >50 21.1
21.1 21.2 21.0
...AAC - 29.4 32.9 35.4 >50 22.3
22.5 22.4 22.0
_
...AAT - 28.5 31.6 35.7 >50 21.4
21.5 21.5 21.6
_
...AAcAGGA-x 2.6 28.1 31.4 35.4 >50 28.4
30.7 31.3 31.3
...AAuAGGA-x 20 28.1 31.3 35.7 >50 27.9
30.4 31.1 31.2
_
...AAcAxxA 50 27.9 31.4 34.6 >50 28.8
31.9 34.8 37.9
_
...AAuAxxA 400 28.2 31.9 36.1 >50 28.9
32.4 36.3 39.0
"rC/rG"
100 28.7 32.8 35.6 >50 28.1
32.1 35.5 >50
di-primer
"rU/rA"
400 29.6 33.0 36.7 >50 28.6
31.7 35.7 38.5
di-primer
Table 74. Use of blocked-cleavable primers for rare allele detection.
Amplification reactions for the SMAD7 rs4939827 locus were run for 50 cycles
using
an internal non-discriminatory dual-labeled hydrolysis probe for detection
with various
primers as indicated (the 3'-end sequence is shown). P.a. RNase H2 was added
at the
amounts indicated per 10 1..EL. Human DNA that was a mismatch at the SNP site
relative to the primers was either present at 0 or 200 ng (66,000 copies) per
reaction;
DNA that was a match at the SNP site relative to the primers was present at 2
ng (666
copies), 0.2 ng (66 copies), 0.02 ng (6 copies), or 0.0 ng per reaction.
Reactions were
run in triplicate and average Cp values are shown. The location of the
mismatch in the
primer compared to the target nucleic acid is underlined.
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[0588] A person of ordinary skill in the art can appreciate that the double
interrogation of 3 '-end primer contructs could be varied and still maintain
the important
functional requirement of preventing self-priming of the forward and reverse
primers
while still providing an adequate RNase H2 cleavage site. For example, one
alternative
construct can be "RxDDDD" wherein a spacer is placed next to the
ribonucleotide and
the ribonucleotide isdirectly over the mismatched site. In another embodiment,
the
forward and reverse primers can have different contructs. For example, the For
primer
can be a "RDxxD" construct while the Rev primer is "RxDDDD". In another
embodiment, the mismatch can at a DNA base adjacent to the RNA base. For
example,
the primer can contain a "RDxxD" construct, wherein the mismatch is located at
the
underlined DNA base.
EXAMPLE 36 Optimizing magnesium cation levels can improve
mismatch discrimination
[0589] The present example demonstrates that the mismatch discrimination of
allele-specific PCR using blocked-cleavable primers with RNase H2 can be
improved
by varying concentrations of Mg ions in the buffer.
[0590] It appreciated that varying magnesium levels in PCR buffers can
affect the
efficiency and specificity of the amplification reaction by effects on the
polymerase and
on the T. of primer binding. However, for rhPCR SNP discrimination is mediated
by
the RNase H2 enzyme and no information is known about how magnesium
concentration affects specificity of match versus mismatch cleavage at the RNA
base
by this enzyme. Pyrococcus abyssi RNase H2 show a broad peak in enzyme
activity
from 2-10 mM Mg" ion concentration and activity dramatically drops as free Mg"
concentration drops below 2 mM; the enzyme is at 50% maximal activity at 1 mM
Mg". In rhPCR, dNTPs are typically present at 0.8 mM concentration, and each
dNTP
binds one molecule of Mg', removing it from availability in the reaction
buffer. Thus
a buffer that contains 3 mM Mg' with 0.8 mM dNTPs actually has 2.2 mM free
Mg".
This example examines the effect of varying Mg' ion concentration in rhPCR on
reaction efficiency and quality of SNP discrimination.
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[0591] In previous examples, PCR reactions employing blocked-cleavable
primers
were performed using 3mM MgC12, a concentration which is commonly employed in
qPCR to ensure a robust reaction. In the present example, we demonstrate that
lowering magnesium levels can increase the specificity of the amplification
reaction, as
measured by the ACp between match vs. mismatch reactions in a SNP genotyping
assay.
When performing PCR, researchers typically employ either homemade buffers or
buy a
premade commercial mastermix. It is simple to adjust the magnesium
concentration to
be higher or lower in a homemade buffer. It is easy to increase the magnesium
concentration in a premade buffer, but can be difficult to lower magnesium if
the buffer
comes with magnesium already added at a set concentration in the mixture.
However,
it is possible to lower magnesium in a premade mixture by adding nucleotide
triphosphates (for example, rNTPs). The rNTPs will chelate a single molecule
of
magnesium for each nucleotide triphosphate, effectively reducing the
concentration of
free magnesium in the buffer. In the present example, we demonstrate
improvement in
PCR specificity using blocked-cleavable primers and RNase H2 by directly
adjusting
magnesium content of the buffer or by indirectly adjusting free magnesium
content of
the buffer by adding rNTPs to the reaction mix.
[0592] Method: reduced magnesium in a premade mastermix having a fixed
MgC12 concentration using rNTPs to chelate free magnesium. Quantitative
real-time PCR (qPCR) was performed with 20 ng of human genomic DNA (GM18562
or GM18537, Coriell Institute for Medical Research, Camden, NJ, USA) using
primers
specific for a SNP site in the human SMAD7 gene (rs4939827, NM 005904). DNA
sample GM18562 is homozygous for the C allele at this locus and DNA sample
GM18537 is homozygous for the T allele. Reactions used lx iQ SYBRTM Green
Supermix (Bio-Rad, Hercules, CA, USA), which is a premade commercial mastermix
containing 3 mM MgC12. Either 0, 5, 96, or 384 fmoles of P.a. RNase H2 (final
concentration of 0, 0.5, 9.6, or 38.4 nM in a 10 pt reaction, which equals 0,
2.6, 50, or
200 mU of enzyme). 200 nM of each primer was employed. Control reactions
employed unblocked Forward and Reverse primers, rs4939827 For (SEQ ID NO. 217)
with rs4939827 Rev (SEQ ID NO. 230). Two different versions of blocked primers
specific for each allele were used, either the RDDDDx (see Example 13) or
RDxxD
designs (see Example 28). Blocked-cleavable forward primers were used in
combination with an unmodified reverse primer as follows: rs4939827 C-For
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RDDDDx (SEQ ID NO. 231) with rs4939827 Rev (SEQ ID #230), rs4939827 T-For
RDDDDx (SEQ ID NO. 235) with rs4939827 Rev (SEQ ID #230), rs4939827 C-For
RDxxD (SEQ ID NO. 252) with rs4939827 Rev (SEQ ID #230), rs4939827 T-For
RDxxD (SEQ ID NO. 273) with rs4939827 Rev (SEQ ID # 230). In addition, either
0.5
or 1 mM rATP (diluted in water) was added to some reactions. All
oligonucleotides
used in this Example are shown in Table 75. Cycling was performed on a Roche
LightCycler 480 (Roche Applied Science, Indianapolis, IN, USA) as follows: 95
C
for 3 minutes followed by 75 cycles of 95 C for 10 seconds and 60 C for 30
seconds.
All reactions were performed in triplicate. The initial 3 minute incubation at
95 C
before thermocycling commences allows for reactivation of the hot-start DNA
polymerase.
Table 75: Synthetic oligonucleotide primers for rs4939827, SMAD7
Name Sequence SEQ ID NO.
rs4939827 Rev CTCACTCTAAACCCCAGCATT SEQ ID NO. 217
rs4939827 For CAGCCTCATCCAAAAGAGGAAA SEQ ID NO. 230
rs4939827
CAGCCTCATCCAAAAGAGGAAAcAGGA-SpC3 SEQ ID NO. 231
C-For RDDDDx
rs4939827
CAGCCTCATCCAAAAGAGGAAAuAGGA-SpC3 SEQ ID NO. 235
T-For RDDDDx
rs4939827
CAGCCTCATCCAAAAGAGGAAAcA(SpC3-SpC3)A SEQ ID NO. 252
C-For RDxxD
rs4939827
CAGCCTCATCCAAAAGAGGAAAuA(SpC3-SpC3)A SEQ ID NO. 273
T-For RDxxD
DNA bases are uppercase and RNA bases are lowercase; SpC3 = C3 spacer
(propanediol)
[0593] Results are shown in Table 76. Use of unmodified primers resulted in
efficient amplification reactions and no difference was seen between reactions
containing or not containing rATP or with or without P.a. RNase H2 enzyme.
When
amplification was performed in the presence of rATP, an increase in mismatch
discrimination was observed (increase in ACp) that varied with the
concentration of
rATP present in the reaction for the RDxxD design primers but not the RDDDDx
design primers. No amplification occurred when using blocked-cleavable primers
if
RNase H2 was not added to those reactions (not shown).
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Table 76: Cp and ACp values seen in qPCR with addition of rATP to vary Mg"
concentration using various For primers with an unmodified Rev primer.
For Primer [RNase H2] 0 mM rATP 0.5 mM rATP 1 mM rATP
CC TT ACp CC TT ACp CC TT ACp
rs4939827
0 mU 22.7 23.7 22.6 23.7 22.8
23.8
For
rs4939827
2.6 mU 22.7 23.8 22.8 23.9 23.4
24.3
For
rs4939827
C-For 2.6 mU 23.0 34.1 11.1 23.0
33.6 10.7 23.0 35.1 12.1
RDDDDx
rs4939827
T-For 2.6 mU 36.4 25.2 11.2 37.1
26.2 11.0 41.2 29.1 12.1
RDDDDx
rs4939827
C-For 50 mU 22.8 45.5 22.7 22.9
56.2 33.4 23.3 67.4 44.1
RDxxD
rs4939827
T-For 200 mU 45.4 24.3 21.1 49.1
24.7 24.4 59.7 25.6 34.1
RDxxD
[0594] Method:
directly varying magnesium concentration in the reaction mix.
Quantitative real-time PCR (qPCR) was performed with 20 ng of human genomic
DNA
(GM18562 or GM18537, Coriell Institute for Medical Research, Camden, NJ, USA)
using primers specific for a SNP site in the human SMAD7 gene (rs4939827,
NM 005904). DNA sample GM18562 is homozygous for the C allele at this locus
and
DNA sample GM18537 is homozygous for the T allele. Reactions used 0.4 U of a
hot-start Taq DNA polymerase (iTaqTm, Bio-Rad, Hercules, CA, USA). Reactions
contained iTaqTm buffer with 3 mM, 2.5 mM, 2 mM, or 1.5 mM MgC12 Reactions
containing 3 mM MgC12 + 1 mM rATP were also tested, which should simulate use
of 2
mM MgC12 without rNTPs. In addition, reactions contained 200 nM of each
primer,
200 nM of a 5' -nuclease assay detection probe (SEQ ID NO. 283), and 0 or 768
fmoles
of P.a. RNase H2 (final concentration of 0 or 76.8 nM in a 10 iiiL reaction,
or 0 or 400
mU of the enzyme). Control reactions employed unblocked Forward and Reverse
primers, rs4939827 For (SEQ ID NO. 217) with rs4939827 Rev (SEQ ID NO. 230).
RDxxD design blocked-cleavable For primers were employed with an unmodified
Rev
primer in the following combinations: rs4939827 C-For RDxxD (SEQ ID NO. 252)
with rs4939827 Rev (SEQ ID #230), rs4939827 T-For RDxxD (SEQ ID NO. 273) with
rs4939827 Rev (SEQ ID # 230). All oligonucleotides used in this experiment are
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shown in Table 77. Cycling was performed on a Roche LightCycler 480 (Roche
Applied Science, Indianapolis, IN, USA) as follows: 95 C for 3 minutes
followed by 85
cycles of 95 C for 10 seconds and 60 C for 30 seconds. All reactions were
performed
in triplicate. The initial 3 minute incubation at 95 C before thermocycling
commences
allows for reactivation of the hot-start DNA polymerase.
Table 77: Synthetic oligonucleotide primers
Name Sequence SEQ ID NO.
rs4939827 Rev CTCACTCTAAACCCCAGCATT SEQ ID NO. 217
rs4939827 For CAGCCTCATCCAAAAGAGGAAA SEQ ID NO. 230
rs4939827
CAGCCTCATCCAAAAGAGGAAAcA(SpC3-SpC3)A SEQ ID NO. 252
C-For RDxxD
rs4939827
CAGCCTCATCCAAAAGAGGAAAuA(SpC3-SpC3)A SEQ ID NO. 273
T-For RDxxD
SMAD7 Probe FAM-CTCAGGAAACACAGACAATGCTGGG-IBFQ SEQ ID NO. 283
DNA bases are uppercase and RNA bases are lowercase; SpC3 = C3 spacer
(propanediol); FAM = 6-carboxyfluorescein; IBFQ = Iowa BlackTM FQ fluorescence
quencher
[0595] Results are shown in Table 78. When magnesium levels were lowered in
the reaction buffer, an increase in mismatch discrimination was observed. As
predicted,
the increase in mismatch discrimination using 2 mM Mg2+ was similar to the
increase
observed when 1 mM rATP was added to reactions containing 3 mM Mg2+. Use of
unmodified primers resulted in efficient amplification reactions and no
difference was
seen between reactions containing varying amounts of the Mg2+ or rATP. No
amplification occurred when using blocked-cleavable primers if RNase H2 was
absent
from the reactions (not shown).
Table 78: Cp and ACp values seen in qPCR with different Mg concentrations.
For Primer [rATP] [MgC12] C/C T/T ACq
rs4939827
For 24.0 24.2
rs4939827
1 mM 3 mM
C-For RDxxD 24.9 59.5 34.6
rs4939827
T-For RDxxD 78.7 30.5 48.3
rs4939827
0 mM 3 mM 24.1 23.9
For
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rs4939827
24.1 44.3 20.2
C-For RDxxD
rs4939827
47.6 24.7 22.9
T-For RDxxD
rs4939827
24.5 24.1
For
rs4939827
2.5 mM 24.7 46.0 21.2
C-For RDxxD
rs4939827
51.7 24.8 26.9
T-For RDxxD
rs4939827
23.9 23.7
For
rs4939827
2 mM 25.7 71.6 45.9
C-For RDxxD
rs4939827
69.5 26.9 42.6
T-For RDxxD
rs4939827
24.9 23.8
For
rs4939827
1.5 mM 26.0 55.4 29.5
C-For RDxxD
rs4939827
>85 51.3 >33.7
T-For RDxxD
[0596] Thus magnesium concentration can substantially affect the ACp values
obtained for SNP discrimination when blocked-cleavable primers are employed in
PCR-based genotyping. Titration of magnesium levels may be needed to optimize
performance of individual primer pairs between different loci studied, and
such
experiments can be readily performed by one with skill in the art.
EXAMPLE 37 Rare-allele detection can be improved with
magnesium optimization.
[0597] The present example demonstrates that the ability to detect "rare
alleles" in
a mixed-allele DNA sample using blocked-cleavable primers in PCR can be
improved
by optimizing the magnesium concentration present in the reaction buffer.
[0598] Example 36 demonstrated that the discrimination achieved between
matched and mismatched blocked-cleavable primers in allele-specific PCR assays
can
be improved by directly lower the magnesium levels or indirectly with the
addition of
rNTPs, which lowers the free magnesium levels. The present example extends
this
method to rare allele detection, using RDxxD design primers (see Examples 28
and 35).
In this case a commercial SYBR-Green mastermix was employed where magnesium
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concentration was 3 mM as provided by the manufacturer; rNTPs were added to
the
reaction mix to lower free magnesium levels.
[0599] Blocked-cleavable primers specific for a single-nucleotide
polymorphism
(SNP) in the human SMAD7 gene (rs4939827, NM 005904) were designed for each
allele using the RDxxD design. Control reactions employed unblocked Forward
and
Reverse primers, rs4939827 For (SEQ ID NO. 217) with rs4939827 Rev (SEQ ID NO.
230). RDxxD design blocked-cleavable For primers were employed with an
unmodified Rev primer in the following combinations: rs4939827 C-For RDxxD
(SEQ
ID NO. 252) with rs4939827 Rev (SEQ ID #230), rs4939827 T-For RDxxD (SEQ ID
NO. 273) with rs4939827 Rev (SEQ ID # 230). To set up a rare allele detection
experiment, DNA for one allele is added at high amounts (to simulate wild type
DNA)
and small amounts of the second allele are added (to simulate a rare mutant
DNA). In
this scenario, the "wild type" DNA is a mismatch to the blocked-cleavable For
primer
while the "mutant" (rare allele) DNA is a match to the blocked-cleavable For
primer.
Quantitative real-time PCR (qPCR) was performed in the presence of 200 ng (-
66,000
genomic equivalent copies) or 0 ng of "wild type" human genomic DNA (either
GM18562 or GM18537, Coriell Institute for Medical Research, Camden, NJ, USA)
included in the reaction as background. GM18562 and GM18537 are homozygous for
two alleles of the rs4939827 SNP (C or T). GM18562 is homozygous for the C
allele at
the rs4939827 locus and GM18537 is homozygous for the T allele. In addition to
the
background genomic "wild-type" DNA, 2, 0.2, 0.02 or 0 ng (660, 66, 6, or 0
genomic
equivalent copies) of "mutant" human genomic DNA from the allele of the
rs4937827
SNP (C or T) opposite to one in the background DNA was added to the reaction.
Reactions also contained lx iQ SYBRTM Green Supermix (Bio-Rad, Hercules, CA,
USA), and either 192 (for rC containing primers) or 768 (for rU containg
primers)
fmoles of P.a. RNase H2 (final concentration of 19.2 or 76.8 nM in a 10 iaL
reaction,
which is the equivalent of 100 or 400 mU of the RNase H2 enzyme,
respectively). 200
nM of each primer was used in each reaction. In addition, either 0 or 1 mM
rATP
(diluted in water) was added to each reaction. All oligonucleotides used in
Example 37
are shown in Table 79. Cycling was performed on a Roche LightCycler0 480
(Roche
Applied Science, Indianapolis, IN, USA) as follows: 95 C for 3 minutes
followed by 65
cycles of 95 C for 10 seconds and 60 C for 30 seconds. All reactions were
performed
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in triplicate. The initial 3 minute incubation at 95 C before thermocycling
commences
allows for reactivation of the hot-start DNA polymerase.
Table 79: Synthetic oligonucleotide primers employed in rare allele detection
experiments
Name Sequence SEQ ID NO.
rs4939827 Rev CTCACTCTAAACCCCAGCATT SEQ ID NO. 217
rs4939827 For CAGCCTCATCCAAAAGAGGAAA SEQ ID NO. 230
rs4939827
CAGCCTCATCCAAAAGAGGAAAcA(SpC3-SpC3)A SEQ ID NO. 252
C-For RDxxD
rs4939827
CAGCCTCATCCAAAAGAGGAAAuA(SpC3-SpC3)A SEQ ID NO. 273
T-For RDxxD
DNA bases are uppercase and RNA bases are lowercase; x = C3 spacer
(propanediol)
[0600] Results are shown in Tables 80, 81, 82, and 83 below. At the
1:10,000
rare-allele discrimination level (200 ng "wild type or mismatch" template with
0.02 ng
"mutant or match" template), the addition of 1 mM rATP increased the ACp (in
this
case defined as the Cp measured in the 1:10,000 samples with the reaction
background
where no "mutant" T-allele is present) from 7.0 cycles (39.5 ¨ 32.5 = 7.0,
Table 80) to
12.6 cycles (46.0 ¨ 33.4, Table 81). When DNAs were switch and the C allele
was used
as the rare mutant, addition of 1 mM rATP increased the ACp from 1.8 cycles
(36.7 ¨
34.9 = 1.8, Table 82) to 18.4 cycles (53.6 ¨ 35.2 = 18.4, Table 83).
Importantly,
reaction efficiency was not compromised and the Cp where positive fluorescent
signals
were first seen were similar between the 3 mM Mg and the 3 mM Mg' plus 1 mM
rATP reactions.
[0601] Use of unmodified primers resulted in efficient amplification
reactions and
no significant difference was seen between reactions containing rATP. No
amplification occurred when using blocked-cleavable primers if RNase H2 was
not
added to the reactions (not shown).
Table 80: Cps resulting from a PCR rare allele detection experiment using C-
allele as
wild type with 3 mM Mg".
Mismatch
template 200 ng (66,000 copies) 0 ng (0 copies)
C allele
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Match 2 ng 0.2 ng 0.02 ng 2 ng 0.2 ng 0.02 ng
0 ng (0 0 ng (0
template (660 (66 (6 (660 (66 (6
copies) copies)
T allele copies) copies) copies) copies) copies)
copies)
rs4939827
19.5 19.5 19.4 20.2 26.0 30.2 35.3 >65
For
rs4939827
T-For 26.4 29.7 32.5 39.5 26.4 30.1 34.4 >65
RDxxD
Table 81: Cps resulting from a PCR rare allele detection experiment using C-
allele as
wild type with 3 mM Mg" plus 1 mM rATP.
Mismatch
template 200 ng (66,000 copies) 0 ng (0 copies)
C allele
Match 2 ng 0.2 ng 0.02 ng 2 ng 0.2 ng 0.02 ng
0 ng (0 0 ng (0
template (660 (66 (6 (660 (66 (6
copies) copies)
T allele copies) copies) copies) copies) copies)
copies)
rs4939827
19.4 19.3 19.4 19.4 26.1 29.5 35.2 >65
For
rs4939827
T-For 26.9 30.7 33.4 46.0 26.4 30.0 34.9 >65
RDxxD
Table 82: Cps resulting from a PCR rare allele detection experiment using T-
allele as
wild type with 3 mM Mg".
Mismatch
template 200 ng (66,000 copies) 0 ng (0 copies)
T allele
Match 2 ng 0.2 ng 0.02 ng 2 ng 0.2 ng 0.02 ng
0 ng (0 0 ng (0
template (660 (66 (6 (660 (66 (6
copies) copies)
C allele copies) copies) copies) copies) copies)
copies)
rs4939827
19.8 19.8 19.8 19.9 26.2 30.0 33.9 >65
For
rs4939827
C-For 26.6 30.0 34.9 36.7 26.5 30.0 33.9 >65
RDxxD
Table 83: Cps resulting from a PCR rare allele detection experiment using C-
allele as
wild type with 3 mM Mg" plus 1 mM rATP.
Mismatch
template 200 ng (66,000 copies) T allele) 0 ng (0 copies) T allele
T allele
Match 2 ng 0.2 ng 0.02 ng 2 ng 0.2 ng
0.02 ng 0 ng (0
0 ng (0
template (660 (66 (6 (660 (66 (6
copies) copies)
C allele copies) copies) copies) copies) copies)
copies)
rs4939827
20.1 20.3 20.2 20.3 27.0 30.8 33.9 >65
For
rs4939827
C-For 27.6 30.9 35.2 53.6 27.7 31.1 34.4 >65
RDxxD
[0602] This example demonstrates that adjusting magnesium concentration in
the
buffer can improve discrimination and detection limits in a PCR rare-allele
detection
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assay using blocked-cleavable primers and RNase H2 and that detection limit
can
reliably reach levels of 1:10,000.
EXAMPLE 38 Use of the non-ionic detergent Brij -58 in PCR with
blocked-cleavable primers and RNase H2
[0603] Example 18
demonstrated the benefit of including a non-ionic detergent in
the reaction mix when performing PCR using blocked-cleavable primers and
Pyrococcus abyssi (P.a.) RNase H2. The present example demonstrates that the
detergent Brij -58 (also known as either polyethylene glycol hexadecyl ether,
or
polyoxyethylene (20) cetyl ether) can substitute for other non-ionic
detergents, such as
Triton X-100, in PCR using blocked-cleavable primers and Pyrococcus abyssi
(P.a.)
RNase H2.
[0604]
Quantitative real-time PCR (qPCR) was performed in 10 pL reactions with
20 ng of human genomic DNA (GM18562 or GM18537, Coriell Institute for Medical
Research, Camden, NJ, USA) using primers specific for a site in the human
SMAD7
gene (rs4939827, NM_005904). DNA sample GM18562 is homozygous for the C
allele and GM18537 is homozygous for the T allele. Reactions utilized either
0.5 U
(10.8 ng/11.1 nM/111 fmol) of native Tag DNA polymerase. Final reaction buffer
conditions used were 10 mM Tris-HCL (pH 8.4 at 25 C), 50 mM KCL, and 3 mM
MgC12. Reactions also used 1 pL of P.a. RNase H2 (5 fmoles; final
concentration of
0.5 nM in a 10 pL reaction, or 2.6 mU of the enzyme), diluted in RNase H2
dilution
buffer without detergent (20 mM Tris-HCL pH 8.4, 100 mM KCL, 0.1 mM EDTA, and
% glycerol). One pL of water (no detergent) or one pL of 0.001%, 0.01%, 0.1%
Brij -58 or 0.1% Triton X-100 was added to each 10 pL final reaction volume.
200 nM
of each primer and a 5' nuclease detection probe were also used in each
reaction.
Control reactions employed unblocked Forward and Reverse primers, rs4939827
For
(SEQ ID NO. 217) with rs4939827 Rev (SEQ ID NO. 230). An RDDDDx design
blocked-cleavable forward primer (see Example 13) was used in combination with
an
unmodified reverse primer as follows: rs4939827 C-For RDDDDx (SEQ ID NO. 231)
with rs4939827 Rev (SEQ ID NO. 230). A SMAD7 5'-
nuclease
fluorescence-quenched probe was included for detection at 200 nM (SEQ ID NO.
283).
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Oligonucleotides used in this Example are shown in Table 84. Cycling was
performed
on a Roche LightCycler 480 (Roche Applied Science, Indianapolis, IN, USA) as
follows: 95 C for 3 minutes followed by 45 cycles of 95 C for 10 seconds and
60 C for
30 seconds. All reactions were performed in triplicate.
Table 84: Synthetic oligonucleotide primers and probe
Name Sequence SEQ ID NO.
rs4939827 Rev CTCACTCTAAACCCCAGCATT SEQ ID NO.
217
SEQ ID NO.
rs4939827 For CAGCCTCATCCAAAAGAGGAAA
230
rs4939827 C-For SEQ ID NO.
CAGCCTCATCCAAAAGAGGAAAcAGGA-SpC3
RDDDDx 231
ID
SMAD7 Probe FAM-CTCAGGAAACACAGACAATGCTGGG-IBFQ SEQ
283 NO.
DNA bases are uppercase and RNA bases are lowercase; x = C3 spacer
(propanediol);
FAM = 6-carboxyfluorescein; IBFQ = Iowa BlackTM FQ fluorescence quencher
[0605] Results are
shown in Table 85 below. Cp and ACp values were obtained
from PCR using either no detergent, 0.0001%, 0.001%, 0.01% final concentration
Brij -58, or 0.01% Triton X-100 detergents. Use of no detergent or 0.0001%
Brij -58
resulted in no amplification with either unmodified or blocked primer designs.
Increasing the concentration of Brij -58 to 0.001% or higher final
concentration
resulted in efficient amplification reactions with both unmodified and blocked
primer
designs. Concentrations of Brij -58 higher than 0.001% showed no significant
difference in amplification efficiency between the different Brij -58
concentrations
compared with reactions performed using 0.01% of the non-ionic detergent
Triton
X-100. This result demonstrates that both the Taq DNA polymerase and the P.a.
RNase H2 enzymes are active under these conditions. No amplification occurred
when
using blocked-cleavable primers if RNase H2 was not added to the reactions
(not
shown).
Table 85: Cp and ACp values for quantitative rhPCR reactions using Brij -58 or
Triton
X-100 detergents.
Detergent Primers C/C T/T ACp*
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Unblocked Rev >95 >95
No Detergent
rC DDDDx For >95 >95
Unblocked Rev >95 >95
0.0001% Brij-58
rC DDDDx For >95 >95
Unblocked Rev 24.2 25.0
0.001% Brij-58
rC DDDDx For 24.7 37.3 12.7
Unblocked Rev 24.2 25.1
0.01% Brij-58
rC DDDDx For 24.7 36.8 12.1
0.1% Triton Unblocked Rev 24.2 25.0
X-100 rC DDDDx For 24.5 36.9 12.4
*ACp = Cp match (C/C) minus Cp mismatch (T/T)
[0606] These results demonstrate that the Brij -58 may be employed as a non-
ionic
detergent in PCR using blocked-cleavable primers and RNase H2.
EXAMPLE 39: Use of blocked-cleavable primers with RNase H2 in PCR with a
high-fidelity 3'-exonuclease DNA polymerase
[0607] The present example demonstrates compositions of blocked-cleavable
primers that function well with high fidelity proof-reading DNA polymerases.
[0608] Many DNA polymerases, like that obtained from Thermus aquaticus (Taq
DNA polymerase), have a 5'-exonuclease activity but lack 3'-exonuclease
activity.
These enzymes are often robust but do not have a "proof-reading" function and
are
generally consider to be "low fidelity". Polymerases in this class are
typically
employed for routine PCR-based sequence detection assays and were the employed
in
most of the earlier Examples above. Other polymerases, like those obtained
from
Pyrococcus furiosus (Pfu DNA polymerase), Thermococcus kodakarensis (KOD DNA
polymerase), etc. lack a 5'-exonuclease activity but have a 3'-exonuclease
proofreading
activity. These enzymes are considered "high fidelity" polymerases. High
fidelity
polymerases are often employed in DNA cloning or in Next Generation Sequencing
(NGS) applications where even rare mutations introduced by the polymerase are
detrimental. However, polymerases with 3 '-exonuclease activity can remove the
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terminal bases at the 3 '-end of a primer or end-blocking groups, such as the
C3 spacer
group employed as a terminal blocking moiety in the RDDDDx primer design;
removing the blocking group allows for primer function in the absence of RNase
H2
cleavage, thereby eliminating the many benefits obtained from using blocked-
cleavable
primers as taught herein. It is desirable to have methods that enable use of
blocked-cleavable primers for applications such as cloning, gene synthesis,
NGS, etc.
where high fidelity amplification is desired.
[0609] Methods. Quantitative real-time PCR (qPCR) was performed with 2e3
copies of a synthetic oligonucleotide DNA template (SEQ ID NO. 144) using
primers
specific for this template. Reactions used lx Phusion'-HF buffer (NEB,
Ipswich, MA,
USA), and 0, 2.5, 5, 9.6, 19.2, 192, 288, 384, or 768 fmoles of P.a. RNase H2
(final
concentration of 0, 0.25, 0.5, 0.96, 1.92, 19.2, 28.8, 38.4 or 76.8 nM in a 10
litL reaction,
or 0, 1.3, 2.6, 5, 10, 100, 150, 200, or 400 mU of the enzyme, respectively).
800 laM
deoxynucleotides and 200 nM of each primer were included in each reaction and
1.5
mM of additional MgC12 was added to each reaction, resulting in a final
concentration
of 3 mM MgC12. 0.2 U of the Phusion DNA polymerase (NEB, Ipswich, MA, USA)
was employed in each amplification reaction. Phusion is a high fidelity DNA
polymerase which has 3'-exonuclease activity. For detection, SYBR Green
(Molecular Probes, Eugene, OR, USA) was added to a final concentration of
30,000
fold diluted versus the stock reagent (dilutions were made fresh in dH20).
Control
reactions employed unmodified primers (Syn-For, SEQ ID NO. 68 and Syn-Rev, SEQ
ID NO. 69). Two different types of blocked-cleavable primers were employed,
the
RDDDDx and RDxxD designs, both perfect match to the target synthetic template.
The
two blocked-cleavable reverse primers were used in combination with an
unmodified
forward primer (Syn Rev rU DDDDx, SEQ ID NO. 116 with Syn-For, SEQ ID NO. 68;
Syn Rev rU DxxD, SEQ ID NO. 284 with Syn-For, SEQ ID NO. 68). Oligonucleotides
used in this experiment are shown in Table 86. PCR was performed on a BioRad
CFX384Tm Real Time System (Bio-Rad, Hercules, CA, USA) as follows: 95 C for 3
minutes followed by 40 cycles of 95 C for 10 seconds and 60 C for 30 seconds.
All
reactions were performed in triplicate.
Table 86: Synthetic oligonucleotides employed in Example 39
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Name Sequence SEQ ID NO.
AGCTCTGCCCAAAGATTACCCTGACAGCTAAGTG
Synthetic SEQ ID NO.
GCAGTGGAAGTTGGCCTCAGAAGTAGTGGCCAGC
Template 144
TGTGTGTCGGGGAACAGTAAAGGCATGAAGCTCAG
Syn-For AGCTCTGCCCAAAGATTACCCTG SEQ ID NO.
68
Syn-Rev CTGAGCTTCATGCCTTTACTGT SEQ ID NO.
69
Syn Rev rU SEQ ID NO.
CTGAGCTTCATGCCTTTACTGTuCCCC-SpC3
DDDDx 116
Syn Rev rU SEQ ID NO.
CTGAGCTTCATGCCTTTACTGTuC(5pC3-5pC3)C
DxxD 284
DNA bases are uppercase and RNA bases are lowercase; SpC3 = C3 spacer
(propanediol)
[0610] Results are
shown in Tables 87 and 88 below. As expected, the unmodified
control primers performed well with the high-fidelity 3 '-exonuclease Phusion
DNA
polymerase and showed uniform amplification efficiency with or without added
RNase
H2 enzyme. The RDDDDx design blocked-cleavable primer (SEQ ID NO. 116)
showed full efficiency amplification in the absence of RNase H2 and reduced
amplification efficiency when RNase H2 was present. Using Taq DNA polymerase,
the reverse is seen: no amplification occurs in the absence of RNase H2 and
efficient
amplification occurs in the present of even small amounts of RNase H2 (usually
100%
efficiency by 2.6 mU enzyme), Example 13). This unexpected observation
suggests
that the 3'-exonuclease activity of the Phusion DNA polymerase can remove the
C3
terminal blocking group on this primer, so primer extension and PCR proceeds
without
RNase H2 cleavage. Further, there appears to be an adverse interaction between
the
Phusion DNA polymerase and RNase H2 such that reduced amplification efficiency
occurred when both enzymes are present with the RDDDDx class of blocked-
cleavable
primer (note: no loss of efficiency is seen using unmodified primers when
RNase H2 is
added).
Table 87: Cp values resulting from qPCR performed with an unmodified forward
primer and various reverse primers using a high-fidelity DNA polymerase.
Reverse SE ID 0 mU 1.3 mU 2.6 mU 5 mU 10 mU
Q
Primer RNase H2 RNase H2 RNase H2 RNase H2 RNase H2
SEQ ID
Syn-Rev 25.1 25.2 25.2 25.2 25.3
NO. 69
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Syn Rev rU SEQ ID
25.6 39.4 37.8 32.8 29.5
DDDDx NO. 116
[0611] In contrast
to these results, the RDxxD blocked-cleavable primer did not
support PCR in the absence of RNase H2. Thus this design of primer is not
unblocked
by the 3'-exonuclease activity of the Phusion DNA polymerase. Primer cleavage
and
PCR occurred in the presence of RNase H2. Lower efficiency PCR was seen using
low
doses of RNase H2 (100 mU per reaction) and near parity with the unblocked
control
primers was achieved using 400 mU RNase H2 per reaction.
Table 88: Cp values resulting from qPCR performed with an unmodified forward
primer and various reverse primers using a high-fidelity DNA polymerase.
Reverse SE ID 0 mU 100 mU 150 mU 200 mU 400 mU
Q
Primer RNase H2 RNase H2 RNase H2 RNase H2 RNase H2
SEQ ID
Syn-Rev 25.1 25.2 25.2 25.2 25.3
NO. 69
Syn Rev rU SEQ ID
NO. 284 >40 31.3 29.1 27.8 26.6
DxxD
[0612] We conclude
that the RDxxD design of blocked-cleavable primer is
preferred over the RDDDDx design when using high fidelity 3'-exonuclease DNA
polymerases, such as Phusion. Higher concentrations of RNase H2 may be
necessary
to achieve reaction efficiencies equal to reactions performed using unmodified
primers
or is typically seen using Taq DNA polymerase.
EXAMPLE 40: Improved design of blocked-cleavable primers for use in PCR
with RNase H2 using a high fidelity 3'-exonuclease DNA polymerase
[0613] The present
example demonstrates utility of a new design of
blocked-cleavable primer, called "RDDDDxxD", in RNase H2-dependent PCR
(rhPCR) in combination with a high-fidelity DNA polymerase possessing 3'
exonuclease activity.
[0614] Previous
Example 39 demonstrated that the RDxxD design of
blocked-cleavable primer worked well using a high fidelity 3'-exonuclease DNA
polymerase while the RDDDDx design did not perform as well. In general, RDxxD
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blocked-cleavable primers require higher concentrations of RNase H2 in PCR to
reach
peak amplification efficiency (i.e., to achieve parity with reactions
performed using
unmodified primers) whereas the RDDDDx design requires use of lower
concentrations of RNase H2. At times it can be advantageous to employ RDDDDx
type primers with low RNase H2 concentrations. The present example
demonstrates
performance of a variant of the RDDDDx primer, the new RDDDDxxD primer, which
can function well with high fidelity 3 '-exonuclease DNA polymerases in PCR
using
relatively low concentrations of RNase H2. See Example 26 for a description of
other
properties of this design of blocked-cleavable primer.
[0615] Quantitative real-time PCR (qPCR) was performed with 20 ng of human
genomic DNA (GM18537, Coriell Institute for Medical Research, Camden, NJ, USA)
using primers specific for a site in the human SMAD7 gene (rs4939827, NM
005904).
GM18537 DNA is homozygous T/T at this locus. The Phire DNA polymerase
(Thermo Scientific, Pittsburgh, PA, USA) is a high fidelity 3'-exonuclease DNA
polymerase and was employed in the experiments performed in this example.
Reactions used lx Phire buffer (Thermo Scientific, Pittsburgh, PA, USA), and
0, 2.5,
9.6, 19.2, 96, 192, 384, 768 or 1152 fmoles of P.a. RNase H2 (final
concentration of 0,
0.25, 0.96, 1.92, 9.6, 19.2, 38.4 76.8, or 115.2 nM in a 10 i.it reaction, or
0, 1.3, 5, 10,
50, 100, 200, 400, or 600 mU of the enzyme, respectively). 800 M
deoxynucleotides
and 200 nM of each primer were included in each reaction. 1.5 mM of
supplemental
MgC12 was added to each reaction, resulting in a final concentration of 3 mM
MgC12.
0.1 U of the Phire DNA polymerase was used in each amplification reaction and
SYBR Green (Molecular Probes, Eugene, OR, USA) was added to a final
concentration of 30,000 fold dilution (from the stock dye) to enable
detection. Three
types of blocked-cleavable primers were tested, including the RDDDDx, RDxxD,
and
the new RDDDDxxD designs. Sequences are shown in Table 89 below. Unmodified
rs4939827 For and rs4939827 Rev primers (SEQ ID NOs. 217 and 230) served as
the
control reaction. Blocked-cleavable For primers of the three designs were
individually
tested in combination with an unmodified Rev primer, including pairs:
rs4939827
T-For RDDDDx (SEQ ID NO. 235) with rs4939827 Rev (SEQ ID NOs. 217);
rs4939827 T-For RDxxD (SEQ ID NO. 273) with rs4939827 Rev (SEQ ID NOs. 217);
and rs4939827 T-For RDDDDxxD (SEQ ID NO. 285) with rs4939827 Rev (SEQ ID
NOs. 217). PCR was performed on a Bio-Rad CFX384TM Real Time System
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(Bio-Rad, Hercules, CA, USA) as follows: 98 C for 3 minutes followed by 40
cycles of
98 C for 10 seconds and 60 C for 30 seconds. All reactions were performed in
triplicate.
Table 89: Synthetic oligonucleotides employed in Example 40
Name Sequence SEQ ID NO.
rs4939827 Rev CTCACTCTAAACCCCAGCATT SEQ ID NO.
217
rs4939827 For CAGCCTCATCCAAAAGAGGAAA SEQ ID NO.
230
rs4939827
CAGCCTCATCCAAAAGAGGAAAcAGGA-SpC3 SEQ ID NO.
235
T-For RDDDDx
rs4939827
CAGCCTCATCCAAAAGAGGAAAuA(SpC3-SpC3)A SEQ ID NO.
273
T-For RDxxD
rs4939827
T-For
CAGCCTCATCCAAAAGAGGAAAuAGGA(SpC3-SpC3)C SEQ ID NO. 285
RDDDDxxD
DNA bases are uppercase and RNA bases are lowercase; SpC3 = C3 spacer
(propanediol)
[0616] Results are
shown in Tables 90 and 91. The RDDDDx blocked-cleavable
primer supported PCR with the high fidelity 3' -exonuclease Phire DNA
polymerase in
the absence of RNase H2, which suggests that the 3' -block is removed by the
3' -exonuclease proofreading activity of the high fidelity polymerase so that
PCR
proceeds without the need for RNase H2 cleavage/activation. Identical results
were
reported in Example 39 using the high fidelity DNA polymerase Phusion ,
suggesting
that similar behavior could be expected from all DNA polymerases of this
class. Also
similar to results from the prior Example, reduced amplification efficiency
was seen
when RNase H2 was added to the amplification reaction, suggesting that some
antagonism occurs between the high fidelity DNA polymerase and the RNase H2
enzymes with the RDDDDx design primer. The RDxxD blocked-cleavable primer did
not support PCR in the absence of RNase H2. Thus this design of primer cannot
readily
be unblocked by the 3' -exonuclease activity of the Phire DNA polymerase.
Primer
cleavage and PCR occurred in the presence of RNase H2. Lower efficiency PCR
was
seen using low doses of RNase H2 (100 mU per reaction) and near parity with
the
unblocked control primers was achieved using 400-600 mU RNase H2 per reaction.
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Therefore results for both the RDDDDx and RDxxD blocked-cleavable primers were
similar when using the two different high fidelity DNA polymerases, Phusion
and
Phire .
[0617] The new design
RDDDDxxD primer showed no amplification in the
absence of RNase H2, indicating that the high fidelity 3 '-exonuclease DNA
polymerase
was unable to remove the 3' -blocking effect present in this primer design.
Further,
good amplification efficiency was achieved using relatively low concentrations
of
RNase H2, indicating that there was no or minimal antagonism between the
polymerase
and the RNase H2 enzymes for this primer design.
Table 90: Cp values resulting from qPCR performed with an unmodified forward
primer and various reverse primers using a high-fidelity DNA polymerase.
0 mU 1.3 mU 5 mU 10 mU 50 mU
Primer SEQ ID
RNase H2 RNase H2 RNase H2 RNase H2 RNase H2
rs4939827 SEQ ID
23.0 23.2 23.0 23.3 22.6
For NO. 230
rs4939827
SEQ ID
T-For 23.7 >40 38.8 36.4 28.5
NO. 235
RDDDDx
rs4939827
SEQ ID
T-For >40 33.6 28.8 25.9 24.5
NO. 285
RDDDDxxD
Table 91: Cp values resulting from qPCR performed with an unmodified forward
primer and various reverse primers using a high-fidelity DNA polymerase.
0 mU 100 mU 200 mU 400 mU 600 mU
Primer SEQ ID
RNase H2 RNase H2 RNase H2 RNase H2 RNase H2
rs4939827 SEQ ID
23.0 23.2 23.0 23.3 22.6
For NO. 230
rs4939827
SEQ ID
T-For >40 29.1 25.9 24.4 23.3
NO. 273
RDxxD
[0618] We conclude that
both RDDDDxxD and RDxxD blocked-cleavable primer
designs support PCR when using high fidelity 3' -exonuclease DNA polymerase
enzymes. The RDDDDxxD design requires lower input RNase H2 concentrations
while the RDxxD requires higher input RNase H2 concentrations. This approach
enables use of the blocked-cleavable primer strategy for applications that
require high
fidelity DNA replication, such as DNA cloning, gene synthesis, or NGS.
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Additional Acknowledgements
[0619] All references, including publications, patent applications, and
patents,
cited herein are hereby incorporated by reference to the same extent as if
each reference
were individually and specifically indicated to be incorporated by reference
and were
set forth in its entirety herein.
[0620] The use of the terms "a" and "an" and "the" and similar referents in
the
context of describing the invention (especially in the context of the
following claims)
are to be construed to cover both the singular and the plural, unless
otherwise indicated
herein or clearly contradicted by context. The terms "comprising," "having,"
"including," and "containing" are to be construed as open-ended terms (i.e.,
meaning
"including, but not limited to,") unless otherwise noted. Recitation of ranges
of values
herein are merely intended to serve as a shorthand method of referring
individually to
each separate value falling within the range, unless otherwise indicated
herein, and each
separate value is incorporated into the specification as if it were
individually recited
herein. All methods described herein can be performed in any suitable order
unless
otherwise indicated herein or otherwise clearly contradicted by context. The
use of any
and all examples, or exemplary language (e.g., "such as") provided herein, is
intended
merely to better illuminate the invention and does not pose a limitation on
the scope of
the invention unless otherwise claimed. No language in the specification
should be
construed as indicating any non-claimed element as essential to the practice
of the
invention.
[0621] Preferred embodiments of this invention are described herein,
including the
best mode known to the inventors for carrying out the invention. Variations of
those
preferred embodiments may become apparent to those of ordinary skill in the
art upon
reading the foregoing description. The inventors expect skilled artisans to
employ such
variations as appropriate, and the inventors intend for the invention to be
practiced
otherwise than as specifically described herein. Accordingly, this invention
includes
all modifications and equivalents of the subject matter recited in the claims
appended
hereto as permitted by applicable law. Moreover, any combination of the
above-described elements in all possible variations thereof is encompassed by
the
invention unless otherwise indicated herein or otherwise clearly contradicted
by
context.
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