Note: Descriptions are shown in the official language in which they were submitted.
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Improved Selective Ligation and Amplification Assay
Technical Field
The present invention relates to assays for amplifying and identifying target
sequences of nucleic acids involving a combined ligation and amplification
protocol, and
the use of nanoliter sampling arrays to perform such assays.
Sack~round Art
Genetic variations are increasingly being linked to a multitude of disease
conditions and predispositions for disease, including cancer, multiple
sclerosis,
autoimmune diseases, cystic fibrosis, and schizophrenia. The ability to
identify genetic
variations rapidly and inexpensively will greatly facilitate diagnosis, risk
assessment, and
determination of the prognosis for such diseases and predispositions for these
diseases.
One possibility for identifying genetic variations involves combining
selective
ligation and amplification techniques, disclosed in U.S. Patent No. 5,593,840
to
Bhatnagar et al. and U.S. Patent No. 6,245,505 to Todd et al, both of which
are hereby
incorporated by reference herein. Both patents disclose the use of at least
three primers,
two of which are complementary to adjacent regions of the 3'-end of one strand
of a
target nucleic acid sequence which, after hybridization, can be ligated and
then extended.
In Todd et al., the third primer is a random sequence, complementary to the
random
sequence at the 3'-end of the downstream primer (that ligates to the upstream
primer) and
identical to the random sequence on the 5'-end of the first primer. In
Bhatnagar et al., the
third primer is complementary to the upstream primer, and also to the opposite
strand of
the target sequence. In both cases, there must be complementarity at the 3'-
end of the
third primer to allow amplification to occur.
A heat-stable polymerase is used to amplify the target nucleic acid sequence,
and
both the ligation and amplification reactions can be carned out in the same
reaction
mixture. An optional gap between the adjacent primers may be present, which
may be
filled by a polymerase to allow successful ligation of the adjacent primers.
Such a system
allows identification of genetic variability in target nucleic acid sequences,
and
identification of multiple alleles.
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Summary of the Invention
In a first embodiment of the invention, there is provided an improved assay of
the
type for amplifying a specific target nucleic acid sequence, wherein the
target sequence
comprises an internal SNP of interest, the assay being a selective ligation
and
amplification method of the type using a controlled-temperature reaction
mixture
including the target sequence, ligatable first and second primers having at
least a portion
substantially complementary to first and second segments of the target
sequence,
respectively, and a third primer that is substantially complementary to a
random sequence
l0 segment of the first and second primers, wherein the improvement comprises:
distinguishing in a single-tube reaction system between one or more SNPs in
one or more
target sequences of nucleic acid using two unique probes designed to hybridize
to the
target nucleic acid sequences with SNPs of interest, each hybridizable probe
having a
different fluorescent tag that is quenched until incorporation of the probe
into amplified
15 target nucleic acid product.
In some embodiments of the improved assay, of the type for amplifying a
specific
target nucleic acid sequence, wherein the target sequence comprises one or
more SNPs of
interest that are not at an end of the target sequence, the assay being a
selective ligation
and amplification method of the type using a thermocycled reaction mixture
including the
2o target sequence, a first primer having at least a portion of its 3'-end
substantially
complementary to a first segment at a first end of the target sequence, a
second primer
having at least a portion of its 5'-end substantially complementary to a
second segment at
a second end of the target sequence, the 5'-end of the second primer being
adjacent to or
within two to four bases of the 3'-end of the first primer wherein a
nucleotide
25 complementary to the SNP of the target sequence is present at either the 3'-
end of the first
primer or at the 5'-end of the second primer, and a third primer that is
substantially
complementary to a random sequence segment at the 3'-end of the second primer
and to a
substantially similar sequence at the 5'-end of the first primer, at least
four different
nucleotide bases, a thermostable polymerise and a thermostable ligase, wherein
the
30 improvement comprises distinguishing in a single-tube reaction system
between one or
more SNPs in one or more target sequences of nucleic acid using two unique
probes
designed to hybridize to the target nucleic acid sequences with SNPs of
interest, each
hybridizable probe having a different fluorescent tag that is quenched until
incorporation
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of the probe into amplified target nucleic acid product. The first
hybridizable probe with
first fluorescent tag has a unique random sequence that hybridizes to a first
amplified
target nucleic acid generated by the third primer from a ligated first primer-
second primer
product having a first SNP of interest on the 3'-end of the first primer, the
first
hybridizable probe thereby becoming incorporated into amplified opposite
strand target
nucleic acid product to give a first fluorescent signal. The second
hybridizable probe
with second fluorescent tag has a unique random sequence that hybridizes to a
second
amplified product generated by the third primer from a different ligated first
primer-
second primer product having a second SNP of interest on the 3'-end of the
first primer,
the second hybridizable probe thereby becoming incorporated into amplified
opposite
strand target nucleic acid product to give a second fluorescent signal.
In a preferred embodiment, the random sequences of the first and second
hybridizable probes are unique sequences, such that specific incorporation of
each of the
hybridizable probes into amplified target nucleic acid preferentially occurs
after ligation
of the first primer-second primer product having the particular SNP of
interest that the
hybridizable probe was designed to detect. Upon incorporation of the
hybridizable probe
into amplified product, fluorescence occurs, making detection of the amplified
product
distinguishable from non-specific background products. Additionally, the
random
sequence of the third primer is also a unique sequence, optimized for PCR to
reduce non-
specific amplified products that may be generated in the presence of human or
other
species chromosomes to a sufficiently low level that such non-specific
products do not
interfere with detection of amplified products having a SNP of interest.
Alternatively, the two hybridizable probes do not contain fluorescent tags,
but are
simply additional primers designed to distinguish different ligated products
having
different SNPs of interest. Detection of amplified product with a SNP of
interest is then
done using additional hybridizable probes, similar to the additional primers,
but are
developed in a manner not to interfere with amplification. These hybridizable
probes
have a fluorescent tag, or alternatively, each have a different fluorescent
tag, and upon
hybridizing to amplified product, fluoresce, thereby allowing detection of
amplified
product.
In another embodiment of the invention there is provided an improved assay of
the type for amplifying a specific target nucleic acid sequence, wherein the
target
sequence comprises a SNP of interest that is not at an end of the target
sequence, the
assay being a selective ligation and amplification method of the type using a
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thermocycled reaction mixture including the target sequence, a first primer
having at
least a portion of its 3'-end substantially complementary to a first segment
at a first end of
the target sequence, a second primer having at least a portion of its 5'-end
substantially
complementary to a second segment at a second end of the target sequence, the
5'-end of
the second primer being adjacent to the 3'-end of the first primer wherein a
nucleotide
complementary to the SNP of the target sequence is present at either the 3'-
end of the first
primer or at the 5'-end of the second primer, and a third primer that is
substantially
complementary to a random sequence segment at the 3'-end of the second primer
and to a
substantially similar sequence at the 5'-end of the first primer, at least
four different
nucleotide bases, a thermostable polymerise and a thermostable ligase, wherein
the
improvement comprises homogeneously detecting amplified target sequence using
a dye
specific for binding to double-stranded (ds) DNA that fluoresces upon binding
target
sequence. In a preferred embodiment, the random sequence of the third primer
is a
unique sequence, optimized for PCR such that no non-specific products are
generated in
the presence of human or other species chromosomes. In some embodiments,
primers
may be affixed on, within or under a biocompatible material such as a wax-like
coating
on the surface of the through-holes by drying the primers after application to
the through-
holes, wherein the biocompatible material may comprise, for example, a
polyethylene
glycol (PEG) material.
Alternatively, assays in accordance with the present invention may. use a
thermostable polymerise that lacks 5' to 3' exonuclease activity, or a
thermostable
polymerise that lacks 3' to 5' exonuclease activity, or a thermostable
polymerise that
lacks both 5' to 3' and 3' to 5' exonuclease activity. Examples of
thermostable
polymerises which lack 5' to 3' exonuclease activity include Stoffel fragment,
IsisTM
DNA polymerise, PyraTM exo(-) DNA polymerise, and Q-BioTaqTM DNA polymerise.
Examples of thermostable polymerises which lack 3' to 5' exonuclease activity
include
Taq polymerise, SurePrimeTM Polymerise, and Q-BioTaqTM DNA polymerise. An
example of a thermostable polymerise which lacks both 5' to 3' and 3' to 5'
exonuclease
activity is Q-BioTaqTM DNA polymerise. Suitable dyes include SYBR°
Green I and
SYBR° Green II, YOYO"-1, TOTO°-1, POPO°-3, ethidium
bromide, or any other dye
that allows rapid, sensitive detection of amplified target nucleic acid
sequence using
fluorescence.
In another embodiment, there is provided a nanoliter sampling array comprising
a
first platen having at least one hydrophobic surface and having a high-density
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microfluidic array of hydrophilic through-holes. In this particular
embodiment, each
through-hole contains at least a first primer having at least a portion of its
3'-end
substantially complementary to a first segment at a first end of a potential
nucleic acid
target sequence a second primer having at least a portion of its 5'-end
substantially
complementary to a second segment at a second end of the potential nucleic
acid target
sequence, the 5'-end of the second primer being adjacent to the 3'-end of the
first primer
upon binding to the potential nucleic acid target sequence.
In addition, the sampling array may further comprise a second platen having at
least one hydrophobic surface and having a high-density microfluidic array of
hydrophilic
through-holes wherein the first and second platen are fixedly coupled such
that the
through-holes of each are aligned.
In yet another embodiment, there is provided a method of identifying a SNP in
a
target sequence of nucleic acid, the method comprising providing a first
sample platen
having a high-density microfluidic array of through-holes, each through-hole
having a
first primer having at least a portion of its 3'-end substantially
complementary to a first
segment at a first end of the target sequence, a second primer having at least
a portion of
its 5'-end substantially complementary to a second segment at a second end of
the target
sequence, the,5'-end of the second primer being adjacent to the 3'-end of the
first primer,
and third primer that is substantially complementary to a random sequence
segment at the
3'-end of the second primer and to the 5'-end of the first primer, introducing
a sample
containing a target sequence of nucleic acid having a SNP of interest to the
array,
introducing reagents to the through-holes in the array, the reagents including
a
thermostable polymerase, a thermostable ligase, and at least four different
nucleotide
bases, thermocycling the array, and detecting amplified target sequence. In a
preferred
embodiment, primers 1 and 2 are designed with a possible match to the target
strand SNP
located at either the 3'-end of the 5' primer (the first primer) or located at
the 5'-end of the
3' primer (the second primer). When the first and second primers hybridize to
the target
strand, adjacent to each other and flanking the SNP, ligation of the primers
only occurs if
there is a successful match to the SNP by one of the primers. In this way, the
ligation is
3o selective and so selective amplification of the desired target sequence
containing the SNP
of interest also occurs. As described above, in some embodiments, primers may
be
affixed on, within or under a biocompatible material such as a wax-like
coating on the
surface of the through-holes by drying the primers after application to the
through-holes,
wherein the biocornpatible material may comprise, for example, a polyethylene
glycol
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(PEG) material.
In addition, the method of identifying a SNP in a target sequence of nucleic
acid
may additionally comprise using a thermostable polymerase that lacks 5' to 3'
exonuclease activity, and detecting amplified target sequence using a dye
specific for
binding to double-stranded (ds) DNA that fluoresces upon binding target
sequence.
Alternatively, detecting may comprise using first primers and second primers
designed to
generate amplified target sequences with differential melting curves to
distinguish
individual amplified target sequences by differences in melting temperatures
(Tms), or
may comprise using a probe specific for hybridizing across a ligation junction
formed
l0 between the first primer and second primer after binding to the target
sequence wherein
the probe specific for hybridizing across the ligation junction has a
fluorescent group 'and
a fluorescence-modifying group, or using a probe containing a fluorescent
group and a
fluorescence-modifying group specific for hybridizing to a region of the
target sequence
wherein upon extension of the probe, the fluorescence-modifying group is
excised and
i5 . the fluorescent group fluoresces. Additionally, detection may be done
using a probe
specific for hybridizing to any unique sequence in the amplified target
nucleic acid, the
probe having a fluorescent group and a fluorescence-modifying group such that
the upon
' hybridization the probe fluoresces, allowing detection of the amplified
target nucleic acid.
Other means of detection comprise the use of amplification primers which match
2o the random sequence of primer 2 wherein the primers are labeled with a
fluorescent group
that only fluoresces when incorporated in a PCR product, similar to LuxTM
primers
known in the art. In such an embodiment, the fluorescent group is quenched by
secondary structure before incorporation into double-stranded product, such
that prior to
incorporation, a sequence in the primer/probe binds to a complementary
sequence in the
25 primer/probe containing the fluorescent group, quenching the fluorescent
group. In
another embodiment, primers 1 and 2 are Fluorescence Resonance Energy
Transfter
(FRET) partners, such that when hybridized to the amplified target sequence,
produced
only after primers 1 and 2 are ligated and amplified, they fluoresce.
Yet another embodiment provides a kit for use in identification of amplified
target
30 nucleic acid sequences, the kit comprising a sample platen having one
hydrophobic
surface and having a high-density microfluidic array of hydrophilic through-
holes. In the
array of the kit, each through-hole contains at least a first primer having at
least a portion
of its 3'-end substantially complementary to a first segment at a first end of
potential
nucleic acid target sequence, and a second primer having at least a portion of
its 5'-end
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substantially complementary to a second segment at a second end of the
potential nucleic
acid target sequence, the 5'-end of the second primer being adjacent to the 3'-
end of the
first primer upon binding to the potential nucleic acid target sequence. The
kit also
comprises a reagent platen having a high-density microfluidic array of through-
holes,
each through-hole containing a third primer that is substantially
complementary to a
random sequence segment at the 3'-end of the second primer and to a
substantially similar
sequence at the 5'-end of the first primer, at least four different nucleotide
bases, a
thermostable polymerase, and a thermostable ligase. In the kit of this
embodiment, the
reagent platen has a structural geometry that corresponds to the sample
platen, thereby
allowing delivery of reagent components and target nucleic acid sample to the
primers in
the sample platen. In other embodiments, the thermostable polymerase may lack
5' to 3'
exonuclease activity.
Brief Descriution of the Drawings
The foregoing features of the invention will be more readily understood by
reference to the following detailed description, taken with reference to the
accompanying
drawings, in which:
Fig. 1-A, shows a double-stranded target nucleic acid sequence with a single
nucleotide polymorphism (SNP).
2o Fig. 1-B1 shows a denatured 3' to 5' target strand with primers 1 and 2
hybridized
adjacent to the SNP, the base complementary to the SNP located at the 3'-end
of primer 1
and shows the random sequence (RS) of primer 3 hybridized to 3'-end of the
ligated Pl-
P2 product.
Fig. 1-B2 shows a denatured 3' to 5' target strand with primers 1 and 2
hybridized
adjacent to the SNP, the base complementary to the SNP located at the 5'-end
of primer 2
and shows the random sequence (RS) of primer 3 hybridized to 3'-end of the
ligated Pl-
P2 product.
Fig. 1-C shows a denatured 5'-3' target nucleic acid strand being extended by
un-
ligated primer P1.
3o Fig. 2-A shows a double-stranded target nucleic acid sequence with a single
nucleotide polymorphism (SNP).
Fig. 2-B shows primers P1 and P2 hybridized to a denatured target strand of
nucleic acid (the 3' to 5' strand) wherein a base complementary to the SNP in
the target
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strand is present on the 3'-end of Pl, and each of primers Pl and P2 contain a
random
sequence at their 5'-end and 3'-end, respectively.
Fig. 2-C shows ligated Pl-P2 product being amplified by primer P3 to produce
P3-amplified product.
Fig. 2-D shows P3-amplified product being amplified by primer P3 to produce
P3-ampflied product (3' to 5').
Figs. 2-El and 2-E2 show exponential amplification of P3-amplified product (5'
to 3') and P3-amplified product (3' to 5'), respectively.
Fig. 3 shows a cartoon of the dye SYBR~ Green I binding to double-stranded
amplified target nucleic acid and fluorescing.
Fig. 4-A shows upstream primer A-B, downstream primer C-D, and general
extension primer D' with a target nucleic acid having a SNP of interest in a
single-tube
reaction system for distinguishing between one or more SNPs in one or more
target
sequences of nucleic acid, the single-tube reaction system also containing
upstream
primer F-E and a second nucleic acid target with a second SNP of interest.
Fig. 4-B shows ligation of upstream primer A-B with downstream primer C-D
when successful match-up occurs with a first SNP of interest in a first target
sequence of
nucleic acid, and also shows ligation of upstream primer F-E with down stream
primer C-
D when successful match-up occurs with a second SNP of interest in a second
target
2o sequence of nucleic acid present in the same tube.
Fig. 4-C shows extension of ligation products A-B-C-D and F-E-C-D by general
extension primer D'.
Fig. 4-D shows hybridization of hybridizable probe A with fluorescent tag 1 to
extended product A'-B'-C'-D' and hybridization of hybridizable probe F with
fluorescent
tag 2 to extended product F'-E'-C'-D'.
Fig. 4-E shows incorporation and amplification of a first target nucleic acid
with a
first SNP of interest by hybridizable probe A, triggering fluorescence of
fluorophore 1 in
a first amplified product, and incorporation and amplification of a second
target nucleic
acid with a second SNP of interest by hybridizable probe F, triggering
fluorescence of
3o fluorophore 2 in a second amplified product.
Fig. 5A shows upstream primer A-B, downstream primer C-D, and general
extension primer D' with a target nucleic acid having a SNP of interest in a
single-tube
reaction system for distinguishing between one or more SNPs in one or more
target
sequences of nucleic acid , the single-tube reaction system also containing
upstream
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primer F-E and a second nucleic acid target with a second SNP of interest in
an
alternative embodiment of the single-tube reaction system of Fig. 4.
Fig. 5S shows ligation of upstream primer A-B with downstream primer C-D
when successful match-up occurs with a first SNP of interest in a first target
sequence of
nucleic acid, and also shows ligation of upstream primer F-E with down stream
primer C-
D when successful match-up occurs with a second SNP of interest in a second
target
sequence of nucleic acid present in the same tube.
Fig. 5C shows extension of ligation products A-B-C-D and F-E-C-D by general
extension primer D'.
Fig. SD shows hybridization of primer A with no fluorescent tag to extended
product A'-B'-C'-D' and hybridization of primer F with no fluorescent tag to
extended
product F'-E'-C'-D'.
Fig. SE shows amplification of a first target nucleic acid with a first SNP of
interest by primer A to produce a first amplified product, and amplification
of a second
target nucleic acid with a second SNP of interest by primer F, to produce a
second
amplified product.
Fig. 5F shows a competing reaction to the amplification reactions in Fig. SE,
wherein incorporation and low-efficiency production of a first target nucleic
acid with a
first SNP of interest is carried out by hybridizable probe A, triggering
fluorescence of
2o fluorophore 1 in a first product, thereby allowing detection of a first
amplified target
nucleic acid, and wherein incorporation and low-efficiency production of a
second target
nucleic acid with a second SNP of interest is carried out by hybridizable
probe F,
triggering fluorescence of fluorophore 2 in a second product, thereby allowing
simultaneous detection of a second amplified target nucleic acid.
Fig. 6 shows a typical high-density sample array of through-holes according to
the
prior art.
Detailed Description of Specific Embodiments
Definitions. As used in this description and the accompanying claims, the
following terms shall have the meanings indicated, unless the context
otherwise requires:
"Target nucleic acid," "target nucleic acid sequence" or "potential target
nucleic
acid sequence" means any prokaryotic or eukaryotic DNA or RNA including from
plants,
animals, insects, microorganisms, etc. It may be isolated or present in
samples which
contain nucleic acid sequences in addition to the target nucleic acid sequence
to be
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amplified. The target nucleic acid sequence may be located within a nucleic
acid
sequence which is longer than that of the target sequence. The target nucleic
acid
sequence may be obtained synthetically, or enzymatically, or can be isolated
from any
organism by methods well known in the art. Particularly useful sources of
nucleic acid
are derived from tissues or blood samples of an organism, nucleic acids
present in self-
replicating vectors, and nucleic acids derived from viruses and pathogenic
organisms
such as bacteria and fungi. Also particularly useful are target nucleic acid
sequences
which are related to disease states, such as those caused by chromosomal
rearrangement,
insertion, deletion, translocation and other mutation, those caused by
oncogenes, and
those associated with cancer.
"Selected" means that a target nucleic acid sequence having the desired
characteristics is located and probes are constructed around appropriate
segments of the
target sequence.
"Probe" or "primer" has the same meaning herein, namely, a nucleic acid
oligonucleotide sequence which is single-stranded. The term oligonucleotide
includes
DNA, RNA and PNA.
A probe or primer is "substantially complementary" to the target nucleic acid
sequence if it hybridizes to the sequence under renaturation conditions so as
to allow
target-dependent ligation or extension. Renaturation depends on specific base
pairing
between A-X (where X is T or U) and G-C bases to form a double-stranded duplex
structure. Therefore, the primer sequences need not reflect the exact sequence
of the
target nucleic acid sequence. However, if an exact copy of the target sequence
is
desired, the primer should reflect the exact sequence. Typically, a
"substantially
complementary" primer will contain at least 70% or more bases which are
complementary to the target nucleic acid sequence. More preferably 80% or the
bases are
complementary, and still more preferably more than 90% of the bases are
complementary. Generally, the primer should hybridize to the target nucleic
acid
sequence at the end to be ligated or extended to allow target-dependent
ligation or
extension.
3o Primers may be RNA or DNA and may contain modified nitrogenous bases which
are analogs of the normally incorporated bases, or which have been modified by
attaching
labels or linker arms suitable for attaching labels. Inosine may be used at
positions where
the target sequence is not known, or where it may be degenerate. The
oligonucleotides
should be sufficiently long to allow hybridization of the primer to the target
sequence and
to
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to allow amplification to proceed. They are preferably 15 to 50 nucleotides
long, more
preferably 20-40 nucleotides long, and still more preferably 25-35 nucleotides
longs. The
nucleotide sequence of the primers, both content and length, will vary
depending on the
target sequence to be amplified.
It is contemplated that a primer may comprise one or more oligonucleotides
which
comprise substantially complementary sequences to the target nucleic acid
sequence.
Thus, under less stringent conditions, each of the oligonucleotide primers
would
hybridize to the same segment of the target sequence. However, under
increasingly
stringent conditions, only that oligonucleotide primer which is most
complementary to
1o the target nucleic acid sequence will hybridize. The stringency of the
hybridization
conditions is generally known to those in the art to be dependent on
temperature, solvent,
ionic strength, and other parameters. One of the most easily controlled
parameters is
temperature and since conditions for selective ligation and amplification are
similar to
those for PCR reactions, one skilled in the art can determine the appropriate
conditions
15 required to achieve the level of stringency desired.
Primers suitable for use in the present invention may be derived from any
method
known in the art, including chemical or enzymatic synthesis, or by cleavage of
larger
nucleic acids using non-specific nucleic acid-cleaving chemicals or enzymes,
or by using
site-specific restriction endonucleases.
2o In order for the ligase of the present invention to ligate the primers
together, the
primers used are preferably phosphorylated at their 5'-ends. This may be
achieved by any
known method in the art, including use of T4 polynucleotide kinase. The
primers may be
phosphorylated in the presence of unlabeled or radiolabeled ATP.
The term "four different nucleotide bases" means deoxythymidine triphosphate
25 (dTTP), deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate
(dCTP); and
deoxyguanosine triphosphate (dGTP) when the context is DNS, and means uridine
triphosphate (UTP), adenosine triphosphate (ATP), cytidine triphosphate (CTP),
and
guanosine triphosphate (GTP) when the context is RNA. Alternatively, dUTP,
dITP
(deoxyinosine triphosphate), rITP (riboinosine triphosphate) or any other
modified base
3o may replace any one of the four nucleotide bases or may be included along
with the four
nucleotide bases in the reaction mixture so as to be incorporated into the
amplified strand.
The amplification steps are conducted in the presence of at least the four
deoxynucleoside
triphosphates (dATP, dCTP, dGTP and dTTP) or a modified nucleoside
triphosphate to
produce a DNA strand, or in the present of the four ribonucleoside
triphosphates (ATP,
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CTP, DTP and UTPO or a modified ribonucleoside triphosphate to produce an RNA
strand from extension of the primer.
The term "adequate detection of desired amplified product" means detection of
at
least a two-fold increase in desired amplified target strand over competing
linear
products.
The term "target sequence detectable above linearly amplified product" means
that target sequence is amplified at least two-fold over that of competing
linearly
amplified non-ligated primer product.
The term "random sequence" as used herein means a sequence unrelated to the
to target sequence or chosen not to bind to the target sequence or other
sequences that might
be expected to be present in a test sample.
The term "biocompatible material" as used herein means that the material does
not prevent biological processes, such as enzymatic reactions, from occurring
when the
biocompatible material is present, does not eliminate biological activity or
required
secondary, tertiary or quaternary. structure of biomolecules, such as nucleic
acids and
proteins, and in general, is not incompatible with biological processes and
molecules.
The term "first and second primers being ligatable upon binding to the nucleic
acid target sequence" as used herein, means that the first and second primers
bind
potential target nucleic acid with the 3'-end of the first primer adjacent to,
or within about
a one- to four- nucleotide gap of, the 5'-end of the second primer, such that
subjecting the
hybridized first and second primers to appropriate enzymatic or non-enzymatic
ligation
conditions, including optionally adding a polymerase activity to fill in the
gap, allows the
first and second primers to be enzymatically or non-enzymatically ligated into
a single
ligated nucleic acid product.
The term "polymerase" as used herein, means any oligomer synthesizing enzyme,
including polymerases, helicases, and other protein fragments capable of
polymerizing
the synthesis of oligomers.
The term "controlled-temperature reaction mixture" as used herein means, any
reaction mixture wherein temperature is controlled by means of a thermocycle
apparatus,
an isothermal apparatus, or any other means known to allow temperature control
of a
reaction, including temperature-controllable environments such as water, oil
and sand
baths, incubation chambers, etc.
The general assay for identifying single-nucleotide polymorphisms (SNPs) that
are not at an end of a target sequence through detection of amplified target
sequences,
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using a dye specific for binding to double-stranded DNA that fluoresces upon
binding
target sequence according to the present invention, is described below and
illustrated in
Figs. 1-5. The assay can be performed in a single-reaction chamber or
container, in a
series of reaction chambers or containers, in a nanoliter sampling array
having a high-
s density microfluidic array of hydrophilic through-holes, or in a kit
comprising such an
array plus necessary reagents. Detection may be homogeneous, and may employ a
polymerase that lacks 5' to 3' exonuclease activity, or a polymerase that
lacks 3' to 5'
exonuclease activity, or a polymerase that lacks both exonuclease activities.
The assay can be done with three (P1, P2, P3) or more (A-B, C-D, F-E, D')
1o primers, and is able to detect one or more SNPs in a single target
simultaneously. In
some versions of the assay, the nucleotide complementary to the SNP of the
target
nucleotide is present at or near the 5'-end of the second primer P2. In other
versions,.the
nucleotide complementary to the SNP of the target nucleotide is present at or
near the 3'-
end of the first primer Pl. In other versions, there are more than one first
primers and
15 second primers, these first and second primers designed to generate
amplified target
sequences having different melting temperatures, such that the assay is able
to distinguish
individual amplified target sequences because of their individual, and
distinct, Tms.
Assays may be done with first and second primers that contain degenerate base-
I
pairing positions which allow hybridization of variable regions in target
sequences
20 . adjacent to the SNP, in this way expanding the general flexibility and
utility of the assay.
Primers 1 and 2, corresponding to 5' and 3' ligation primers, respectively,
may be
fully or partially complementary to the target sequence. Primer 3 is a generic
primer
complementary to a random sequence (RS) located at the ends of primers 1
and/or 2 (see
Figs. 1 and 2). The 3' end of primer 1 and the 5' end of primer 2 can
hybridize either
25 immediately adjacent to each other on the target sequence or can hybridize
on the target
sequence with a separation, or gap, or one or more nucleotides between them
(see Figs. 1-
2 and 4-5). Primers 1 or 2 contain a variant base at or near the 3' end (P1)
or the 5' end
(P2) to enable the primers to bind to SNPS in a target sequence (see Figs. 1-
2). There is
also a 3'-hydroxyl group on P2, to facilitate enzymatic or non-enzymatic
ligation between
3o P1 and P2 or polymerase extension prior to ligation (to fill in any gap).
In addition, the
5'-end of P2 can be modified to prevent undesirable ligation to fragments
other than P1.
Similarly, the 5'-end of Pl is phosphorylated to facilitate ligation with P2,
and the
3' end of P1 may be modified to prevent ligation to fragments other than P2.
Amplification of target nucleic acid is illustrated in Figs. 1 and 2.
Temperature is used to
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denature and anneal target nucleic acid and primers, as required, to allow
selective
extension of ligation of primers Pl and P2.
Detection of single-stranded ligation product is carried out using several
strategies, some employing a dye specific for binding to double-stranded DNA
that is
generated either using hybridization probes which hybridize to single-stranded
amplified
product, or generated after extension and amplification of both the sense and
non-sense
strands of the ligation product. Other detection strategies employ molecular
beacons
attached to hybridizable probes. And still other detection strategies employ
the use of
FRET pairs on hybridizable probes. In some assays, the fluorescent dye is
merely added
1o to the reaction mixture, and change in fluorescence intensity is monitored
to detect ligated
product. In other assays, hybridizable probes are added after generation of
ligation
product which are specific to the ligation product, and which also contain a
molecular
beacon, or a fluorescent group and a fluorescence-modifying group. The
hybridizable
probe may bind to extended ligation product, remaining quenched by the
fluorescence-
modifying group until extended into amplified product, whereupon the
fluorescent group
fluoresces and amplified target sequence is detected (see Fig. 4), or the
hybridizable
probe may be specific for hybridizing across the ligation junction, wherein
the probe is
again quenched until after hybridizing (see Fig. 5). In the assay illustrated
in Fig. 4, one
or more hybridizable probes may be used, each having a distinct fluorophore
and unique
l
2o sequence that hybridizes to and amplifies each of one or more target
nucleic acid
sequences, thereby allowing multiple SNPs to be detected in a single-tube
reaction
system.
Any of the assays may also be carried out in a nanoliter sampling array. The
nanoliter array may comprise one or more platens having at least one
hydrophobic
surface and a high-density microfluidic array of hydrophilic through-holes.
The inner
surfaces of the through-holes may be coated with a biocompatible material such
as a wax-
like polyethylene glycol material, or other biocompatible material. Primers
may be
applied into the through-holes and then dried, either before or after
application of the
biocompatible material coating, thereby affixing the primers on, within or
under the
3o biocompatible material. Target nucleic acids and reagents for processes
used in the
selective ligation and amplification assay can be loaded in liquid form into
the sample
through-holes using capillary action, with typical volumes of the sample
through-holes
being in the range of from 0.1 picoliter to 1 microliter. The interior
surfaces of the
through-holes may also have a hydrophilic surface or be coated with a porous
hydrophilic
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material, or as described above, be coated with a biocompatible material such
as PEG, to
enhance the drawing power of the sample through-holes, attract liquid sample
and aid in
loading.
Kits for performing the assay may also be prepared, comprising one or more
sample platen as described, the primers being affixed within the hydrophilic
sample
through-holes of the microfluidic array, and also comprising reagents required
for the
selective ligation and amplification assay. Target nucleic acid sequences) can
then be
added as desired to perform the assay. If not already provided with the kit,
enzymes
required to carry out the ligation and amplification reactions can also be
added along with
l0 the target nucleic acid sequence(s).
. E~PLES
Example 1. Homogeneous detection of amplified target sequence .
Homogeneous detection of amplified target sequences may be carried out using a
dye specific for binding to double-stranded DNA or RNA. Primers Pl and P2,
upstream
and downstream primers, respectively, do not participate in amplification of
target
sequence, but rather, are responsible for identifying the target sequence
containing a SNP.
When either primer P1 or P2 contains a match to the SNP of interest in the
target
sequence, ligation of P1 and P2 occurs, and then primer P3, the general
extension primer,
amplifies the Pl-P2 product. Consequently, concentrations of primers 1 and 2
are
preferably optimized and adjusted to not interfere with exponential
amplification of the
target sequence such that only linear amplification of competing non-target
sequences
occurs. Examples of ds-DNA- and/or RNA-specific dyes that may be used include
SYBR~ Green I and SYBR° Green II, YOYO°-1, TOTO~-1, POPO~-3
(see Appendix A,
attached hereto), ethidium bromide (EtBr) and any other dye providing adequate
sensitivity and ease of detection of desired amplified product.
In a particular embodiment, a sample target sequence of,nucleic acid,
optionally
containing a single nucleotide polymorphism, is mixed with at least three
primers - a first
upstream primer having at least a portion of its 3'-end substantially
complementary to a
first segment at a first end of the target sequence, a second downstream
primer having at
least a portion of its 5'-end substantially complementary to a second segment
at a second
end of the target sequence, the 5'-end of the second primer being adjacent to
the 3'-end of
the first primer wherein a nucleotide complementary to the SNP of the target
sequence is
present at either the 3'-end of the first primer or at the 5'-end of the
second primer, and a
CA 02549849 2006-06-02
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third general extension primer that is substantially complementary to a random
sequence
segment at the 3'-end of the second primer and to a substantially similar
sequence at the 5'-
end of the first primer. Additionally, at least four different nucleotide
bases, a
thermostable polymerise and a thermostable ligase are included in the reaction
mixture,
the thermostable polymerise preferably one that lacks 5' to 3' exonuclease
activity, such as
the Stoffel Fragment (see Appendix B, attached hereto). Examples of other
thermostable
polymerises which lack 5' to 3' exonuclease activity include IsisTM DNA
polymerise,.
PyraTM exo(-) DNA polymerise, and Q-BioTaq DNA polymerise (see Appendix C,
attached hereto). Alternatively, the assay may use a thermostable polymerise
that lacks 3'
to to 5' exonuclease activity, or a thermostable polymerise that lacks both 5'
to 3' and 3' to 5'
exonuclease activity. Examples of thermostable polymerises which lack 3' to 5'
exonuclease activity include Taq polymerise, SurePrimeTM Polymerise, and Q-
BioTaqTM
DNA polymerise (id.). An example of a thermostable polymerise which lacks both
5' to
3' and 3' to 5' exonuclease activity is Q-BioTaq DNA polymerise (id.).
Addition of a dye
specific for ds-DNA such as SYBR~ Green I, or specific for RNA such as SYBR~
Green
II, allows detection of amplified product, by monitoring fluorescence emission
of dye-
bound nucleic acid product at 520 nm(see Appendix D, attached hereto).
As can be seen in Figure 1-A, a target nucleic acid may contain a SNP within
the
target sequence. Upon denaturation, Primer 1 (P1) and Primer 2 (P2) bind to
the 3' to 5'
2o strand of the target sequence, adjacent to the SNP. There may be a gap of
several
(approximately 2-4) bases between the 3'-end of P1 and the 5'-end of P2, or
there may be
no gap. In Figure 1-B1, the base complementary to the SNP of the target
sequence is at
the 3'-end of P1. Alternatively, the base complementary to the SNP of the
target
sequence may be at the 5'-end of P2, as shown in Fig. 1-B2. The third primer
(P3)
contains a random sequence (RS) complementary to the random sequence of the 3'-
end of
P2, such that after ligation of P1 and P2, P3 binds and extends the ligated
primer product,
thereby amplifying the complementary strand (5'-3' strand) of the target
sequence. As
discussed above, a competing reaction may occur, such that primer P3 binds to
primer P2
and extends this sequence to produce a linear product based on the P2
sequence.
Preferably, concentrations of primers P1 and P2 are adjusted to minimize the
competing
linear reaction. As shown in Figure 1-C, un-ligated primer Pl extends the 3' -
5' strand of
the target sequence.
In another, preferred embodiment shown in Figure 2 (A - E), the first primer
(P1)
also has a random sequence at the 5'-end. When a primer containing the
complement to
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the SNP, either Pl on its 3'-end or P2 on its 5'-end (see Fig. 1-B), binds to
the target
strand (see Fig. 2-B), primers P1 and P2 are ligated, and the third primer
(P3) then binds
to the 3'-end of the ligated Pl-P2 product and produces the (3' to 5') P3-
amplified strand
(Fig. 2-C). At this point, primer P3 now also binds to the (3' to 5') P3-
amplified product
and produces the other (5' to 3') amplified product (see Fig. 2-D). Both
target strands
have now been produced, and can go on to yield exponentially amplified target
sequence
(Fig. 2-E1 and 2-E2). Additionally, detection with a fluorescent dye, such as
SYBR~
Green I (SGI) may be done at temperatures above the Tm of the linear product,
i.e., any
product produced non-exponentially, thereby removing competing signal from any
dye
to bound to linear product. SYBR~ Green I and other dyes that bind to double-
stranded
nucleic acids do not bind to nucleic acids above their Tms because at those
elevated
temperatures, the nucleic acids are denatured. As seen in the cartoon of
Figure 3, a dye
such as SYBR° Green I binds to double-stranded amplified target nucleic
acid with a
concomitant laxge increase in fluorescence. Although SGI is shown in Figure 3
as
intercalating into the amplified target ds-nucleic acid, nothing in the figure
is intended to
suggest either an actual structure, or actual mode of binding, for SGI with ds-
nucleic
acids.
Alternatively, the use of molecular beacon probes, having a fluorescent group
on
one end and a fluorescence-quenching group on the other, may be used. In this
system,
the molecular beacon remains quenched until being bound to amplified product
(see, for
example, Appendix E, attached hereto) because the molecular probe is typically
in a
hairpin conformation with the fluorescent group in close proximity to the
fluorescence-
quenching group, until the probe binds to the target amplified product
(causing the
hairpin structure to unfold, separating the fluorescent group from the
quenching group).
Examples of fluorescence-quenching groups appropriate for embodiments of the
present
invention include the dark quencher dabcyl, and the EclipseTM Quencher from
Epoch
(id.). Examples of appropriate fluorescent groups that may be used in
accordance with
the present invention include Epoch's Yakima YellowTM and Redmond RedTM (id.),
and
any other appropriate fluorescent dye whose fluorescence may be quenched to an
appropriately positioned quencher molecule.
In another embodiment, real-time amplification may be measured using a
TaqMan" probe that is homologous to an internal sequence of the target nucleic
acid, and
having a fluorogenic 5'-end and a quencher 3'-end. During PCR amplification
and
extension, the quencher molecule is removed from the probe by 5'-exonuclease
activity,
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WO 2005/059178 PCT/US2004/041480
releasing the fluorescent reporter molecule from close proximity to the
quencher
molecule on the 3'-end of the probe, thereby producing an increase in
fluorescence
emission as amplified product is produced (see Appendices F and G, attached
hereto). In
this system, a polymerase having 5' to 3' exonuclease activity is required.
Another embodiment utilizes a detection method for real-time amplification
measurement that involves the use of a pair of amplification primers, one of
which
matches the random sequence of primer 2. One of these primers in the pair is
labeled with
a fluorescent group that only fluoresces when incorporated into a PCR product,
similar to
LuxTM primers known in the art (see Appendix H, attached hereto). In such an
1o embodiment, the fluorescent group is quenched by secondary structure before
incorporation into double-stranded product, such that prior to incorporation,
a sequence in
the primer/probe binds to a complementary sequence in the primer/probe
containing the
fluorescent group, quenching the fluorescent group. In another embodiment,
primers 1
and 2 are FRET partners, such that when hybridized to the amplified target
sequence,
. produced only after primers 1 and 2 are ligated and amplified, they
fluoresce (see
Appendices E and also A) and thus permit detection of amplified target
sequence. In a
preferred embodiment, fluorescence detection would be carned out above the
either the
Tm for primer Pl, or above the Tm for primer P2, or alternatively be carried
out above the
Tms of both primers Pl and P2, to avoid background signal from possible
hybridization of
P1 and/or P2 to amplified target.
In another embodiment, primer may be designed to exponentially amplify target
nucleic acid products that are distinguishable by an increase or decrease in
melting
temperature (Tm), wherein the exponentially amplified target sequence is
either stabilized
as indicated by an increase in Tm or de-stabilized, as indicated by a decrease
in Tm,
relative to the melting temperatures of linearly produced non-target product
produced
from non-ligated primers. Variability in the random sequence, or elsewhere in
the
primers, may be used to produce such exponentially amplified target nucleic
acid
sequence distinguishable by melting temperature from the linear product.
In another embodiment, a probe specific for hybridizing across the ligation
junction formed after ligation of the first and second primers may be used.
Such a probe
may have a hairpin conformation with a fluorescent reporter group on one end
and a
fluorescence-quenching group on the other end whereby no fluorescence occurs
when the
probe is not bound across the ligation junction. By optimizing reaction
(conditions, such
as temperature and/or ionic strength, the hairpin would be stabilized by
binding across the
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WO 2005/059178 PCT/US2004/041480
ligation junction, whereupon fluorescence would occur and emission could be
monitored
to detect amplified product.
Example 2. Single-tube reaction system for distinguishing SNPs
One preferred embodiment of the present invention is the single-tube reaction
system shown in Figure 4. Similar to the embodiments shown in Figures 1 and 2
and
discussed above in Example 1, a three-primer system is utilized to identify a
SNP of
interest in a target sequence of nucleic acid. Again, there is an upstream
primer and a
downstream primer that bind to the target nucleic acid, flanking the SNP of
interest. The
l0 3'-end of the upstream primer may be directly adjacent to the 5'-end of the
downstream
primer, or there may be a gap of between about 1 to 4 bases between the 3'-end
of the
upstream primer and the 5'-end of the downstream primer. Either the 3'-end of
the
upstream primer or the 5'-end of the downstream primer may contain the
complement to
the SNP of interest in the target nucleic acid.
Unlike the embodiments shown in Figures 1 and 2, however, the single-tube
reaction system allows simultaneous single-tube identification and distinction
between
one or more SNPs of interest in one or more target nucleic acid sequences of
interest.
This is accomplished by using unique sequences in each of the random sequence
regions
of the upstream primer and the downstream primer (the two which ligate) and
the general
extension primer. As see in Figure 4A, a single-tube reaction system may
contain a first
upstream primer A-B with random sequence A, which identifies a first SNP of
interest in
a first target nucleic acid segment, and a second upstream primer F-E with
random
sequence F, which identifies a second SNP or interest in a second target
nucleic acid
segment, and a general extension primer with random sequence D' complementary
to
random sequence D present in downstream primer C-D, wherein C is common to
both
target nucleic acid segments.
Upon successful identification and binding to a target nucleic acid having a
SNP
of interest, upstream primers A-B and/or F-E will be ligated to downstream
primer C-D,
creating ligation products.A-B-C-D and/or F-E-C-D. If a gap is present between
the 3'-
end of the upstream primer and the 5'-end of the downstream primer, the gap
will first be
filled in by a polymerase activity, followed by ligation to form the ligation
products.
Extension of both ligation products can then occur by general extension primer
D', to
produce extended products A'-B'-C'-D' and F'-E'-C'-D'.
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Next, hybridizable probe A with fluorophore 1 and hybridizable probe F with
fluorophore 2, hybridize to extended products A'-B'-C'-D' and F'-E'-C'-D',
respectively,
which is followed by amplification such that each of the probes with its
particular
fluorescent tag is incorporated into amplified product (A-B-C-D or F-E-C-D),
triggering
fluorescence of either fluorophore 1 or fluorophore 2 or both. In this way,
one or more
SNPs may be identified and distinguished in a single-tube reaction system by
monitoring
the fluorescent signals of the two (or more) fluorophores upon incorporation
into
amplified product.
In another embodiment, an alternative single-tube reaction system for
identifying
and distinguishing one or more SNPs in one or more target nucleic acid
segments is
shown in Figures 5A-5F. Figures 5A through 5C are identical to Figures 4A
through 4C,
in that upstream primers A-B and F-E, downstream primer C-D, and general
extension
primer D' are present in the single-tube reaction system. Again, either the 3'-
end of the
upstream primers may contain the complement to the SNP of interest in the
target nucleic
acids, or the 5'-end of the downstream primer may contain the complement to
the SNP of
interest in the target nucleic acids, and upon binding to the target nucleic
acids, the two
primers may be adjacent, or have a gap of about 1-4 bases between the 3'-end
of the
upstream primer and the 5'-end of the downstream primer, which must be filled
by a
polymerase, before ligation between the upstream and downstream primer can
occur.
2o As shown in Fig. 5D, however, the alternative single-tube reaction system
does
not use hybridizable probes A and F with fluorophores 1 and 2 to amplify
target nucleic
acid, but rather, uses regular primers A and F to amplify extended products A'-
B'-C'-D'
and F'-E'-C'-D' into amplified target nucleic acids products A-B-C-D or F-E-C-
D. Such a
system may be advantageous when a particular target nucleic acid does not
amplify '
efficiently with hybridizable probes that have bulky fluorophores attached to
them. In
this alternative single-tube reaction system, the amplified target nucleic
acids are detected
after amplification, by additional fluorescent-tagged hybridizable probes hyb-
A and hyb-
F, which differ from regular primers A and F in that they are shorter, and
have secondary
structure that dissolve at lower temperatures than the annealing temperatures
of primers A
and F (or fluorescent probes A and F in Figure 4). This allows inefficient
competition
between hyb-A and hyb-F probes and regular primers A and F, in amplification
of
extended products A'-B'-C'-D' and F'-E'-C'-D' into target nucleic acid
products A-B-C-D
or F-E-C-D, but allows enough competing reaction to occur to measure
fluorescence of
CA 02549849 2006-06-02
WO 2005/059178 PCT/US2004/041480
fluorophores 1 and 2, thereby allowing detection and quantitation of amplified
target
nucleic acid product.
Although use of a general extension primer such as D' that is complementary to
a
sequence D in segment C-D common to both target nucleic acid segments is
convenient
in the single-tube reaction systems described above and exemplified in Figs. 4
and 5, it is
not required. It is envisioned that single-tube reaction systems could also be
adapted for
creating ligation products with with A-B and F-E using more than one extension
primer
simultaneously. The selectivity of the first primer A-B for the first SNP and
the second
primer E-F for the second SNP will ensure selective ligation, even with
additional
1o primers being used to generate the C-X product to be ligated.
Upon successful identification and binding to a target nucleic acid having a
SNP
.; of interest, upstream primers A-B and/or F-E will be ligated to downstream
primer C-G
and C-H, respectively, creating ligation products A-B-C-G and/or F-E-C-H. If a
gap is
present between the 3'-end of the upstream primer and the 5'-end of the
downstream
15 primer, the gap will first be filled in by a polymerase activity, followed
by ligation to
form the ligation products. Extension of both ligation products can then occur
by
extension primers G' and H', to produce extended products A'-B'-C'-G' and F'-
E'-C'-H'.
As described above, one or more SNPs may be identified and distinguished in a
single-tube reaction system by a) monitoring the fluorescent signals of two
(or more)
2o fluorophores upon incorporation into amplified product, or b) detecting
fluorescent
signals after amplification, by use of additional fluorescent-tagged
hybridizable probes
hyb-A and hyb-F.
Example 3. A rcanoliter sampling array
25 Another embodiment of the present invention encompasses a nanoliter
sampling
array. Any array presently available in the prior art may be used, but an
array of particular
utility, similar to that described in U.S. Provisional Application Serial No.
60/518,240,
filed November 7, 2003, and US regular application serial no. 10/984,027 filed
on
November 8, 200.4, both of which are hereby incorporated by reference herein,
is one
30 preferred array. In this particular embodiment, the array comprises a first
platen having
at least one hydrophobic surface and having a high-density microfluidic array
of
hydrophilic through-holes. A target nucleic acid sequence is selected, and the
array is
prepared wherein each through-hole in the array contains at least a first
primer having at
least a portion of its 3'-end substantially complementary to a first segment
at a first end of
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WO 2005/059178 PCT/US2004/041480
the nucleic acid target sequence and a second primer having at least a portion
of its 5'-end
substantially complementary to a second segment at a second end of the nucleic
acid
target sequence, the 5'-end of the second primer being adjacent to the 3'-end
of the first
primer upon binding to the potential nucleic acid target sequence. Figure 4
shows such
an array, known in the prior art. Array chip 10 typically may be from 0.1 mm
to more
than 10 mm thick; for example, from 0.3 to 1.52 mm thick, and commonly 0.5 mm.
Typical volumes of the sample through-holes 12 could be from 0.1 picoliter to
1
microliter, with common volumes in the range of 0.2 to 100 nanoliters, for
example,
about 35 nanoliters. Capillary action or surface tension of the liquid samples
may be used
to load the sample through-holes 12. For typical chip dimensions, capillary
forces are.
strong enough to hold liquids in place. Chips loaded with sample solutions can
be waved
in the air, and even centrifuged at moderate speeds, without displacing the
samples.
To enhance the drawing power of the sample through-holes 12, the target area
of
the receptacle interior walls 42 may have a hydrophilic surface that attracts
a liquid
sample. Alternatively, the sample through-holes 12 may contain a porous
hydrophilic
materiel that attracts a liquid sample. In some embodiments, the sample
through-holes in
the array may be coated with a biocompatible material such as polyethylene
glycol, and
the primers may be affixed on, within or under the biocompatible material on
the surface
of the through-holes by drying the primers after application to the through-
holes. To
prevent cross-contamination (crosstalk),,the exterior planar surfaces 14 of
chip 10 and a
layer of material 40 around the openings of sample through-holes 12 may be of
a
hydrophobic material. Thus, each sample through-hole 12 has an interior
hydrophilic .
region bounded at either end by a hydrophobic region.
The through-hole design of the sample through-holes 12 avoids problems of
trapped air inherent in other microplate structures. This approach, together
with
hydrophobic and hydrophilic patterning enable self-metered loading of the
sample
through-holes 12. The self loading functionality helps in the manufacture of
arrays with
pre-loaded reagents, and also in that the arrays will fill themselves when
contacted with
an aqueous sample material.
Example 3. Method for identifying a SNP in a target sequence of nucleic acid.
Yet another embodiment is a method for identifying a single nucleotide
polymorphism (SNP) in a target sequence of nucleic acid. A target sequence of
nucleic
acid is identified, and primers are prepared according to standard methods,
such that two
primers, Pl and P2, are designed to flank an internally-positioned SNP on one
strand of
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WO 2005/059178 PCT/US2004/041480
the target nucleic acid sequence and are designed to be ligated with a
thermally stable
ligase. Primer Pl and P2 are further designed such that the base complementary
to the
SNP in the target sequence is either on the 3'-end of P1, or on the 5'-end of
P2. In this
particular method, a nanoliter sampling array is used. The method comprises
providing a
first platen having a high-density microfluidic array of through-holes is
provided wherein
each through-hole of the array contains a first primer having at least a
portion of its 3'-end
substantially complementary to a first segment at a first end of the target
sequence, and a
second primer having at least a portion of its 5'-end substantially
complementary to a
second segment at a second end of the target sequence. Upon binding to the
target
1o sequence, the 5'-erid of the second primer is adjacent to the 3'-end of the
first primer.
The method further comprises introducing a sample containing the target
nucleic
acid sequence with internal SNP into the array, and introducing reagents into
the through-
holes in the array wherein the reagents include a third primer having a random
sequence
J capable of amplifying ligated primer Pl-P2 product, a thermostable
polymerase, a
i5 thermostable ligase, and at least four different nucleoside triphosphates.
Additional steps
in the method comprise thermocycling the array with primers, target nucleic
acid, and
reagents, and detecting the resulting amplified target nucleic acid sequence.
Optionally,
the thermostable polymerase may lack 5' to 3' exonuclease activity, or it may
lack 3' to 5'
exonuclease activity, or it may lack both 5' to 3' and 3' to 5' exonuclease
activity.
20 It is also envisioned that the detecting step may comprise the use of a dye
specific
for binding to double-stranded DNA or to RNA that fluoresces upon binding
amplified
target sequence. Suitable dyes include SYBR~ Green I, SYBR~ Green II, YOYO~-1,
TOTO~-l, POPO~-3, EtBr, and any other dye capable of providing low-sensitivity
detection of amplified target sequence by fluorescence emission.
25 Alternatively, detection may occur through the addition of probes specific
for
hybridization across the ligation junction of the ligated P1-P2 primer
product, where such
probes contain a fluorescent group and a fluorescence-modifying group such as
a
fluorescence quencher.
In another alternative embodiment, detection may involve the use of a probe
30 containing a fluorescent group and a fluorescence-modifying group such as a
fluorescence quencher that is specific for hybridizing to a region of the
target sequence.
In this particular embodiment, the fluorescence-modifying group is excised
upon
extension of the probe, and the fluorescent group thus fluoresces, allowing
detection of
amplified product.
23
CA 02549849 2006-06-02
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Additional embodiments of the present invention include a kit for use in
identification of amplified target nucleic acid sequences, wherein the kit
provides a
sample platen having one hydrophobic surface and having a high-density
microfluidic
array of hydrophilic through-holes. In one particular kit each through-hole
contains at
least a first primer having at least a portion of its 3'-end substantially
complementary to a
first segment at a first end of potential nucleic acid target sequence, a
second primer
having at least a portion of its 5'-end substantially complementary to a
second segment at
a s ~ and end of the potential nucleic acid target sequence, the 5'-end of the
second primer
being adjacent to the 3'-end of the first primer upon binding to the potential
nucleic acid
target sequence and a reagent platen having a high-density microfluidic array
of through-
holes with each through-hole containing a third primer that is,substantially
complementary to a random sequence segment at the 3'-end of the second primer
and to a
substantially similar sequence at the 5'-end of the first primer, at least
four different
nucleotide bases, a thermostable ligase and a fluorescent dye. In this
particular
embodiment, the reagent platen has a structural geometry that corresponds to
the sample
platen allowing delivery of reagent components and target nucleic acid sample
to the
primers in the sample platen. In some embodiments of the kit, the primers may
be affixed
on, within or under a biocompatible material such as a wax-like coating in the
through-
holes by drying the primers after being applied to the through-holes, wherein
the
biocompatible material may comprise, for example, a polyethylene glycol (PEG)
material. To perform the selective ligation and amplification reaction for
identification of
an amplified target nucleic acid sequence, the user would merely add a sample
containing
the target nucleic acid, a thermostable polymerase, and optionally a buffer
supplied with
the kit to the through-holes.
24
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Section 8.7 - Analysis of DNA Sffiuucture, DNA Binding and DNA Damage
~ P P ~-~d ~ ~c ~1
~~t~~
Updated: August 30, 20U3
Section 8.7 - Analysis of DNA Structure, DNA Binding and DNA Damage
I ~t,s~er-xriePcxr~
Nucleic Acld Conformatir~nal Analyses
A number of conventional dyes have been used to analyze nucleic acid
conformation in vitro and In
vivo:
~ Acridine orange (A-1301, A-3568; Section 8.1) is one of the most popular and
versatile
fluorescent stains for hlstochemistry and cytochemistry and can provide a wide
variety of
information about the in situ content, molecular structure, conformation and
environment of
many nucleic acid-containing cell constituents.
. Fluorescence photobleaching of DNA that has been photolytically labeled with
ethidium
monoazide (E-1374, Section 8.1) permits measurement of slow reorientational
motions.e"
. The fluorescence intensity and binding affinity of the Hoechst dyes appear
to be highly
dependent on the sequence and conformation of the DNA base pairs.tFor example,
staining by Hoechst 33258 (H-1398, H-3569; FluoroPure Grade, H-21491; Section
8.1) can
discriminate parallel and anttparalfel stem regions in hairpin DNA
conformations.
~ The fluorescence lifetime of the PlcoGreen dye (P-7581, P-11495; Section 8.3
bound to
single-stranded DNA is reported to be different when bound to double-stranded
DNA.
Uwle also anticipate that several of our cyanine dyes (Section 8.1) - in
particular the SYTO dyes
(Table 8.3,) - may be useful in these applications because many of these
stains appear to yield
environment-sensitive rnetachromatlc shifts upon binding to nucleic acids.
Fluorescence of the
TOTO-1, YOYO-1, BOBO-1 and POPO-1 dyes I'Table 8.2, Dimeric Cyanine Nucleic
Acid Stains) is
'dependent on nucleic acid secondary structure; a shift to longer-wavelength
emission and a
concomitant drop in quantum yield are observed upon binding of these dyes to
single-stranded
nucleic acids at high dye:base ratios. Most of our unsymmetricai cyanine dyes
show this
spectral shift, and some show sequence selectivity in their fluorescence
intensity as well.
Examining the Behavior of Single Nucleic Acid Mvlecu~es
Once bound to nucleic acids, several of the cyanine dyes in section 8.1 are so
bright that they can
be used to directly visualize single nucleic acid molecules in the
fluorescence microscope (~, 11~).
The YOYO-1 and POPO-3 dyes (Y- 601 P-3584) dyes have also been used to follow
the making
and breaking of single chemical bonds.cA number of laboratories have taken
advantage of the
high sensitivity of these dyes to detect single nucleic acid molecules and to
study btopolymer
behavior:
. Video microscopy has been used to observe relaxation of YOYO-1 dye-stained
phage lambda
DNA multirners, after stretching in a fluid flow,lon a surFace t1~ or with
optical tweezers.
TOTO-1 dye (T-3600) has also been used in this application.?
. Individual YOYO-1 dye-ssDNA~ molecular complexes have been imaged ih
solution by
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section 8.7 - Analysis of DNA Structure, DNA Binding and DNA Damage
fiJuorescence video microscopy.~Y
. Molecular combing, a technique that uses a receding fluid interface to
elongate DNA ,
molecules for optical mapping of genetic loci, was developed using the YOYO-i
dye.k
. Adsorption and desorption of single molecules of YOYO-1 dye-stained phage
lambda DNA
have been observed on fused-silica and Cl8 chromatographic surfaces.("
. The activity of a single Recl3CD enzyme, which unwinds and separates the
strands of dsDNA,
has been studied using YOYO-i dye-stained. dsDNA in conjunction with optical
tweezers and
eplfluorescence microscopy.
Our YOYO-1 dye (Y-3601 has been used to stain DNA manipulated in solution
by~changlng
electronic fields, a technique that could prove valuable in miniaturizing and
automating
analysis of DNA fragments,,
. Staining with the YOYO-1 dye (Y-36(71) was used to observe the interacfiion
of ANA with
various llpasomes d~k and to size plasrnids in a flowing stream.'
~ The YOYO-1 dye was also used to detect radiation-induced double-strand
breaks in. individual
electrostretched bacterial DNA molecules.
. Single-molecule imaging of nucleic acids stained with either YOYO-1 or POPO-
3 or a
combination of the two dyes through collection of the entire fluorescence
spectrum of their
campiex has been reported.
. Highly sensitive sheath-flow techniques have also been developed for
detecting and
discriminating the size of single TOTO-1 dye-DNA molecular complexes.l~
Large fragments of DNA stained with our TOTO-1 dye (T-3600) have been sorted
by flow
cytometry. This extremely rapid analytical method yields a linear relationship
between the
fluorescence intensity and the fragment size over a IO-50 kilobase pair
ranger:
. The POPO-1 (P-3580, Section 8.1) and POPO-3 (P-3584) stains have been used
to sensitively
detect single DNA fragments by flow cytometry using two-photon fluorescence
excitation..
~ The POPO-3 dye P-3584 has been used to study a single chemical reaction
wlth,an
individual DNA molecule. POPO-3 dye-stained DNA molecules stretched taught on
a glass
surface relax when a focused laser beam causes fluorescence-related breakage
of the DNA
backbone, forming a gap that is visible by fluorescence micrascopy.~
. The TOTO-1 (T-3600 , YOYO-1 (Y-3601), POPO-3 (P-3584) and SYBR Green I (S-
7563, 5-
7557, S-7585) dyes have been used to visualize lambda DNA that has been
stretched
between beads with optical tweezers.t"~,
. Fragment sizing on single molecules of dsDNA stained with our PicoGreen
reagent has also
been reported.
. The SYTOx Orange dye S-11368 Is the preferred dye for single-molecule sizing
of DNA
fragments by flow cytometry in an instrument equipped with a Nd:YAG laser.,tl~
~ DAPI (D-1306, D-3571; FluaroPure Grade, D-21490) has also been employed to
detect a
single DNA molecule in solution ,cue and by fluorescence microscopy a and to
detect
femtograms of TUNA in stngle cells and chloroplasts.t~k
The high affinity and bright fluorescence of other cyanlne dimers has allowed
researchers to follow
stained and transfected plasmids or stained virus particles within a cell.f~k
DNA Binding Assays
r~iectraphoretic Mabiiity-Shift (i3andshlft) Assays .
Bandshift assays to analyze DNA-protein interactions are conventionally
performed using
radtaactively labeled DNA fragments. However, use of our high-sensitivity
fluorescent dyes makes
these assays much simpler to pertorm and eliminates radioactive waste issues.
For example, SYBit
Green I nucleic acid get stain (S-7567, S-7563, S-7585; SYBR Green I Nucleic
Acid Gel Stan) has
been used to post-stain gels after electrophoresis and can detect bound and
unbound DNA
fragments with high sensitivity (Figure 8.134?. The SYBR Goid nucleic acid gel
stain IGS-11494,,
26
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Section 8.7 - Analysis of DNA Structure, DNA Binding and DNA Damage
SYBR Gold Nucleic Acid Gel Stain) is potentially even more useful in bandshift
experiments because
of its higher sensitivity.
Molecular Probes has made bandshift assays easy and more convenient with our
Electrophoretic
Mobility-Shift Assay (EMSA) Kit (E-33075). Our EMSA Kit provides a fast and
quantitative
fluorescence-based method to detect both nucleic acid and protein in the same
gel (lii~), doubling
the information that can be obtained from bandshift assays. This kit uses two
fluorescent dyes for
detection - SYBR Green EM9A.nudeic acid gel stain for RNA or DNA and SYPRO
Ruby EMSA
protein gel stain for proteins. Because the nucleic acids and proteins are
stained in the gel after
electrophoresis, there is no need to prelabel the the DNA or RNA with a
radioisotope, biotin or a
fluorescent dye before the binding reaction, and therefore there Is no
possibility. that the label will
interfere with protein binding. Staining takes only about 20 minutes for the
nucleic acid stain, and
about 4 hours far the subsequent protein stain, yielding results much faster
than radioisotope
labeling (which may require multiple exposure times) or chernilumlnescence-
based detection
(which requires blotting and multiple incubation steps). This kit also makes
it possible to perform
ratiametric measurements of nucleic acid and protein in the same band,
providing more detailed
information on the binding interaction. The signal from the two stains is
linear over a broad range,
allowing accurate determination of the amount of nucleic acid and protein,
even in a single band,
with detection limits of N1 ng for nucleic acids and N20 ng for protein . Both
stains can be detected
using a standard 300 nm UV illuminator, a 254-nm epi-illuminator or a laser-
based scanner ().
Digital images can easily be overlaid for a two-color representation of
nucleic acrd and protein in
the gel. The EMSA Kit contains sufficient reagents for 10 nondenaturing
potyacrylamide minigei
assays, including:
. SYBR Green EMSA nucleic acid gel stain
~ SYPRO Ruby EMSA protein gel stain
~ Trichloroacetic acid, for preparing the working solution of SYPRO Ruby EMSA
protein gel stain
. Concentrated EMSA gel-loading solution
. /ac repressor, a DNA-binding protein to be used as a control .
. !ac operator, control DNA
. Concentrated buffer for the !ac repressor:operatar controls
~ A detailed protocol (Elsctroahoretic Mobility Shift Assay (EMSA) Kit)
Fluorescent dyes have also been used to stain the DNA fragments or proteins
before
electrophoresis. For instance, proteins or DNA labeled cavalently with a
reactive fluorescent dye
(hC~,apter 1, Section 8.2) can be easily,tracked during cap111ary
electrophoresis to monitor DNA-
protein interactions.l~~ High-afi'inity nucleic acid stains have also been
used prior to
electrophoresis, although they can potentially Interfere with protein binding
and alter mobility on
the gel. The ethidlum homodimer-Z (EthD-1., E~1,69; Section 8.1), YOYO=1 and
1'0T0-1 dyes have
been shown by several laboratories to be useful tools far labeling DNA prior
to electrophoresis in
bandshlft assays. EthD-1 and TOTO-1 were used to examine interactions between
the binding
domain of the Kluyvemmyces lactic heat shock transcription factor and its
specific binding site.~k
YOYO-1 dye has been used to study the association of E, coif RNA polymerase
with DNA templates
d~ and the binding of a heat-shock transcription factor to its promoter.d~k
All ten of our spectrally
distinct (Figure 8.1), high-affinity dimeric cyanine dyes (Table 8.2) and the
ethidiurn homodlmers
are potentially useful for multlcomponent analysis in this application.
DNA Binding Assays in Solution
Hlolecular beacons exploit fluorescence resonance energy transfer (FRET) to
simplify detection of
nucleic acid hybridization in solution (Section 8.5. Figure 8.104). This
method has also proven
useful for studying DNA-protein interactions In solution. Binding of a
molecular beacon to lactic
dehydrogenase separated the fluorophore from the quencher on the two ends of
the labeled
oligonucleotide, resulting in an increase in fluorescence. The assay is
sufficiently accurate to
27
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'Section 8.7 - Analysis of DNA Structure, DNA Binding and DNA Damage
measure binding constants. A molecular beacon was also used to develop a
solution-based binding
assay for ~x-CP2, which is part of an RNA-binding complex.
Selective Cleavage of Nucleic Acids with a Chemical Nuclease
The thiol-reactive iodoacetamide of i,10-phenanthroline (P-6879, Section 2.3)
is a useful adjunct
reagent for bandshift assays. Conjugation to thiot-containing Ilgands confers
the metal-binding
properties of this important complexing agent on the ligand. For example, the
covalent copper-
phenanthroline complex of oilganucleatides or nucleic acid-binding molecules
in combination with
hydrogen peroxide acts as a chemical nuclease to selectively cleave DNA or
RNA.tThis reagent
can also be conjugated to proteins iv detect nucleic acid binding and targeted
cleavage.d~
Assessing DNA Damage
Comet (Single~Cell Gef Electrophoresis) Assay to Detect Damaged DMA
The comet assay - or single-cell gel electrophoresis assay - is used for rapid
detection and
quantitation of DNA damage from single ceiis.The comet assay is based on the
alkaline tysis of
labile DNA at sites of damage. Cells are immobilized in a thin agarose matrix
on slides and gently
fysed. When subjected to electrophoresis, the unwound, relaxed DNA migrates
out of the cells,
After staining with a nucleic acid stain, cells that have accumulated DNA
damage exhibit brightly
fluorescent comets, with falls of DNA fragmentation or unwinding (). In
contrast, cells with
normal, undamaged DNA appear as round dots, because their intact DNA does not
migrate out of
the cell. The ease and sensitivity of the comet assay has provided a fast and
convenient way to
measure damage to human sperm DNA,tmonitor the sensitivity of tumor cells to
radiation
damage and to assess the sensitivity of molluscan cells to toxins in the
environment. The,
comet assay can also be used in combination with FISH to identify specific
sequences with
damaged DNA.d'
Comet assays have traditionally been performed using ethidium bromide ( -13 , -
3565) to stain
the DNA;dhowever, our YOYO-1 dye (Y-3601) increases the sensitivity of the
assay elghtfold
compared to ethidium bromide and the fluorescence background from unbound YOYO-
1 dye is
negligible. Use of the SYBR Goid and SYBR Green I stains {Section 8.~.)
further Improves the
sensitivity of this assay.
TUNEL Assay for ~'n Situ Detection of Fragmented DNA
To detect fragmented DNA in labeled cells, terminal deoxynucleotidyl
transferase (TdT) along with
a fluorophore-, biotin-, or hapten-labeled dUTP can be added to cells. TdT
adds the labeled
nucleotide to all available 3'-ends - the mare fragmented the DNA, the more 3'-
ends are available
and the brighter the fluorescent signal. Direct TUNEL assays using ChromaTlde
BODIPY FL-14-dUTP
--C 7b14) to visualize DNA fragment ends are four times more sensitive than
TUNEL assays using
fluorescetn-labeled dUTP (~). Terminal deoxynucleottdyl transferase (TdT)-
catalyzed
tncorporatlon of bromo dUTP into nucleic acids of apoptotic cells and
detection of the incorporated
BrdU with an ant)bady conjugate Is the basis of the AP4-BrdU TUNEL Assay Kit
(A-23210, Section
15.5). Indirect TUNEL assays using probes such as biotinylated dUTP or our
ChramaTide DNP-11-
dUTP (C-~sxo, Section 8.2) allow for amplification of the signal with our
fiuorophore- or erizyme-
conjugated streptavidtn conjugates (Section 7.f. Table 7.20) or with anti-DNP
antibody (Section
7~4). Several additional assays for apoptosis can be found in Section 15.5. .
Mtcroplate-Based Assays for DNA Damage
Abasic sites in DNA, generated spontaneously or caused by free radicals,
ionizing radiation or
mutagens like MMS (methyl methanesuifonate), are one of the most common
lesions in DNA and
28
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Section 8.7 - Analysis of DNA Structure, DNA Binding and DNA Damage
are thought to be important intermediates in mutagenesis. A quick and
sensitive microplate assay
for abasic sites can be performed using ARP (A-10550, Ficture 8.137), a
biotinylated hydroxylamine
that reacts with the exposed aldehyde group at abasic sites. Biotins tround to
the abasic sites can
be quantitated with our fluorescent- or enzyme-conjugated streptavldin
complexes d~'k (Section
7~6, Table 7.20). ARP is permeant to cell membranes, permitting detection of
basic sites in living
celis.t~k
The PicoGreen reagent has also been used to simplify denaturatton assays for
DNA damage. Strand
breaks in dsDNA that result from DNA damage can ba quantified by measuring the
relative
amounts of ssDNA and dsDNA in a damaged sample. The relative amounts of dsDNA
to ssDNA can
be assessed by measuring the increase In absorbance at 260 nm or by separating
the two farms of
DNA by alkaline sucrose gradient centrifugatlon,filters,d~ or hydroxyapatlte
chromatography.
~ However, the absorbance-based technique suffers from Ivw sensitivity and
thus requires
relatively large sample sizes k and separation of ssDNA from dsDNA is
laborious. This assay
becomes much simpler and mare sensitive using the PtcoGreen dsDNA quantitatlon
reagent (P-
7581, P-7589. P-11495, -1~ 1496, R-21495; Section 8.3), which preferentially
detects dsDNA in the
presence of ssDNA.sThe dye can be added directly to the sample and the
fluorescence signal
read on a fluorescence-based microplate reader. Thts method makes it possible
to screen large
numbers of very small samples in a high-throughput setting. The PicoGreen
reagent was also used
to develop a homogeneous PCR-based genotyping assay.d~ Because the products do
not need to
be run on a gel, the assay can be easHy adapted for high throughput
particularly using the
RediPIate 96 version of the PicoGreen dsDNA quantitation assay (R-21495,
Section 8.3).
Assays for Enzymes that Modify Nucleic Acids
Get-Based Assays for DNase t)etectlon
Our SYBR Green I stain (S-7563, S-7567, S-7585; SYt3R Green I Nucleic Acid GeI
Stainl has been
used to develop DNase assays that show up to a 64-fold increase in sensitivity
over similar
ethidium bromide-based assays and up to 10,00-fold higher sensitivity than the
traditional UV
hyperchrornidty assay. In a fast and simple assay, a single-length fragment of
ANA can be
incubated with the sample, followed by a short gel electrophoresis. Staining
the gel with the SYBR
Green I dye permits easy detection of less than 10-5 Kunitz units (N5 pg) of
DNase activity.
Even greater sensitivity can be achieved using the single radial enzyme
diffusion (SRED) method,
In which the SYBR Green I stain is mixed with DNA in matted agarose and the
mixture is
poured into a 2 mm thick slab. The sample to be tested is poured into i.5 mm
circular wells
punched out of the solidified agarose slab. As the sample diffuses through the
agarose, the DNase
degrades the DNA, creating dark circles around the sample well that do not
show staining with the
SYBR Green I dye when illuminated with UV light. The radius of these dark
circles is proportional to
the level of DNase activity. This method allows detection of as little as 2 x
IO'' units (N0.1 pg) of
DNase I or 2 x 10-6 (N0.9 pg) of DNase II. A third DNase assay - called the
dried agarose film
overlay (DAFO) method - uses the SYBR Green I stain to detect the presence of
DNase activity in
a polyacrylamide gel, allowing the tdentii~ication of heterogenelties in DNase
species,t~ This
method allows the detection of 4 x 10-6 units (N2 pg) DNase I or DNase II.
Solution-Based Assays for Nuclease Detection .
Contaminating DNases are often responsible for poor resolution of DNA
fragments, degradation of
samples and nicking of supercviled plasmids. Conventional DNase assays detect
DNase activity by
monitoring the increase tn UV absorbahce that occur$ when the base pairs
unstack as the DNA is
degraded. Trits absorbance method, however, is intrinsically insensitive as tt
requires large sample
volumes and relies on small changes in absorbance. In contrast, our dyes far
nucleic acid detection
show a tremendous fluorescence increase upon binding to nucleic acids, but
their fluorescence is
not affected by the presence of a large excess of a nucleotide or very short
oligonucleotides. Thus,
nuclease activity can be easily and accurately measured by the decrease in
fluorescence in the
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Section 8.7 - Analysis of DNA Structure, DNA Binding and DNA Damage
presence of one of these dyes. For instance, the YOYO-1 nucleic acid stain (Y-
3601 has been used
in a fluorescence-based mlcraplate assay for nuclease activity.tk This assay
takes advantage of
the large fluorescence enhancement of the YOYO-i dye upon binding to nucleic
acids and
corresponding Jack of fluorescence in the presence of released nucleotides and
very small nucleic
acid fragments. Other dyes - in particular our PIcoGreen dsDNA quantitation
reagent (P-7581. P-
1 495; Section 8.3) - are likely to be more suitable for this assay.
Similarly, use of the. RIboGreen ,
RNA quantitation reagent R-11490, R-11491; Section 8.3) should allow
ultrasensitive detection of
rlbonuclease (RNase) activity. '
Using a design similar to that of molecular beacons (Section 8.5), the stem
sequence in an
oligonucleotlde hairpin loop can be modified to be a substrate for specific
DNA cleavage agents,
including nucleases. Dubbed a "break light," this substrate shows increased
fluorescence as the
Geavage agent breaks the DNA strand, separating. the fluorophore Pram the
quencher.
An Assay for Reverse Transcrlptase Actt~ity
The EnzChek Reverse Transcriptase Assay Kit (E-22064) is a convenient,
efficient and inexpensive
assay for measuring reverse transcriptase activity (F39ure 8.138). The key to
this method is our
PlcoGreen dsDNA quantitation reagent, which preferentially detects dsDNA or
RNA-DNA
heteroduplexes over single-stranded nucleic acids or free nucleotides. In the
assay, the sample to
be measured is added to a mixture of a long poly(A) template, an oligo(dT)
primer and dTTP.
Reverse transcriptase activity in the sample results In the formation of long
RNA-DNA
hekeroduplexes, which are detected by the PicoGreen reagent at the end of the
assay. In less than
an hour, samples can be read in a fluorometer or micropiate reader with filter
sets appropriate for
fluorescein (FITC). The assay Is sensitive, detecting as little as 0.02 units
of HIV reverse
transcriptase, and has about a 50-fold linear range (Figure 8.139), Because It
is much more rapid
and less expensive than standard isotopic assay or immunoassays, it is
suitable For testing large
numbers of biological samples. The assay's simplicity also makes It useful for
automated high-
throughput screening of reverse transcriptase inhibitors.
The EnzChek Reverse Transcriptase Assay IClC (E-22054) contains:
~ The PicoGreen dsDNA quantitation reagent
~ A lambda DNA standard
. A poly(A) rlbonucleotide template
~ An oligo(dT)16 primer
w TI= buffer, polymerization buffer and an EDTA solution
. A detailed protocol (EnzChek Reverse Transcriiptase Assay Kit)
Sufficient amounts of reagents are provided for approximately 1000
fluorescence micropiate
assays.
Telomerase
In a gel-based assay far detection of telomerase activity (the telomeric
repeat amplification
protocol or TRAP) in human cells,and tumors, SYBR Green I dye staining was
found to be more
sensitive than silver staining and gave results comparable to those achieved
with a radioisotope--
based TRAP assay.tl~ Moreover, unlike the silver stains, the SYBR Green I
stain did not label
proteins carried over From the reaction mixture. The SYBR Gold stain was also
shown to be more
sensitive than silver staining in the TRAP assay, and much easier to use.ti(~-
The SYBR Green I
stain (S-7567, 5-7563, S-7585) has also beep used to develop high sensitivity
assaysto detect
topoasomerase activity.
SUBSTITUTE SHEET (RULE 26)
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section 8.7 ~- Analysis of DNA Structure, DNA Binding and DNA Damage
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pe Research -- Abstracts: Lawyer. et al. 2 (4): 275
~6'.ENOME Appendix S
PCR Methods and Applications, vol 2, 27S-287, Copyright ~ 1993 by Cold Spring
, ~ ~~1 ~s article to a friend ,
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1 Download to Citati~Man_a~er
High-level expression, purification, and
e~izymatic characterization of full-length- Thermos aquaticus
I)NA poiylmerase and a truncated folrm deficient in 5' to 3'
exonuclease activity
FC Lawyer, S Stoffel, RIB Saiki, SY Chang, PA Landre, RD Abramson and DH
Gelfand
Program in Core Research, Roche Molecular Systems, Alameda, California 9450I.
The Thermos aquaticus DNA polymerise I (Taq PoI l) gene was cloned into a
plasmid expression vector
'that utilizes the strong bacteriaphage lambda PL promoter. A truncated form
of Taq Pol I was also
constructed. The two constructs made it possible to compare the full-length
832- amino-acid Taq Pol I
and a deletion derivative encoding a 544-amino- acid translation product, the
Stoffel fragment. Upon
heat induction, the 832-amino-acid construct produced 1-2% of total protein as
Taq Pol I. The induced .
544-amino-acid construct produced 3% of total protein as Stoffel fragment.
Enzyme purification
included cell lysis, heat treatment followed by Polymitt P precipitation of
nucleic acids, phenyl
sepharose column chromatography, and heparin-Sepharose column chromatography.
For full-length 94-
kD Taq Pol I, yield was 3.26 x 10(7) units of activity from 16~ grams wet
weight cell paste. For the 6I-
1cD Taq Pol I Stoffel fragment, the yield was 1.03 x 10(6) units of activity
from 15.6 grams wet weight
cell paste. The two enzymes have maximal activity at 75 degrees C to 80
degrees C, 2-4 mM MgCl2 and
IO- 55 mM KCI. The nature of the substrate determines the precise conditions
for maximal enzyme
activity. For both proteins, MgCl2 is the preferred cofactor compared to
MnCl2, CoCl2, and NiCl2. The
full-length Taq Pol I has an activity half life of 9 min at 97.5 degrees C.
The Stoffel fragment has a half
life of 21 min at 97.5 degrees C. Taq Pol I contains a polymerization-
dependent f to f exonuclease
activity whereas the Stoffel fragment, deleted for the f to 3' exonuclease
domain, does not possess that
activity. A comparison is made among thermostable DNA polymerises that have
been characterized;
specific activities of 292,000 units/mg for Taq Pol I and 369,000 units/mg for
the Stoffel fragment are
the highest.reported.
32
SUBSTITUTE SHEET (RULE 26)
CA 02549849 2006-06-02
WO 2005/059178 PCT/US2004/041480
~ Genome Research -- Abstracts: Lawyer et al. 2 (4): 275
i~
33
SUBSTITUTE SHEET (RULE 26)
CA 02549849 2006-06-02
WO 2005/059178 PCT/US2004/041480
L
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et
34
SUBSTITUTE SHEET (RULE 26)
CA 02549849 2006-06-02
WO 2005/059178 PCT/US2004/041480
~ O ~
E ~ '~ c cu o
Vl O U . p +.r C
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SUBSTITUTE SHEET (RULE 26)
CA 02549849 2006-06-02
WO 2005/059178 PCT/US2004/041480
0
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36
SUBSTITUTE SHEET (RULE 26)
CA 02549849 2006-06-02
WO 2005/059178 PCT/US2004/041480
PAGE INTENTIONNALLY LEFT BLANK
a
m
Z
w
v
c
N'
a
ar
0
a
N
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y
t
01
L
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37
SUBSTITUTE SHEET (RULE 26)
CA 02549849 2006-06-02
WO 2005/059178 PCT/US2004/041480
For general laboratory use.
FOR !N VITRO USE ONLY.
r.~~'BR Green 1 Nucleic Acid Gel Stain
Nighty sensitive fluorescent stain for detecting DNA in agarose and
polyacrylamide gels
Cat. No. 1 988123 2 x 500 wi
Cat. No. 1 988131 500 Lt,l Store at -15 to -25' C
9. Product ovanriaw Product storage! SYBR Green I Is supplied In an anhydrous
DMSO
stability solution and is shipped at ambient temperature.
The unopened vial is stable at -7 5 to -25° C through
Caution Because the expiration
this date printed
reagent an the label.
binds
to
nucleic
acids,
h
should
be
treated ~ Aitquote
as the stock
a solution
potential in 5D ~t
mutagen aliquots,
and
used
with
appropriate
care.
The
DMSO
stock
solution
should
be
handled brown tubes
with should be
particular used.
caution
as
DMSO
is
known
to
anic
molecules
into
tissues.
of
or
ilitate
the
ent
f
g Condition TemperaturgStability
ry
ac
When undiluted -16 8 -12
handling stock to months
the
DMSDStock
solution,
double
gloves,
protective
clothing
and
eyewear
should
be
worn . -25
and C
safe
laboratory
practices
should
be
followed.
diluted stain2-8 por
in C several
ContentsHa, Label contents pH 7.0-8.5 days
Cat buffer
. fr
e
n
1 988 SYBR 2 x 500 wi o1
723 Greenl rc
lene
Stn a
p ~ py
Nucleic10 000 x contatner,protected
Acid concentrate
GelStaln. InDMSD from IighU
1988 SYBR 500 wl
731 Green
t
NucleicActd10000 x concentrate
Gel in DMSO Handlingprotect from
Stain Ilght. ,
recvrnmendatlons
. before
opening,
eachvlal
should
be allowed
to warm
l
t
flu
~
ee
g
s
Tan principleThe 6a
dye ttom of
exhibits he v
a teal.
preferential tion t
affinity th
for it the DMSO
nucfefe so
acids tp dap
and
its store aqueous
fluorescent stain solutions
signal in polypropylene
Is
largely
enhanced
when
bound rather than
to glass. as
DNA the stain
.(more may adsorb
than to glass
one
order
of
magnitude
larger ~rfaces,
than
the
fluorescent
enhancement
or
bound
ethldtum dye is not
bromide). stable in
water alone.
A~piicationSYBR
Green
I
dye
is
a
highly
sensitive
fluorescent
stain
for
detecting
nucleic
acids
In
agarose
and
polyacrylamide 2. Product
gels charaetetistirs
(1,2).
'
The
exceptional
sensitivity
of
SYBR
Green
I
stain
makes 5ensltiviThe detection
it limit usin
useful SYBR Green
for f Is as
those krw as
applications
where D
the
Including
the
detection
amount
of
DNA
is
limiting
, NA usin )112
of nm trans-
low-cycle ,
number 100 pg per
and band oI
tow-target ds
number illumination
DNA with the
Luml Image
F1 Instrument
ampllflcadon from Roche
products; Diagnostics
the (Cat. No.
detection 2 015170).
and
restriction
analysis This is approximately
of 25 -100
low-copy times more
number sensitive
of than
DNA
and
RNAvectors;
and
the
detection
of
products
of
nuclease
protection
and ethidlum
bandshitt bromide
assays, staining.
'
SYBR The detection
Green Ifmit for
1 oligonuoleotides
stain-s stained
superior
sensitivity
allows
replacement with SYBR
of Green 1
radtolsotopes is t~elow
In 5D pmot
some with
applications,
e,g, 312 nm transiilumlnation
RT (Lumi-imager
PGR. F7) or
d(gte: 254 nm eplillumination
SYBR ,
Green
I
stain
van
also
be
applied
as
detection
format ToidcityThe Amas
in mammalian
the mlcrosome
LJghtCyder reverse
InsVUment mutation
But
the
provided
corxenVation
fn
DMSO
is
not
standardized
for assay shows
the slgniflcantty
precise less mutegeniclty
quantfflcation of SYBR
and Green I than
detection ethidlum
of bromide
nucleic (3.4)
acids
with
the
LightCycier:
Please
refer
to
specialized
kits gpeotralSYBR Green
and 1 is maximally
reagents excited
for at 497 nm
this and has
instrument
Sample SYBR characteristicssecondary
mateHat Green broad excitation
1 peaks at
can 284 nrn
detecC and
382 nm. The
emission
of DfVA
stained
with SYBR
double Green I Is
stranded centered
and at520 nm,
stn
to
stranded
DNA
g
RNA
(with DetecUonThe spectral
tower characteristics
sensitivity); of SYBR
for Green I
RNA makes
staining
we
recommend
to
use
SYBR
Green
it
Dllgonucleotides itepb~a with
a wide variety
of get Imaging
~(q~a.
The UV traps-illuminators
detection
limit
for
oligonucteotides
stained
with
SY13R
Green
I
is
1
2
hg
with
254
nm
epi-illuminator - UV epi-illuminators
or
312
nm
translilumination,
argon Ion
lasers.
StainingWithin Nnta: Double
time 30 stranded
minutes, DNA-bound
gels SYBR Green
are t
ready
to
image
or
photo-
graph stain fluoresces
w(thout green under
destaining. UV transillumination,
Gais
Number 5D0 that oontatn
of stainsp,1 DNA with
stock single stranded
solution raglans
is may
sufflc(ent show fluorescence
to that is
prepare more orange
a than green.
total
of
5 liters
of
working
solution.
which
can
be
used
to
stain
more
than
100
agarose
or
polyacryl
minigeis.
otes.s4s.zzoos ysoma Ma cPM ROCI18
38
SUBSTITUTE SHEET (RULE 26)
CA 02549849 2006-06-02
WO 2005/059178 PCT/US2004/041480
~~cluantaga 3.2 Preparation of working solution
Benefit Featuro Caution Since SYBR Green 1 hinds
Fast staining of Half the time to nucleic adds, it should
DNA necessary as
In agarose and for ethtdium be treated as a potential mutagen
bromide. and used with
-
polyacrylamide gelsi~ss than a the DMSO stock solution should be
quarter of appropriate care.
the
time necessary handled with particular caution as
for most DMSO is known to
silver staining.. facilitate the entry of organic
molecules into tissues.
When handling the DMSO stock solution
double
Lower mutageniclty Less mutagenic gloves, protective clothing and eyewear
than ethidium should be
bromide accordingfollowed
to Ames b
f
ti
h
ld b
d
t
l
Test results .
sa
e
ora
ory prac
ces s
ou
e
worn an
a
High sensiUvity Z5-100 times preparation at Dilute SYBR Green
more sensitive I Stock soluUon 1:10 000 In TE,
TBE,
than ethidium SygR Green or TAE buffer. The diluted
bromide solution has to be stored In
working solutions polypropylene containers
or bottles.
o eStalning with SYBR Green 1 is
very pH sensitive.
For optimal sensftivity, verify that
the pH of the staining
3. Procedures and solution at the temperature used
required materials for staining Is
- between 15 and 8 (preferably pH 8.0),
3.1 Before pisposat
you Aswith
begin all nucleic
acid stains,
solution
of SYBR
Green 1
PrestainedWe do should be
gets not poured
recommend through
prepar(ng activated
prestalned charcoal
gels before
with disposal.
The charcoal
must then
be inc)nerated
to
SYBR destroy
Green the dye.
I stain One gram
more of activated
than charcoal
1-2 easily
days
In
advance.
Gels absorbs
evious the dye
stained from 10
with liters
ethidlum of freshly
bromide prepared
can
b
e stained working
with solution:
SYBR
Green
I followln
g
subs
quentiy
the
standard
protocol
for
poststalning.
There
may
be
some
decreases
in
senskivity
when
compared
to
a gel
stained 3.3 Staining
only DNA following
with electrophoresis
SYBR
Green
I.
HlectrophoieslsPerform
elecVophoresis
on
an
agarose
gel
ar
denaturing Additional
polyarxytamide UV trans-
gel or epi-illuminators,
using: or respective
Imaging
TBE equipment
[89 and instruments,
mM such as
Tris Lumi-Imager
base, F1, argon
89 ion
mM
boric
acid,1
mM
EDTA,
pH 8j reagents
or required
lasers
or respective
imaging
instrument
TAE clear polyproylene
[40 contalnerfor
mM staining
Tris-acetate,1
mM
EDTA,
pH
8]
buffer
Note: TBE buffer
po or
not
add
any
SDS
to
the
electrophoresis
buffer TAE buffer
as
this
will
dramatically
reduce
staining
efficiency.
- Procedure
Please
refer to
the following
table for
the protocol.
Additional Note: For
buffers reaching
TBE sharp bands
[89 and low
mM Tris background
base,
89 mM
boric
acid,
required1 mM 'stain tt~e
for EDTA, gel directly
pH after electrophoresis.
B)
or
stainingTE [10
mM
Tris-HCt,1
mM
EDTA,
pH
8]
or
TAE StepAction
[40
mM
Tris-acetate,1
mM
EDTA,
pH
8]
buffer.
1 Place
the
gel
in a
fitting
polypropylene
container.
Handlingin the Notes
following There
table is no
please need
find to wash
information urea
about or
instructfansworking formaldehyde
for conditions out
for of the
successful) gels
staining. prior
staining.
proper 2 Add enough
staining staining
solution
to cover
the
Gel t]se clear potyproytene et.
container.for .
containersta(ning gels with protect
SYBR Green 1. the
staining
container
from
light
by
f0 ' Never use covering
polyvinylchloride/ it with
polysterene ar aluminum
glass container , foh
for the or placing
staining of agarose in the
or acrylamide dark.
gels.
Stain gets in a 3 Stain
fitting container. the
gel
for
approx.
30 min
under
constant
The size of the C.
container should agitation
be gently
at 15-25
not larger than 4 Ilituninatlon
the gel. of the
stained
gel:
Protect the staining
container from You can nm lime
light use.
WorkingMake sure that
the pH of the ultraviolet312 1-20 s
working
solutionsolution was adjusted trans-
to pH 7.0 to 8.5
pliute 1:10 000 illumination
In running buffer,
do not use water epI 25A 1-1.5
or minutes
AgarosePreferably use eluminatlon,320 (required
0,8196 agarose for max.
gels,
gets increase of in for greater sensitivity
gal concenVattan
increases
background. sensWvity especially
po not use agarose with
gels thicker than a
hand-help
lamp]
1 cm. If you use
thicker gels the
diffusion
of the stain into the Lumi312 .1-20
the gel is decreased s
and
the background lmager
is increased. F1
'
5 Photograph
the
gel
with
Polaroid
661
black
and
white
print
film
using
a SYBR
Green
gel
stain
photographic
filter.
Note:
Stained
gels
have
negligible
background
fluorescence,
allowing
long
film
exposure
using
an f-stop
of 4.5
Is adequate.
Roche Molet~ular.Biochexnicals
39
SUBSTITUTE SHEET (RULE 26)
CA 02549849 2006-06-02
WO 2005/059178 PCT/US2004/041480
~.4 Precasting gels with SYBR Green ii stain 3.5 Staining DNA belore
Electrophoresis
Gener,~l in the following.table please find the features of General
precast gels. .
See
references
(5,6)
for
general
methods
on
how
to
stain
DNA
before
electrophoresis.
It
may
be
necessary
to
optimize
the
protocols
In
these
references
for
the
DetectionThe DNA detection specific
Itmlt for gels application.
precast
limitwith SYBR Green We
I may be slightly have
poorer stained
(on the order 30 1
to 40 pg/band). mg
molecular
weight-marker
DNA
with
1:10
ODO
dilution
of
SYBR
Green
I
in
a
total
volume
MigrationThe rate of migration of
of DNA fragments 16
ml.
SYBR
Green
I
has
also
been
tested
as
a
of In gels containing prestaining
DNA SYBR Green I stain label
for
DNA
templates
In
bandshift
fragmentsmay be sfgniticandy assays,
slower than the and
rate has
been
prove
to
be
useful
in
this
of migration of application.
the same fragments
In a
get containing ,
no dye.
MobilityThe mobility of ProcedurePlease
DNA smaller molecules refer
tends to to
l the
d following
table.
of arger
be affecte
more than that
of
fragmentsfragments. StepAction
'
1 Incubate DNA with
a 1:10 000 dilution
of the
dye (In TE, TBE or
TAE) for at least
15 min prior
Procedure to electrophoresis.
The final
dilution
of the
SYBR
Green
t is
best
,
determined ' Follow el etectro
empirically, 2 hores(s as usual
as there g p
may be
some
nan
.
linear
effects
on the
migration
of different
fragment
site.
StepAction
1 Dilute
SYBR
Green
I stock
reagent
1:10
000 into
the gel 3.6 Removing
solutionJust SYBR Green
prior 1 stain
to pouring from double-stranded
the gel. DNA
The liquid
should
be as
cool
as possible
when
the dye Procedure
is added. At least
Boiling 99.996
and near of SYBR
boiling Green
temperatures I can
be removed
from
double-stranded
destroy DNA by
the ability simple
of SYBR ethanol
Green precipitation.
I to
stain
nucleic gyppeon
acid.
Do not
heat
SYBR
Green
I in
the
microwave.
2 Follow 1 Bring a solution of
gel electrophoresis DNA stained with
as usual. SYBR
Green ! reagent up
to 100 mM NaCI and
add
3 Illumination 2.5 volumes of absolute
of the or 9596 ethanol.
stained
get:
2 In
b
20
i
t
i
cu
You can nm Time e
use... n on
ce
a
m
- Centrifuge mixture
for at least 1D m1n
in
ultraviolet3121-20 a microfuge at 9'C.
s
trans-itlurnination Remove the ethanol
and wash the pellet
epi eluminatlon,2541-1.5 once w(th 70-9596
or minutes ethanol.
for greater320(required 3 Dry the pellet and
for resuspend double-stranded
sensitivity max. . DNA In buffer
sensitivity.
especially
with
a hand-help
lamp)
the Lumi 3121-20
fmager s
F1
A Photograph
the gel
with
Polaroid
667 black
and
white
print
film
using
a SYBR
Green
get stain
photographic
filter
Alote:
Stained
gels
have
negligible
background
nuarescence,
allowing
tong
film
exposure
using
an f-slap
of 4.5
is adequate.
Roche Molecular BJochemicals
SUBSTITUTE SHEET (RULE 26)
CA 02549849 2006-06-02
WO 2005/059178 PCT/US2004/041480
r4. References E-mail Adress Country
1 Schneeberger, C, et a1.1995. PCR Methods & App. 4. 234-238.
2 3hrger, V., et e! 1994. 8lomed Products 18. 68.
3 MutaUon Res.113,173 (1983)
4 PNA570, 2261 (1973)
MeUz Enrymol 217, 414 (1993)
6 Nudelc AcklS Rea 20, 2803 (1992)
aigenUnablochem~rocha.comArgentina
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WO 2005/059178 PCT/US2004/041480
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SUBSTITUTE SHEET (RULE 26)
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'F~~t~~~ 1~'t~~rt~rt~tiQ~i
~, Rcaa=lmae PCR'1'aqman
/~ ppendix c
OVERVIEW
Real-time quantitative PCR is a powerful tool that can be used for gene
expression
analysis, genotyping, pathogen detectionlquantitation, mutation screening and
DNA
quantltation. At the BRC, we use the ABi Prisrn 7900 Real Time Quantitative
PCR
instrument (TaqMan~) to detect accumulatiorw of PCR product, allowing easy and
accurate quantitation in the exponential phase of PCR reactions.
The ABI 7900 instrument continuously measures PCR product accumulation using a
dual-labeled flourogenic oligonucleotide probe called a TaqMan~ probe. This
probe is
labeled with two different flourescent dyes, the 5' terminus reporter dye and
the 3'
terminus quenching dye. The sequence of the oligonucleotide probe is
homologous to an
internal target sequence present in the PCR amplicon. When the probe is
intact, energy
transfer occurs between the two flourophors, and the fluorescent emission is
quenched.
During the extention phase of PCR, the probe is cleaved by 5' nuclease
activity of Taq
polymerase. Therefore, the reporter is no longer in proximity to the quencher,
and the
increase in emission intensity is measured.
Tha ABI 7900 Prism software aXamines, the fluorescence intensity of reporter
and
quencher dyes and calculates the increase in normalized reporter emission
intensity
over the course of the amplification. The results are , then plotted versus
time,
represented by cycle number, to produce a continuous measure of PCR
amplification.
To provide precise quantification of initial target in each PCR reaction, the
amplification
plot is examined at a point during the early fog phase of product accumulation
above
background (defined as the threshold cycle number or GT). Differences in
threshold
cycle number are used to quantify the relative amount of PCR target contained
within
each well.
Primers, Probes, and Reagents .
It is ess~ntial to have a well thought out experimental design for Real Time
PC.R. Good
primer and probe design is imperative. The BRC will design your probes and
primers
using Primer Express, the industry gold standard. Primers should be
synthesized and
purified at the BRC. This service is charged at our consultant rate of $50/hr.
We require
purified primers. Probes should ' be synthesized by Biosearch Technology.
(wwinr.biosearchtech.com). Black hole quench probes give the most consistent
data.
Average probe cost is $250.
If you plan to perform your own Taqman~ reactions, Applied Biosystems provides
a
number of kits specific to applications. See their web site
www.AppliedBiosystems.com..
Pacfl~ty Acknowledgement
so
SUBSTITUTE SHEET (RULE 26)
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Re~-'~'irrie'~PCR"'~aqman
The Taqman~ facility requests an aclmowledgement in the Methods section of any
publications resulting from this data. An example is "Real time quantitative
PCR was
conducted by the Biomolecular Resource Center at the University of California,
San
Francisco." Additionally, if your project required special attention by a
specific person at
the BRC, an example would be "Technical expertise was provided by (specific
name of
BRC personnel) of the Biomolecular Resource Center at the University of
California, San
Francisco."
Biomolecular Resource, Center Genetic Analysis
Faci 1 ity
UCSF, Science 983, San Francisco, CA 94143-0541
Phone: (415) 514-0101 x1; FAX: 502-7649
Email: dnaC~3cgl.ucsf.edu
sl
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l~~Qendt~t N
n~
fife ~~~hnr~~c~gi~~
tn~~t~uction manual
LUXT"" FluorogeniC Primers
For real-dime PCR and RT-PCR
Vexsion E
22 September 2003
25-D546
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Table of Contents
Introduction
...............................................................................
................:......................1
Designing and Ordering Custom LUX Primers
..............................................................3
Certified LUX Primer Sets for Housekeeping
Genes.....................................................5
Storing and Reconstituting
Primers............................................................:...........
..........5
Real-Time
PCR............................................................................
....................................7
Multiplex Real-Time
PCR............................................................................
...................11
Two-Step Real-Time RT-
PCR.............................................,..............................
............12
One-Step Real-Time RT-PCR
..........................................................................:....
........14
Troubleshooting ... .
...............................................................................
.......................18
Accessory Products
...............................................................,...............
........................19
Purchaser
Notification...................................................................
............'.....................20
Technical
Service........................................................................
....................,..............21
23
..
.
References
...............................................................................
...........
. ~ ......................
~
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lnt~rodrction
wefView LUX"' (Light Upon e_Xtension) Primers are an easy to use, highly
sensitive, and
efficient method for performing real-time quantitative PCR (qPCR) and
RT-PCR (qRT-PCR). LUX"' Primers combine high specificity and multiplexing
capability with simple design and streamlined protocols. LUX"' Primers
require no special probes or quenchers, and are compatible with melting curve
analysis of real-tune qPCR products, allowing the differentiation of amplicons
and primex dimer artifacts by their melting temperatures. You can-custom-
design LUX"' Primers' from a target sequence using Web- or desktop-based
software, or order predesigned and validated Certified LUX"' Primer Sets for
Housekeeping Genes.
Each primer pair in the LUX"' system includes a fluorogenic primer with a
fluorophore attached to its 3' end, as well as a corresponding unlabeled
primer. The fluorogeruc primer has a short sequence tail of 4-6 nucleotides on
the 5' end that is complementary to the 3' end of the primer. The resulting
hairpin secondary structure provides optimal quenching of the fluorophore
(see the figure below). When the primer is incorporated into the double-
stranded PCR product, the fluorophore is dequenched and the signal increases
by up to lt?-fold.
i:UX~" Primer . Itetative fluorescence:
Reaction .
0.1
Hairpin primer
1 rl~
0.4
Single-stranited primer
1.0
Extended primer
(double~nded DNA)
Labelitlg Each fluorogeruc LUX"' primer is labeled with one of two reporter
dyes-P.A.M
(ti-carboxy-fluorescein) or ~OE (trcarboxy-4', 5'-dichIoro-2', T-dimethoxy
fluorescein). Additional reporter dyes will be available in the future.
Continued on next page
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IfltPOdUCtiol1, Continued
Applications LUX"" Primers can be used in real-time PCR and RT-PCR to quantify
x00 or
fewer copies of a target gene in as little as 1 pg of template DNA or RNA.
They
have a broad dynamic range of 7-S orders.
Multiplex applications use separate FAM and JOE-labeled primer sets to
detect two different genes in the same sample. Typically, a custom-designed
FAM-labeled primer set would be used to detect the gene of interest, and a
JOE-labeled Certified LUX"" Primer Set would be used to detect a
housekeeping gene as an internal control.
ltlstfuit'1B11t ' LUX'" Primers axe compatible with a wide variety of real-
time PCR
Compatlblllty instruments, including but not limited to the ABI PRTSM~
7700/7000/7900 and
GeneAmp~ 5700, the Bio-Rad iCycler'", the Stratagene Mx4000"", the Stratagene
Mx3000"", the Cepheid Smart Cycler'a, the Corbeu Research Rotor-Gene, and
the Roche LightCycler~.
ABI PRISM is a registered trademark of Applere Corporation. GeneAmp is a
registered trademark of
Roche Molecular Systems, Inc. LightCycler is a registered trademark of Idaho
Technologies, Ina
iCycler, Mx4000, Mx3000, Rotor-Gene, and Smart Cycler are trademarks of their
respective
companies.
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~Deaigning and Ordering Custom LUXTM Primers
LUX'~ Desig~et' To design and order custom LUX"" Primers for your genes of
interest, visit the
Primer Design Invitrogen LLJX"" Web site at wwvw.invitrogen.com/L11?( and
follow the link to
Software the LUX"' Designer software. The software is available as either a
Web-based
application or a Microsofl'~ Windows~ compatible download. Follow the step-
by-step instructions in the software to submit your target sequence and
generate primer designs.
LUX'" Designer will automatically generate one or more primer designs based
on each sequence you submit and the selected design parameters. The design
software includes algorithms to minimize primer self complementarity and
interactions between primers. It also assigns rankings to the generated
designs--based on primer melting temperature, hairpin structure, self
annealing properties, etc.-to aid in selection.
When the designs have been generated, you can review them, select a design,
select the fluorophore labels, and place your order.
Guid811~es fol' When you submit a target sequence containing your gene of
intexest, keep in
Submiftitlg a mind the following design criteria:
°TePget Sequence , ~e optimal amplicon length for real-time PCR ranges
fxom 80 to 200
bases. You can specify a minimum, optimal, and maximum amplicon
length when you submit the sequence.
~ The target sequence should be at least 10 bases longer than the minimum
amplicon size you select. The longer the sequence, the more likely that
an optimal primer design can be developed.
The sequence must contain only standard ILTPAC (International Union of
Pure and Applied Chemistry) letter abbreviations. '
~ When you select the design parameters, the default melting temperature
range is 60-b8°C. Do not change this default unless the design engine
finds
no primers in this range. For primers in this range, PCR annealing
temperatures from 55° to 64°C axe appropriate.
When you first submit a sequence, the Disable Score-Based Rejection checkbox
should not be checked; the resulting scores provide an important measure of
primer suitability. Scores in the range of 0.0-4,0 are acceptable. If no
primers
with a score of 4.U or lower can be generated from a sequence, you can disable
score-based rejection and redesign the primers. Note that if you select a
primer
with a highex score, the efficiency of the reaction may be less than optimal.
See
the LUX"' Designer Help for additional guidance.
SeleCtillg a~Primec After you submit your sequence, LUX'" Designer will first
generate one or
Design more designs for the labeled primex. The labeled primer can be either
the
forward or the reverse prinner. After you select a design for the Labeled
primer,
you will be prompted to select a design for the corresponding unlabeled
primer.
Continued on next page
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Designing and Qrdering Custom LUXTM Primers, continua
Selecting Labels After you have selected a primer set (labeled and unlabeled)
for a particular
sequence, you can specify the particular label and synthesis scale. Custom
LUX"' Primers axe provided ize 50 nM or 200 nM synthesis scale,
When selecting labels in a multiplex reaction, we recommend using the FAM
label for your gene of interest and the JOE label for the housekeeping gene
that
you will use as the internal control. Certified LUXT" Primer Sets for
Housekeeping Genes are recommended for the JOE-labeled control gene.
Placing the Order After you have selected the label and synthesis scale, you
can submit your
order to Invitrogen using the Web site or by e-mail or fax. Each.primer order
will be shipped durectly from Invitrogen's Custom Primer Pacilities. Labeled
primers are supplied in an amber tube; unlabeled primers are supplied in a
clear tube. '
Each primer ordered from lnvitrogen's Custom Primer Facilities comes with a
Certificate of Analysis (COA) verifying the amount and sequence.
PI'OdUCt Custom LUX"' Primers are tested pest synthesis by optical density
(OD) ratio
Qualification measurements and mass spectroscopy to ensure efficient dye
labeling and
correct molecular weight and composition.
See the Certificate of Analysis shipped with each primer for more information.
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Storing and Reconstituting Primers
Primer Storage atld Store primexs at 20°C in the dark LU7C"' Primers
are stable for:
Stability , >t2 months when stoxed at-20°C in lyophilized form.
~ >6 months when stored at 20°C in solution.
Stability can be extended by storing at 70°C.
ReGOllstitutitlg Custom LUX"' Primers are provided lyophilized in 50-nmole or
200-nmole
Primers synthesis scale. To reconstitute primers, centrifuge the tube for a
few seconds
to collect the oligonucIeotide in the bottom of the tube. Carefully open, add
an
appropriate volume of TE buffer or ultrapure water, close the tube, rehydrate
for 5 minutes, and vortex fox 15 seconds.
We recommend that you rehydrate primers at concentrations greater than
ltlvl. To prepare a 100 ExM primer stock solution, multiply the primer
amount in nmoles by ten to determine the volume of diluent in p1.
.After reconstitution, store the pr'smer stock at-20°C in the dark,
where it will
be stable for 6 months or more.
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Cerfiified LU~~' Primer Sets for Housekeeping Genes
CeCEifled LUX~ Certified LUX"' Primer Sets for Housekeeping Genes are
predesigned primer
Primer Sets for . sets for genes that are commonly used as internal controls
for normalizing ,
HOUSekeeplllg real-time RT-PCR experinnents. These primer sets have been
optimized and
Genes functionally validated to provide accurate, reproducible results using
standard
LUX'" protocols. They are supplied ready to use in TS buffer.
Each Certified LUX"' Primer Set includes a FAM- or JOE-labeled LUX'"' primer
and a corresponding unlabeled primer. Each primer (labeled and unlabeled is
supplied at 100 Etl and a concentration of 10 ltM. Available sets axe listed '
below. For additional informs#ionD visit r-irtvit:~~en.comlLLJX.
GenBank Forwardl Cat. Cat, RelativeCDS 1'CR
Product AccessionReverse no. no. reaslon LocationProduct
no. Label FAM Ot3label Size
label Ran
a
Human
eves
l8SrRNA X032Q5 Forward 115FiM-Ol115f1~I-02++f+ n!a l0I-150b
h ACTlN Nlvt<001101Forward 101H-01101H-02+++ Bxons 101-I5D
2/3 b
hATPSaseNI~Iv.OD1686Forward 108I3-OllOBH-02+++ n/a 101-150
b
h132M 1VM_0020Q8Forward I13H-Ol113H-02+++ Exans 101-150
if b
2
hGAPI~H t3M_002046Forward 100H-01100H-02++t- Exons 151-200
4/5 h
hPGK1 NM_000291Porward 109H-Ol109Ii-02+++ nla 50-i00
hPPlA NM_021130Forward 106H-01lOdH-02+++ Faeons2/350-100b
hRPL4 NM~000968Reverse lOt3H-Ol103H-02+++ E~tons8/910I-150b
hEEFIG NM_001404Forward 147H-01107H-02++ nJa 50-IODb
hHPRTI NM U00194Reverse 105H-01105H-02++ Hxons 50-100
5/6 b
hSDHA NM_D04168Forward 102H-Ol102H-02++ fixons 50-100
' 12/13 b
hTPRC NM_003234Forward 111H-0I111H-02++ Futons 101-150
10/11 b
hGI3S 13~OD0181Forward 112H-01I12H-02+ Rxons 101-150
7/8 b
hHHMS NNiv000190Forward 110H-01I10H-02+ Facons 50-lOD
2/3 b
hTBP NM_003194Porward 104H-01104Fi-02+ Bxons 101-150
3/4 b
hLtBC NIv~021009Forward 114H-Ol114H-02+ n/a 5D-l0U
b
Mouseirat
eves
185 rRNAX03205 Forward 115HM-0i11513M-02t+++ n(a 10I-150
b
m ACTIN NM UO?S93Forward 101M-01101M-02t++ fixons 1DI-150
2/3 b
mB2M X01838 Forward 113M-01iI3M-02+++ n/a 50-100h
mBfiPiG AP321I26Ponvard 107M-01107M-02+++ n/_a 101-150_
mGAFDFI Ntv1'008084Forward LOOM-01100M-02+++ Exons 151-200
4/5 b
mPGKt N1V1_,008828Forward 109M 109M-(12t++ Exons 101-I50
01 1!2 b
mPPIA. NM_008907Reverse 106M-Ol106M-02+++ Exons 50-100
1/2 b
mRPh NM_02Z510Forward 103M-01103M-02+++ Exons 151-200b
2!3
irs-ipRT1NI~013556Forward 105M-Ol105MD2++ Fxons 50-100b
6/7
mSDHA AF095438Forward 102M-Ol102M-02++ Exons 50-100b
6/9
mATPSaseMt~016774Porwerd 108M-01I08M-02t n/a 50-1006
mGUS NM_OI0368Forward 112M-01112M-02+ Bxons 50-1DD
7/8 b
mHBMS XM_129404Reverse 110M-01110M-02+ Fxons 50-IOD
4/5 b
mTBP NM-013684Forward 104M-011021V1-02+ Exons 101-150
3/4 b
mTPRC NIv~01I638Forward 111M-01111M-02+ Exons I01-I50
2/3 b
Dros
hila
enea
d185rRNAAYD37174Reverse 1151-01115D-02++++ n1a 101-150
. b
dActln NM~079486Forward 101D-01'lOlD~02+++ Exons2/35p-lODb
.
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Reat-Time PCR
Int1'oduCtion . This section provides guidelines and protocols for performing
real-time PCR
using LUX'" Primers.
Ten'ipiate The target template for real-time PCR is linear single-stranded or
double
SpeCifiCations stranded DNA, cDNA, or circular DNA (such as plasmids). The
amount of
DNA typically ranges from 10Z to 10' copies or 1 pg to 20 itg of template.
See page 12 far instructions on generating cDNA using reverse transcription as
part of two-step real-time RT PCR.
Primer Far optimal PCR conditions, primer iitrations of 50-500 nM per primer
are
Concentration recommended. The sample reactions on pages 9-10 use 200 nM of
each primer,
equivalent to 1 ~xI of a 10 uM primer solution.
Magnesium The optimal Mg*'' concentration for a given target/primer/polymerase
.Concentration combination can vary between 1 mM and 10 mM, but is usually in
the range of
3 mM. See the sample reactions on pages 9-10.
dNTP The optimal concentration of dATP, dCTP, dGTP, and dTTP is 200 iaM each.
If
COnCentt'atiott dUTP is used in place of dTTP, its optimal concentration is
400 ~M.
Enxyme We recommend using a "hot-start" DNA polymerase, preferably one that
has
SpeCi~CattonS been optimized for real-time PCR. Platinum~ Quantitative PCR
SuperMix
UDG (Catalog no.11930-01~ is a 2X-concentrated, ready-to-use mixture
containing all coxriponents except primers and template. It uses Platinum Tag
DNA polymerase and has been specifically formulated to provide optimal
performance in, real-time PCR systems.
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R.eaf-Tfme PCR, continues
instrument LUX'" Primers are compatible with a wide variety of real-time PCR
Specifications instruments with various detection capabilities. See page 2 for
a partial list of
compatible instruments. A protocol for instruments that use PCR tubes/plates
is provided on page 9. A protocol for the LightCycler~ is provided on page 10.
At a n~ixumum, the instrument used to perform real-time PCR with LUX"'.
Detect fluorescence at each PCR cycle
>3xcite and detect FAM-labeled LUXn' Primers near their
excitation/emission wavelengths of 490/520 nm, and/or
~ Bxcite and detect jOE-labeled LU?C"' Pximers near their
excitation/ennission yvavelengths of 520J550 nm
Please refer to the specific instrument's user manual fox operating
instructions.
itlstt'llmetlt Please follow the manufacturer's instructions for configuring
your real tune
Settings PCR instrument for use with LUX"' Primers. Note the following
settings:
~ LUX'" Primers are compatible with standard melting curve analysis, if
your instrument allows that option. Program your instrument accordingly.,
~ The quencher setting on the insirument should reftect~the fact that LUX"'
Primers do not contain a quencher.
~ We recommend the use of ROX Reference Dye (Cat. no. 12223-023) for
normalization of well-to-well variation with instruments That are
compatible with this option. Adjust your instrument settings accordingly.
Continued on next page
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Rest-Time PCR, Continued
Protocol for The following protocol uses Platinum~ Quantitative PCR SuperMix-
UDG with
(nstrUtnent5 Using ROX reference reagent. It has been optimized for use with
real-time PCR
PCR Tubes or instruments that use PCR tubes or plates. A protocol for the
Roche
Piates LightCycler~ is provided on the following page.
Note: The following protocol uses a 50-Etl reaction volume; smaller volumes
may be used, depending on the requirements of your instrument. Before
proceeding, see the real-time PCR guidelines on the previous pages. For
multiplex reactions, see the guidelines on page 11.
1. To reduce well to-well variafi.on, prepare a Master Mix of all the reaction
ingredients except template. The following table provides Master Mix
volumes for one reaction and 50 reactions (scale up or down as needed):
Comp,Qnent o1/1 Vol/50 rxiis
Platinum Quantitative PCR SuperMix-UDG' 25 ~l 1250 ~1
ROX Reference Dye 1 E.~l 50 ~.il
Labeled LI7X"' Primer (10 uM) 1 gel 50 ~.il
Unlabeled primer {10 uM) 1 w1 50 N.1
Sterile distilled water2 to 40 p1 to 2000 Nl
'60 U/ml Platinum's Tag DNA polymerase, 40 mM Tris-HCl (pH 8.4),100 mM TCCI,
6 rnl4l MgClz, 400 f.~M dGTP, 400 ECM dATP, 400 tiM dCTP, 800 ~M dUTP,
40 U/ml UDG, and stabilizers.
zor use DNase/RNase Free DistHled Water (Cat. No.10977-015).
2. Program the real-time PCR instrument as follows:
3-Step Cycling (recommended) 2-Step Cycling (optional)
50°C, 2 min hold (TJDG treatment) 50°C, 2 min .hold (UDG
treatment)
95°C, 2 min hold 95°C, 2 min hold
45 cycles of: 45 cycles of:
95°C, x5 s , 95°C,15 s
55°C, 30 s 60-65°C, 30-45 s
72°C, 30 s
Melting Curve Analysis (optional)
Refer to instrument documentation
3. Add 40 ~.~1 of the Master Mix to an optical PCR tube or each well of a
96-well PCR plate.
4. Add 10 Eti of template diluted in TE or sterile dHzO to the tube or each
well of the 96-well PCR plate. Cap or seal the tube/plate.
5. Gently mix and make sure that all components axe at the bottom of the
tube/plate wells. Centrifuge briefly if needed.
6. Place reaction in the real-time PCR instrument and run the program.
Collect and analyze results.
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Real-Time PC-R, contir~uea
P1'otOCOI for the q'he following protocol uses Platinum~ Quantitative PCR
SuperMix-UDG and
Roche LightCycler~ has been optimized for the Roche LightCycler~. Consult the
LightCyclef~
documentation for detailed instructions on preparing the capillary tubes and
operating the instrument. FAM-labeled LUX'" Primers are also compatible
with Roche enzyme mixes.
Note: jOE-labeled LUX"' Primers are not compatible with the current version
of the LightCycler~; use FAM-labeled primers only. The following protocol
uses a 20-pt reaction volume. Before proceeding, see the real-time PCR
guidelines on the previous pages.
1. To reduce well-to-well variation, prepare a Master Mix of all the reaction
ingredients except template. The following table provides volumes for one
reaction and 34 reactions (scale as needed):
Component Vol/1 rxn Vo1/34 rxns
Platinum~ Quantitative PCR SuperMix-UDG' 10 Eel , 340 Evl
FAM labeled LUX'" Primer (10 uM) 1 pi 34 E.vl
Unlabeled primer (10 ~ 1 N.l 34 Eel
Bovine serum albumen (5 mg/ml)z 1 ~.il 34 Eel
Platinum~ Tag DNA Polymerase3 0.12111 y,tl
Sterile distilled water to 18 pi to 612 lsl
'60 U/ml Platinum~ Tag DNA polymerase, 40 mM Tris-HCI (pH 8.4),100 mM KCI,
6 mM Mgt'1z, 400 ).tM dGTP, 400 ltM dATP, 400 ~M dCTP, 800 itM dUTP,
40 U/ml UDG, and stabilizers.
zValidated with non-acetylated Ultrapure BSA (10% solution) from Panvera (Cat.
nos. P2489 and P2046).
3Tota1 units of Platinum~ Tag DNA Polymerase in the reaction is 1.2 (including
0.6 U
in Platinum~ Quantitative PCR SuperMix-UDG
'or use DNase/RIVase Free Distilled Water (Cat. No.10977-015).
2. Set the fluorescence on the Roche LightCycler~ to the P1 channel. .
3. Program the instrument as follows: . '
Thermal Cycling Melting Cwrve Analysis (optional)
Program choice: Amplification Program choice: Melting curve
Analysis mode: Quantification Analysis mode: Melting curves
Cycling: Cycling:
50°C, 2 min hold (UDG treatment) 95°C, 0 s
95°C, 2 min hold 55°C,15 sec
45 cycles of: 95°C, 0 (ixtcxease 0,1°C/s with
94°C, 5 s continuous acquisition)
55°C,10 s (single acquire) 40°C, 0 s
72°C,10 s
4. Add 18 ~1 of Master Mix to each capillary tube of the LightCycle~.
5. Add 2 E.vl of template to each tube, and cap the tube.
6. Centrifuge the tubes at 700 x g for 5 seconds.
Place the reaction tubes in the rotor of the LightCyclez~ and run the
prograxn. Collect and analyze results.
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Multiplex Real-Time PCR
MuitipleX In multiplex real-time PCR, different sets of primers with different
labels are
Real Time PCR used to amplify separate genes on the template DNA. Multiplexing
with LUX"'
Primers offers simplified kinetics when compared with probe based
technologies, because only two oligos are used per target.
LUX'" Primers have been tested in multiplex reactions using a FAM-labeled
primer set for the gene of interest and a jOE-labeled set for a housekeeping
gene used as an internal control to normalize between different reactions. We
recommend using Certified LUX'" Primer Sets for F3ousekeeping Genes for the
internal control.
In a standard multiplex reaction, you can include the additional primers at
the
same volumes and concentration as the primers in a singleplex reaction, as
shown in the example mixture below:
Component Volume
Platinum~ Quantitative PCR SuperMix-UDG (2X) 25 E,~I
ROX Reference Dye (50X) 1 ~.~I
Template 10 ~I
Forward primer 1 (FAM label)1 i.i,I
(10 ~
Reverse primer 1 (10 ~.iM) 1 ial
Forward primer 2 QOE label)1 ~.~1
(10 liM)
Reverse primer 2 (1D ~.M) 1 N.I
Sterile distilled water to 50 ~l
,
Reduce the volume of wafer to compensate for the additional primer volume.
All other reaction volumes remain the same.
Follow the thermal cycling guidelines provided in Protocol for Instruments
Using PCR Tubes or Plates on page 9. If you have difficulty performing the
multiplex reaction using these guidelines, see the optimization hints below:
Optimizing If you notice a decline in real-lime PCR efficiency in your
multiplex real-time
Muitipiex PCR, you can optimize the reaction by performing the steps listed
below.
Collditiolls Note: We recommend that you perform one optimization step and
then repeat
the reaction to test for efficiency before moving on to the next step:
1. Reduce the primer concentration of the gene with the highest abundance
(typically the housekeeping gene) to 1 /4 the primer concenixation of the
other gene. For example, in a standard 50 ~I reaction, you would add the
primers for the less abundant gene at 1 NI each, and add the primers for
the more abundant gene at 0.25 NI each.
2. Increase fine MgCl2 in the reaction from 3 mM to 5 mM.
3. Double the amount of polymerise enzyme (to 0.06 U per pi of reaction
volume). If you are using Platinum~ Quantitative PCR SuperMix-UDG,
add Platinurn~ Tag DNA polymerise stand-alone enzyme (Catalog no.
10966-018) to double the amount of enzyme.
4. Increase the dNTP concentrations in the reaction to 400 l.iM each.
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Two-Step Reat-Time RT-PCR
IntroduCtlott For real time RT-PCR applications, vve recommend a two-step
protocol so that
the RT and PCR modules can be optimized separately for maximum efficiency
and specificity.
This section provides an optimized protocol for performing reverse
transcription as part of a two-step real-time RT-FCR protocol. You can use the
resulting cDNA in the real-time PCR reaction on pages 7-10.
Templets ~ The target template for real-time RT-PCR is RNA-usually total
cellular RNA
SpeCifICatiofls or mRNA. The amount of RNA typically varies from 1 pg to 100
ng of
template per assay. The purity and integrity of the RNA have a direct impact
on results. RNase and genomic DNA contamination are the most common
problems, and purification methods should include RN 'se inhibitors and
DNase digestion to minimize these.
We recommend using the Micro-to-Midi Total RNA Purification System
(Catalog no. 12183-018) or TRIzoI~ reagent (Catalog no. 15596-026) to isolate
total RNA. High-quality total RNA can be purified from as little as 100 cells
up
to 10' cells or 200 mg of tissue.
To isolate mRNA, we recommend using the FasfiTrack~ 2.0 mRNA Isolation
Kit (Catalog no. K1593-02).
EnZyl'118 We recommend using Superscript'" II or Superscript'" 1TI RT for the
reverse
SpeCi#ICations transcription reaction. The sample protocol on page 13 uses the
Superscript"'
First-Strand Synthesis System for RT-PCR (Catalog no.11904-018), available
from Invitrogen, which includes all components needed for the first strand
synthesis reaction except the RNA.
Removing We recoxnrnend that you decrease the genomic DNA content in the RNA
Gel7omiC DNA f1'otll sample by performing a digest with DNase I, Amplification
Grade (Catalog
RNA Samples no.18068-015), as described below. The DNase I digest is designed
for up to
1 Fg of RNA; for larger amounts of RNA, increase volumes accordingly.
Combiune the following in a tube on ice:
Component Conc. Volume
RNA template - x E.il
DNase reaction buffer 10X 1 1,i1
DNase I, Amplification1 U/ul 1 lil
Grade
DEPC-treated ddHzO to 10 E.~l
1. Incubate at room temperature fox 15 min.
2. Add 1 ~.~1 of 25-mM EDTA solution to the reaction mixture and incubate at
65°C for 10 min to inactivate the DNase I.
Continued on next page
67
SUBSTITUTE SHEET (RULE 26)
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Two~Step Real-Tir~r~e RT-PCR, continues
ReVePSe The following protocol can be used with either the SuperScript'" First-
Strand
Transcription Synthesis System for AT-PCR (with SuperScript~" II RT) or the
SuperScript"' III
PI'OtOCO) First Strand Synthesis System for RT-PCR (with SuperScript'" III
RT). The
protocol has been optimi2ed for LUX"' Primers. Follow this protocol to
generate cDNA, which can then be used in real-time PCR (see pages 7 10).
1. Combine the following a tube on ice.
kit components in For multiplex
reactions, a master mix
without RNA may be prepared:
Oligo(dT),~-,s (0.5 pg/iil)
or
Oligo(dT)ZO (50 mM)" 0.5 ltl
Random hexamers (50 ng/~1) 0.5 ~.i
RNA (up to 1 fig) x lti
lOx Buffer 2 ~1
25 mM MgClz 4 ~1
mM dNTP 1 ~1
0.1 M DTT 2 Etl
RNase~UT"' (40 U/1.~1) 1 itt
SuperScript"' II RT (50
U/Etl)
or SuperScript'" III RT 1 i,il
(200 U/NI)
DBPC-treated ddHzO to 20 01
'Ollgo(dT juac is recommended
for use with SuperScript"'
lI RT; oligo(dTko is
recommended for use with
5uperScript"' 1TI RT
2. Incubate tube at 25C
for 10 min.
3. Incubate tube at 42C
for 50 min.
4. Terminate the reaction
at 70C at 20 min, and then
chill on ice.
5. Add 1 E.~l (2 U) of E.
coTi RNaseH and incubate
at 37C for 20 min.
Store the reaction at 20C
until use. Use 2,8 0.1
of cDNA for real-time PCR,
as
described on pages 7 10.
68
SUBSTITUTE SHEET (RULE 26)
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C)ne-Step Real-Time RT-PCR
Int1'oduEtion This section provides information and a protocol for performing
one-step xeal-
time RT-PCR using LUX'" Primers. One-step RT PCR is a complex reaction in
which both reverse transcription and PCR are carried out in the same tube.
The one-step reaction described in this section uses the SuperScript'" III
Plafinum~ One-Step Quantitative RT-PCR System for superior specificity and
sensitivity with LUX"' Primers.
Primer For optimal PCR, pximer titrations of 50--500 nM per primer are
recommended.
Concentration! The 50-~1 sample reaction on page 16 uses 200 nM of each
primer, equivalent
to 1. g1 of a 10 ~IVI primer solution. See also the Important note below.
t ~ In one-step RT PCR, the reverse primer drives the xeverse ixanscription
w, theN.p r
reaction. We have found that doubling the concentration of the reverse primer
"' from 200 nM to 400 nM can in some cases decrease the cycle threshold for
detecting a given target concentration, and thus increase sensitivity. See
. pages 3-4 fox guidance on primer design.
Template The target template for one-step teal-time RT PCR is RNA usually
total
SpeCificationS cellulax RNA or mRNA. The amount of template typically ranges
from 1 pg to
100 ng per assay. The purity and integrity of the RNA have a dirrect impact on
results. RNase and genomic DNA contamination are the most common
problems, and purification methods should be designed to avoid these.
We recommend using the Micro-to-Midi Total RNA Puxification System
(Catalog no 12183-018) or TRIzol~ reagent (Catalog no.15596-026) to isolate
total RNA. High-quality total RNA can be purified from as little as 100 cells
up
r to 10' cells or 200 rng of tissue.
To isolate mRNA, we recommend using the FastTraclc~ 2.0 mRNA Isolation
Kit (Catalog no. K1593-02).
EllZyhle The one-step RT PCR enzyme mix should be optimized for real-time PCR.
We
Specifications recommend usiung the SuperScript"' III Platinum~ One-Step
Quantitative RT
PCR System (Catalog nos.11732-020 and -088), which uses a SuperScript'" III
RT/Platinurn° Tag enzyme mix. It has been optimized for use in
zeal-tune
fluorescent PCR systems. See the sample reactions on pages 16-17.
Megtlesluto The optimal MgClz concentration for a given
target/primer/polymerase
Concentration combination can vary between 1 mM and 10 mM, but is usually in
the range of
3 mM (see the sample xeaction on page 16).
dNTP The optimal concentration of dATP, dCTP, dGTP, and dTTP is 200 ~M each.
If
COttce~ttt'ation dUTP is used in place of dTTP, its optimal concentration is
X00 i.iM.
Continued on next page
69
SUBSTITUTE SHEET (RULE 26)
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One-step Real-Time RT-PCR, continued
lnStPUmellt LUX'" Primers are compatible with a wide variety of real-time PCR
Specifications instruments with various detection capabilities. See page 2 for
a partial list of
compatible instruments. A one-step real-time RT PCR protocol for instruments
that use PCR tubes/plates is provided on page 16. A protocol for the
LightCycle~ is provided on page 17. At a minimum, the instrument used to
perform one-step real-time RT-PCR with LU?C"' Primers must be able to:
~ Detect fluorescence at each PCR cycle
~ Bxcite and detect FAM-labeled LUX"' Primers near their
excitation/emission wavelengths of 490/520 nm, and/or
~ Bxcite and detect ]OE-labeled LUX"' Primers near their
excitation/emission wavelengths of 520/550 nm
Ittstl'umerit Please follow the manufacturers instructions for configuring
your real-time
Settings PCR instrument for use with LUX"' Primers. Note the following
settings:
~ LUX"" Primers are compah'ble with standard melting curve analysis, if
your instrument allows that option. Program your instrument accordingly.
~ The quencher setting on the instrument should reflect the fact that LUX'"
Primexs do not contain a quencher.
~ We recommend the use of ROX Reference Dye (Catalog no.12223-023) for
normalization of well-to-well variation with instruments that are
compatible with this option. Adjust your instrument settings accoxdingly_
Pxogram the instrument to perform cDNA synthesis irnrn, ediately followed
by PCR amplification.
Removing We recommend that you decrease the genamic DNA content in the .RNA
GettOmiC DNA fPOlri sample by performing a digest with DNase I, Amplification
Grade (Catalog
RNA Samples no.18068-015), as described below. The DNase I digest is designed
fox up to
1 ~,g of RNA; for larger amounts of RNA, increase volumes accordingly.
Combine the following in a tube on ice:
Component Conc. Volume
RNA template - ~ x g1
DNase reaction buffer 10X , 1 g1
DNase I, Amplification Grade 1 1 ~1
U/ul
DEPC-treated ddHzO to 10 ~.~1
1. Incubate at room temperature
for 15 min.
2. Add 1 1.r1 of 25-mM EDTA solution
to the reaction mixture and incubate
at
65C for 10 min to inactivate the
DNase I.
To verify the absence of genomic DNA iun the RNA sample, prepare a control
reaction identical to the reactions on pages 16-17, using 2 U of Platinum~ Tag
DNA polymerise (Catalog no.10966-018) in place of the
SuperScript~° III
RT/Platinum~ Tag Mix.
Continued on next page
SUBSTITUTE SHEET (RULE 26)
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One-Step Real-Time RT-PCR, Continued
Protocol fob The following protocol using the SuperScript'" III Platinum~ One-
Step
ltlStl'tlmet7ts Using ~~~~dve RT PCR System has been optimized for LilX"'
Prinners. Further
PCR Tubes or op~a0on may be required.
Plates Note: Keep all components, reaction mixes and samples on ice. After
assembly, transfer the reaction to a thermal cycler preheated to the cDNA
synthesis temperature and immediately begin RT PCR. We recommend
performing the cDNA synthesis reaction at 50°C, but higher temperatures
(up
to 60°C) may be required for high GC content templates.
RNase inhibitor proteins, such as RNaseOLlT'" (Catalog no.10777-019), may be
added to the reaction to safeguard against degradation of RNA.
1. The following table provides Master Mix volumes for a standard 50-~,~1
reaction size. Note that preparaiion of a master mix is crucial in
quantitative applications to reduce pipetting errors.
Component Voll1 rxn Vol/100 rxns
SuperScript''" III RT/Platinum° Tag Mix 1 ltl 100 Etl
2X Reaction Mixt 25 pl 2500 E.tl
ROX Reference Dye (optional) 1 ul 100 Etl
Labeled LU?C~' Primer (10 EtM} 1 ul 100 let
Unlabeled primer (10 ttM)a 1 ul 100 E.tl
RNaseOUT'" (optional) 1 itl 100 Id
Sterile distilled water to 40 ul to 4000 pl
~SuppIied at 2X concentration includes 0.4 mM of each dNTP and 6 mM MgSO~
Seethe Important note on primer concentration on page 14.
2. Program the instrument with the following thermal cyclingprotocol (for
cDNA synthesis, use a 15-min incubation at 50°C as a starting point):
cDNA synthesis:
54°C for 25 min hold
PCR:
95°C for 2 min hold
40-50 cycles of:
95°C,15 s
60°C, 30 s
Melting Curve Analysis (optional)
Program according to instrument instructions
3. For each reaction, add 40 ~.l of the master mix to a 0.2-ml microcentrifuge
tube or each well of a 96-well PCR plate on ice.
4. Add 10 ul of sample RNA (1 pg to 1 ttg total RNA) to each tube/plate well,
and cap or seal.
5. Gently mix and make sure that au components axe at the bottom of the
tube/plate wells. Centrifuge briefly if needed.
6. Place reactions in a preheated thermal cycler programmed as described
above. Collect data and analyze results.
71 Continued on next page
SUBSTITUTE SHEET (RULE 26)
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One-Step Reaf-Time RT-PCR, cont~ntaea
PfotnCOl fof the The following protocol using the SuperScriptT" III Platinum~
One-Step
RoChe LightCyClet~ ~~B~dve RT-PCR System has been optimized for LUX'" Primers
and the
Roche LighfCycler~. Further optimization may be required. FAM-labeled
LUX'" Primers are also compatible with Roche enzyme mixes.
Note: ]OB-labeled primers are not compatible with the current version of the
LightCyclex'~; use FAM labeled primers only.
After assembly, transfer the reaction to a thermal cycler preheated to the
cDNA synthesis temperature and immediately begin RT-PCR. We recommend
performing the cDNA synthesis reaction at 50°C, but higher temperatures
(up
to 60°C) may be required fox high GC content templates.
RNase inlu-bitor proteins, such as RNaseOUT"" (Catalog no.1OT77-019), may be
added to the reaction to safeguard against iiegradation of RNA.
1. The following table provides Master Mix volumes for a standard 20,u1
reaction size. Note that preparation of a master mix is crucial in
quantitative applications to reduce pipetting errors.
Component Vol 1 rxn Voll34 rxns
SuperScript~' III RT/Platinum~ Taq Mix 0.8 E.t1 27.2 ttl
2X Reaction Mixl 10 ).1l 340 u)
FAM labeled LUX"° Primer (10 p,M)2 1 N.1 34 it)
Unlabeled primer (10 ~' 1 u) 34 NI
Bovine serum albumin (5 mg/mI)' 1 ~l 34 N.1
Sterile distilled water to 18 p) to 612 N.1
'Includes 0.4mM of each dIVTP and 6 mM MgSOa
zIn the I,ightCycle~ reaction, the LUX'" Fiuorogenie Primer must be PAM
labeled.
35ee the Ianportant note on primer concentration on page I4.
"Validated with non-acetylated Ulbrapure BSA (10°I° solution)
from Panvera (Cat.
pos. P2489 and P2046)
2. Set the fluorescence on the Roche LightCycler~ to the F1 channel.
3. Program the instrument as follows:
Thermal Cycling Melting Curvy Analysis (optional)
Program chofce: Amplification Program choice: Melting curve
Analysis mode: Quantification Analysis triode: Melting curves
Cycling: Cycling:
45°C, 30 min hold (cDNA synthesis) 95°C, 0 s
95°C, 2 min hold 55°C,15 sec
50 cycles of: 95°G, 0 (increase 0.1°C/s with
95°C, 5 s continuous acquisition)
55°C,10 s (single acquire) 40°C, 0 s
72°C.10 s
4. Add 18 p) of Master Mix to each capillary tube of the LightCyclex'a on ice.
5. Add 2 NI of sample RNA (1 pg to 1 ttg total RNA) to each capillary tube
and cap the tube.
6. Centrifuge the tubes at 700 x g fox 5 seconds.
7. Place the reaction tubes in the rotor of the LightCyclez'a and run the
program. Collect and analyze results.
72
SUBSTITUTE SHEET (RULE 26)
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Troubleshooting
Problem Cause Solution
Signal in controls with no DNA contamination Hnsure that amplification
reactions are assembled
template in a DNA-free environment. Use of aerosol-
resistant barrier tips is recommended. Take care
to avoid cross-contamination between primers or
template DNA in different reactions. Run PCR
product on an agarose gel in an area separate
fxom the reaction assembly area to confirm
product.
Amplification of PCR Analyze PCR product on an agarose gel in an area
carryover products separate from the reaction assembly area.
Use Platinum~ Quantitative PCR SuperMix-UDG
as specified in the sample protocols on pages 9-
10. Since dU'TP is substituted for dTTP in the
reaction cocktail, any amplified DNA will contain
uraal. UDG prevents rearnplification of PCR
carryover products by removing uracil residues
from single or double stranded DNA. dU-
containing DNA that has been digested with UDG
is unable to serve as template in future PCRs.
UDG is inactivated at high temperature during
PCR thermal cyding, thereby allowing
amplification of genuine target sequence(s).
Primer dimers Perform melting curve analysis of the PCR
product; identify dimers by lower melting point
temperature. Confirm that primer designs have
low scores (0.0-4.0) to minimize self annealing_
Redesign primers if necessary. When redesigning
primers, note that you can first try redesigning
only the unlabeled primer to save the cost of the
LUX'" primer.
No or low signal Tnstruments setting not Confirm that the cycling parameters
are correct,
optimal the quencher is set to none, and the reference dye
setting is correct.
Primer/template sequences do Confirm that the sequences match.
not match '
Primer designs are not optimal Confirm that the primer design scores are
within
the O.t~-4.0 xange and the optimal melting
temperatures have been specified. Redesign
primers if necessary. When redesigning primers,
note that you can first try redesigning only the
unlabeled primer to save the cost of the LUX"'
primer.
Poor standard curve and $eaction is not optimized Reoptimize reacfion
conditions. Prepare primer
dynamic range titrations if necessary.
Reference dye not used Use ROX Reference Dye as specified.
73
SUBSTITUTE SHEET (RULE 26)
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Accessory Products
P!'oduCts The following products are available for use with >a'CTX"' Primers
in real-time
PCR and RT-FCR protocols:
Product unt Cataloe
no.
Platinum Quantitative PCR SuperMix-UDG100 rxns11730-017
500 rxns11730-025
SuperScript'" III First-Strand50 rxns 18080-051
Synthesis System for
RT-PCR
Superscript'" III Platinum~ 100 rxns11732-020
Or,e-Step Quantitative RT 500 rxns11732-088
PCR System
Platinum~ Taq DNA Polymerase 104 rxns10966-018
250 rxns10966-026
50Drxns 10966-034
5,000 10966-083
x~ms
Micro-to-Midi Total RNA Purification50 rams 12183-018
System
TRTzoh' Reagent 100 ml 15596-026
200xnI 15596-018
Micro-FastTrack'" 2.0 mRNA 20 rxns K1520-02
Isolation Kit
ROX Reference Dye 500 Itl 12223-023
DNase I, AmpliC~cation Grade 100 U 18068-015
(1 Ul~.~t)
RNaseOUT'" Recombinant Ribonuclease5,000 10777-019
Inhibitor U
(40 U/Ni)
mM dNTP Mix 100 Ol 18427-013
DEPC-treated water 4 x 1.2510813-012
ml
74
SUBSTITUTE SHEET (RULE 26)
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Purchaser Notificafiion
limited Use The purchase of this product conveys to the
Label buyer the non-transferable right to use the
License NO. pm'~ased amount of the product and components
114: of the product in research
LUXE P1UOPO~eIIiCconducted by the buyer (whether the buyer is
an academic or for-profit entity). The
Primer buyer cannot sell or otherwise transfer (a)
this product (b) its components or (c)
materials made using this product or its components
to a third party or otherwise use
this product or its components or materials
made using this product or its components
fox commercial purposes. The buyer may transfer
information or materials made
through the use of this product to a scientific
collaborator, provided that such transfer
is not for the commercial purposes of the buyer,
and that such collaborator agrees in
. writing (a) to not transfer such materials to
any third party, and (b) to use such
transferred materials and/or informakion solely
for research and not for commercial
purposes. Commercial purposes means any activity
by a party for consideration and
may include, but is not limited to: (I) use
of the product or its components in
manufacturing; (2) use of the product or its
components to provide a service,
information, or data; (3) use of the product
or ifs components for therapeutic,
diagnosiac or prophylactic purposes; or (4)
resale of the product or its components,
whether ox riot such product or its components
are resold for use in research. '
Invitrogen Corporation will not assert a claim
against the buyer of infringement of
patents owned by Invitrogenbased upon the manufacture,
use ox sale of a therapeutic,
clinical diagnostic, vaccine or prophylactic
product developed in research by the buyer
in which this product or its components was
employed, provided that neither this
product nor any of its components was used in
the manufacture of such product. If the
purchaser is not willing to accept the limitations
of this limited use statement,
lnvifrogen is wDling to accept return of the
products with a full refund. For
information on purchasing a license to this
product for purposes other than researeh,
contact Licensing Department,1600 Faraday Avenue,
Carlsbad, California 92008.
Phone (760) 603-7200. Fax (760) 602-6500.
Limited Use Label 'rlus product is optimized for use in the Polymerase Chain
Reaction (PCR) covered by
LICSf7S~ P10. 4: patents owned by Roche Molecular Systems, Inc. and P.
Hoffmann-La Roche, Ltd.
PrOdUCtS fOr PCR (~~Roche"). No license under these patents to use the PCR
process is conveyed expressly
which do not or by implication to the purchaser by the pur~ase of this
product. A license to use the
include an r1 h~ PCR process for certain research and development activities
accompanies the purchase
Y J of certain reagents From licensed suppliers such as Invitrogen, when used
in
t0 perform PCR conjunction with an Authorized Thermal Cycler, or is available
from Applied
Biosystems. Further information on purchasing licenses to practice the PCR
process
may be obtained by contacting the Director of Incensing at Applied Biosystems,
850
Lincoln Centre Drive, Foster City, California 94404 or at Roche Molecular
Systems, Inc.,
1145 Atlantic Avenue, A3ameda, California 94501.
SUBSTITUTE SHEET (RULE 26)
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Technical Service
WoPld Wide Web Visit the Invitxogen Web Resource using your World Wide Web
browser. At the
site, you can:
~ Get the scoop on our hot new products and special product offers
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~ Bxplore our catalog with full color graphics
~ Obtain citations for Invitrogen products
~ Request catalog and product literature
Once connected to the Internet, launch your Web browser-(Internet Bxplorer 5.0
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76
SUBSTITUTE SHEET (RULE 26)
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Technical Service, Continued
Limited Warranty Invitrogen is committed to providing our customers with high-
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Invitrogen warrants that all of its products will perform according to the
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No
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77
SUBSTITUTE SHEET (RULE 26)
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References
Ailenberg, M., and Silverman, M. (2000) Controlled hot start and improved
speaficity in carrying out
PCR utilizing touch-up and Loop incorporated primers (TULIPS). BioT'echnigues
29,1018-1024.
Bustin, S. A. (2000) Absolute quantification of mRNA using real-lime reverse
transcription polymerase
chain reaction assays. J. Mol. Endocrinol. 25,169-193.
Cardullo, R. A., Agrawal, S., Flores, C., Zamecnik, P. C., and W olf, D. E.
(2988) Detection of nucleic acid
hybridization by nonradiative fluorescence resonance energy transfer. Proc.
Natl. Acad. Sci. USA 85,
8790-8794.
Crockett, A.~., and Wittsver, C.T. (2001) Fluorescein-labeled oligonucleofides
for real-time pcr: using
the inherent quenching of deoxyguanosine nucleotides. Anal. Biochem. 290, 89-
97.
Higuchi, R., Focklei, C., Walsh, P.S., and Griffith, R (1992) Simultaneous
amplification and detection of
specific DNA sequences. Biotechnology .20, 413-417.
Higurhi, R., Fockler, C., Dollinger, G., and Watson, R. (1993) Kinetic PCR
analysis: real-time monitoring
of DNA amplification reactions. Biotechnology I2,1026--1030.
Holland et al. (1991) Detection of specific polymerase chain reaction product
by utilizing the 5'-3'
exonuclease activity of Thermus aquaticus DNA polymerase. Proc. Natt. Acad.
Sci. LISA 88, 7276-
7280.
Kaboev, O. K., Luchkina, L. A., Tret'iakov, A. N., and Hahrmand, A.R. (2000)
PCR hot start using
primers with the structure of molecular beacons (hairpin-like structure).
Nucleic Acids Res. 28, e94.
Knemeyer, j.P., Marme, N., and Sauer, M. (2000) Probes for detection of
specific DNA sequences at the
single-molecule Level. Anal. Chem. 72, 3717 3724.
Murchie, A. I. H., Clegg, R. M., von Kitzing, E., Duckkett, D. R.,1?iektnann,
S., and Lilley D. M. J. (1989)
Fluorescence energy transfer shows that the four-way DNA junction is a right
handed cross of
antiparallel molecules. Nature 341, 763-766.
Myakishev. M.V., Kluipin, Y., Hu, S., and Hamer, D. H. (2001) High-throughput
SNP genotyping by
allele-specific PCR with universal energy-transfer-labeled primers. Genorile
Res.11,163--169.
Nazaxenko, L, Lowe, 8., Darfler, M., Ikonomi, P-, Schuster, D., and
Rashtchian, A. (2002} Multiplex
quantitative PCR using self quenched primers labeled with a single
fluorophore. Nucl. Acids Res.
30, e37
Nazarenko, L, Pixes, R., Love, B., Obaidy, M., and Rashtchian, A. (2002)
Effect of primary and
secondary structure of oligodeoxyribonudeotides on the fluorescent properties
of conjugated dyes.
Nucl. Acids Res. 30, 2089-2095
Nazarenko, LA., Bhatnagar, S.K., and Hohman, R.J. (1997) A closed tube format
for amplification and
detection of DNA based on energy transfer, Nucleic Acids Res. 25, 2516-2521.
Nuovo, G. J., Hohman, R. j., Nardone, G. A., and Nazarenko I. (1999) In situ
amplification using
universal energy transfer-labeled primers. J. Histochem. Cytochem. 47, 273-
279.
Todd, A. V., Fuery, C. J., Impey, H. L., Applegate, T. L. and Haughton, M.A.
(2000) DzyNA-PCR: use of
DNAzymes to detect and quantify nucleic acid sequences in a real-time
fluorescent format Clin.
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~2002 2003 Invitrogen Corporation. All rights reserved.
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