Note: Descriptions are shown in the official language in which they were submitted.
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LOW-TEMPERATURE CYCLE EXTENSION OF DNA
WITH HIGH PRIMING SPECIFITY
BACKGROUND OF THE INVENTION
The genetic material of all known living organisms is deoxyribonucleic acid
(DNA), except in certain viruses whose genetic material may be ribonucleic
acid (RNA).
DNA consists of a chain of individual deoxynucleotides chemically linked in
specific
sequences. Each deoxynucleotide contains one of the four nitrogenous bases
which may
be adenine (A), cytosine (C), guanine (G) or thymine (T), and a deoxyribose,
which is a
pentose, with a hydroxyl group attached to its 3' position and a phosphate
group attached
to its 5' position. The contiguous deoxynucleotides that form the DNA chain
are
connected to each other by a phosphodiester bond linking the 5' position of
one pentose
ring to the 3' position of the next pentose ring in such a manner that the
beginning of the
DNA molecule always has a phosphate group attached to the 5' carbon of a
deoxyribose.
The end of the DNA molecule always has an OH (hydroxyl) group on the 3' carbon
of a
deoxyribose.
DNA usually exists as a double-stranded molecule in which two antiparallel DNA
strands are held together by hydrogen bonds between the bases of the
individual
nucleotides of the two DNA strands in a strictly matched "A-T " and "C-G"
pairing
manner. It is the order or sequence of the bases in a strand of DNA that
determines a gene
which in turn determines the type of protein to be synthesized. Therefore, the
accurate
determination of the sequence of the bases in a DNA strand which also
constitutes the
genetic code for a protein is of fundamental importance in understanding the
characteristics of the protein concerned.
The process used to determine the sequence of the bases in a DNA molecule is
referred to as DNA sequencing. Among the techniques of DNA sequencing, the
enzymatic method developed by Banger et al. (1) is most popular. It is based
on the
ability of a DNA polymerase to extend a primer annealed to the DNA template to
be
sequenced in the presence of four normal deoxynucleotide triphosphates
(dNTPs),
namely, dATP, dCTP, dGTP and dTTP, and on the ability of the nucleotide
analogs, the
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dideoxynucleotide triphosphates (ddNTPs), namely, ddATP, ddCTP, ddGTP and
ddTTP,
to terminate the extension of the elongating deoxynucleotide polymers at
various lengths.
In enzymatic polymerization reactions using double-stranded DNA templates, it
is
necessary to denature the target DNA fragments. To that end, heating a double-
stranded
DNA, usually to 95°C, denatures the molecule to create two
complementary single-
stranded DNA fragments. In an enzymatic DNA polymerization reaction, after a
primer
annealed to its complementary sequence on a single-stranded template has been
extended
to form a new DNA strand, the latter can be separated from its template when
heated to
95°C, which is above its melting temperature. The single-stranded
template is again
available for annealing with an oligonucleotide primer upon cooling, ready for
another
cycle of enzymatic DNA synthesis in the presence of a functioning DNA
polymerase and
dNTPs. Usually a heat-resistant DNA polymerase, which can survive the heating
to 95°C
and is active at temperature between 55 and 72°C, is employed in the
system so that no
fresh enzyme needs to be added to initiate each cycle of DNA synthesis after
denaturing
at high temperature. When a primer is mixed in excess with a template and the
temperature cycles repeat for a plurality of times, the number of the extended
single-
stranded target fragments increases one fold per cycle. When a set of ddNTPs,
including
all four A, C, G and T bases, or their analogs, is present as chain
terminators in the
reaction system, numerous single-stranded DNA fragments of various lengths,
all having
the same primer at their 5'end and terminating with a specific ddNTP or its
analog, which
may be labeled with a fluorescent dye as a reporter, at the 3'end are
generated. This
forms the basis of automated DNA cycle sequencing with fluorescent dye-labeled
DNA
terminators.
When a set of two primers (one forward and one reverse) complementary to the
two ends of a target sequence on a double-stranded DNA template is used in the
above-
described DNA cycle sequencing system, the newly extended DNA strands can
serve as
additional templates in the subsequent cycle of DNA synthesis. Hence the copy
number
of the target sequence fragments is amplified exponentially if the heating
cycle is
repeated for phzrality of times. This forms the basis of the Polymerase Chain
Reaction
(PCR).
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In practice, both automated cycle sequencing with fluorescent dye-labeled
terminators and PCR have depended on the use of heat-resistant DNA
polymerases, such
as Thermus aquaticus DNA polymerase (Taq) and its equivalents, which can
survive the
heating temperature of 95°C. However, heat-resistant polymerases are
usually associated
with low processivity, and may lose their sequence-specific polymerase
activity under
certain unpredictable conditions, especially when GC-rich DNA segments (that
is,
segments containing a significantly higher content of guanine and cytosine,
relative to the
content of thymine and adenine) in a template are to be amplified or to be
sequenced.
Therefore, attempts have been made to develop conditions suitable for low
temperature
cycle sequencing and for low temperature cycle PCR using thermolabile DNA
polymerases, which, in general, have higher fidelity and higher processivity
than the
heat-resistant DNA polymerases. For example, in U.S. Patent No. 5,432,065,
there is
described the use of glycerol or ethylene glycol-at a final concentration of
40% (v/v~
to lower the melting temperature of the template DNA and to extend the primer
at
temperatures below 80°C in a cycle primer extension reaction, in
conjunction with a
DNA polymerase from Bacillus caldotenax and from a Klenow fragment. However,
it
was later found that even at such a high concentration of glycerol, the Klenow
DNA
polymerase was not useful for low temperature cycle primer extension. Lowering
the
glycerol concentration toll % (v/v) in the reaction mixture with the addition
of proline
appeared to protect the Klenow polymerase activity in cycle PCR at the
temperature
range between 70°C and 37°C. (Iakobashvili and Lapidot)
Significantly, neither of these
procedures for low temperature cycle primer extension has been shown to
generate high
quality sequence-specific PCR products, or has been shown to generate reaction
products
suitable for DNA sequencing. In the Iakobashvili and Lapidot report, the PCR
products
generated in low-temperature cycle primer extension have not proved to be
sequence-
specific, especially when primers of 20-25 base pairs (bp) in length were
used. Although
the application was said to be successful for cycle extension of long primers
(such as 30-
35 by in length) using the Klenow polymerase at the low temperature range, the
system
has not been shown to generate useful sequence-specific amplification products
from
such long primers. Since most primers used for DNA cycle sequencing and for
PCR are
shorter than 30 by in length, there is a need for a low-temperature cycling
system with
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which sequence-specific extension of primers of shorter than 30 by (preferably
about 20
bp) can be achieved to generate useful amplification DNA products for
sequencing and
for further molecular analysis.
SUMMARY OF THE INVENTION
The invention described in this application has provided such a system. With
the
above issues in mind, the inventors have developed methods for extending a
primer or a
pair of primers in cycle DNA amplification for automated cycle sequencing and
PCR. In
particular, the methods contemplate moderately thermostable DNA polymerases in
the
presence of a low concentration of glycerol or ethylene glycol, or the
mixtures thereof, as
an agent to reduce the melting temperature of DNA (that is, the temperature at
which the
double-strands of DNA are denatured). The inventors observed that at a certain
concentration range, glycerol and/or ethylene glycol not only reduced the
melting
temperature of the DNA template, but also increased the polymerization
activity of the
moderately thermostable DNA polymerases. In these enzymatic reaction systems,
the
temperature range of cycling is between 70°C and 37°C-much lower
than what is
usually required for denaturing DNA. In addition, the methods use highly
processive,
moderately thermostable DNA polymerases preferably derived from Bacillus
stearothermophilus, Bacillus caldotenax or Bacillus caldolyticus. These
polymerases
have an optimum reaction temperature at 65°C, bnt are rapidly
inactivated above 70°C;
thus, they axe quite useful as the polymerizing enzymes for the cycle primer
extension to
overcome some of the shortcomings of the heat-resistant DNA polymerases, such
as Taq
and its corresponding mutants, and of the heat-labile DNA polymerases, such as
the
Klenow fragment. The moderately thermostable DNA polymerases may be in their
natural state (e.g., purified from the organisms), or modified.
Thus, in a broad embodiment, the invention contemplates a method for extending
a primer (or a pair of primers) using an enzymatic cycle primer extension
reaction at low
cycling temperatures (that is, temperatures below about ~0°C), in a
reaction mixture
composition comprising between about 10% and about 20% (and preferably about
15%)
(vlv) glycerol, ethylene glycol, or a mixture thereof, in the presence of a
moderately
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thermostable (also referred to as mesophilic) DNA polymerase. (By "enzymatic
cycle
primer extension reaction", it is meant that in excess of primer over
template, the limited
number of template molecules can be used repeatedly for DNA polymerization
catalyzed
by a functional DNA polymerase when the temperature of the reaction mixture
fluctuates
repeatedly between the levels required for denaturing, annealing and primer
extension in
cycles. By "moderately thermostable DNA polymerase, it is meant polymerases
that have
an optimum reaction temperature at 65°C, and which are rapidly
inactivated above 70°C.)
For instance, DNA template may be mixed with a primer (or a pair of primers)
and a
natural or a modified form of a moderately thermostable DNA polymerase from
one of
Bacillus stearothe~mophilus, Bacillus caldotenax or Bacillus caldolyticus, in
a solution
containing between about 10% and about 20% (v/v) (preferably about 15% (v/v))
glycerol, ethylene glycol, or a mixture thereof. The reaction may be carried
out under
conditions that the cycle reaction temperature fluctuates between a melting
temperature
of about 70°C and a cooling (or annealing) temperature of about
37°C, so that the DNA
polymerase repeatedly extends the primer or pair of primers at the
tempterature between
about 45°C and 50°C. The method may include the further step of
repeating the cycle
primer extension reaction, as many times as is desired.
In another embodiment, copies of a selected segment of a double-stranded DNA
are amplified in the presence of a forward primer and a reverse primer (where
both may
be of various lengths) to the template by repeated heating and cooling (or
annealing)
cycles (such as, for instance, in a PCR). Here, again, the reaction is run at
low
temperatures (that is, temperatures below about 80°C), in a reaction
mixture composition
comprising between about 10% and about 20% (and preferably about 15%) (v/v)
glycerol, ethylene glycol, or a mixture thereof, in the presence of one of the
moderately
thermostable DNA polymerases described above. The reaction may be carried out
under
conditions that the reaction temperature fluctuates between a melting
temperature of
about 70°C and a cooling (or annealing) temperature of about
37°C, so that the DNA
polymerase repeatedly extends the forward and reverse primers at the
temperature of
between about 45°C and 50°C. The method may include the further
step of repeating the
reaction, as many times (e.g., cycles) as is desired.
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In a preferred embodiment, the DNA polymerase is one of those described in
U.S.
Patent No. 5,747,298, U.S. Patent No. 5,834,253 or U.S. Patent No. 6,165,765.
Preferably
the DNA polymerase has an amino acid sequence that shares not less than 95%
homology
of a DNA polymerase isolated from Bacillus stearothermophilus, Bacillus
caldotenax or
Bacillus caldolyticus.
In a fixrther embodiment, molecules of a single primer of various lengths are
extended with specific nucleotide terminations in the presence of ddNTPs or
their analogs
for cycle sequencing.
The invention also contemplates a method for extending the molecules of a
single
primer amiealed to a single-stranded copy of the doubled-stranded DNA product
amplified in vitro without prior isolation or purification for direct cycle
sequencing. For
instance, a diluted crude amplified reaction product (preferably generated
with a low-
temperature PCR reaction catalyzed by a moderately thermostable DNA polymerase
as
described herein) is used as a template and mixed with an excess amount of a
sequencing
primer, the four standard ddNTP terminators (ddATP, ddGTP, ddTTP and ddCTP)
fluorescently labeled (or their corresponding analogs fluorescently labeled),
a moderately
thermostable DNA polymerase (preferably one with a reduced innate selective
discrimination against incorporation of a subset of dye-labeled ddNTPs), a
suitable
concentration of dNTPs (dATP, dGTP, dTTP and dCTP), and a composition
comprising
a buffer in a solution containing between about 10% and about 20% (preferably
15%)
(v/v) of glycerol, ethylene glycol, or mixture thereof. A standard cycle
primer extension
reactions) may then be run at a temperature below 80°C for a sufficient
number of times
to extend the sequencing primer molecules to desired varying lengths, which
extended
molecules will be terminated specifically by fluorescently labeled ddNTPs or
their
corresponding analogs. Preferably, the cycle reaction temperature fluctuates
between a
melting temperature of about 70°C and a cooling/annealing temperature
of about 37°C.
In one preferred embodiment, the method of sequencing a DNA strand may
comprise the steps of:
i) hybridizing a primer to a DNA template to be sequenced; and
ii) extending the primer using one of the above-described DNA polymerases, in
the presence of a solution containing between about 10% and about 20% (vlv)
(preferably
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about 15% (v/v)) glycerol, ethylene glycol, or a mixture thereof, adequate
amounts of the
deoxynucleotide bases dATP, dGTP, dCTP and dTTP, and the four
dideoxynucleotide
terminators or their analogs, whereby the cycle reaction temperature
fluctuates between a
melting temperature of about 70°C and a cooling or annealing
temperature of about 37°C,
and under such conditions that the DNA strand is sequenced. Preferably one of
the
deoxynucleotides is radioisotope-labeled, or the primer molecules are
fluorescent dye-
labeled, and more preferably all dideoxynucleotide terminators are fluorescent
dye-
labeled.
In another embodiment, the invention entails a dry or liquid ready-to-use
reaction
mixture or kit suitable for use in a low-temperature cycle primer extension
reaction at
temperatures below about 80°C. This reaction mixture or kit comprises a
moderately
thermostable DNA polymerase (such as one of those described above) that is pre-
mixed
with at least one enzymatic DNA primer extension reaction component suitable
for use in
DNA amplification or for specific extension terminations with
dideoxyribonucleotide
analogs. The reaction mixture is preferably pre-distributed into
microcentrifuge tubes or
in multiple-well plates, such as, for instance, those that are suitable for
large-scale
automated PGR or for large-scale automated DNA sequencing. This ready-to-use
reaction mixture or kit can be stored at room temperature between about
22°C and about
25°C for at least eight weeks without losing its specific
polymerization activity for DNA
primer amplification or extension terminations.
Further objects and advantages of the invention will become apparent from the
description and examples below.
BRIEF DESCRIPTION OF THE DRAWINGS
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Figure 1 is a graph illustrating the effect of glycerol on 5'-3'
polymerization
activity on Bst-II DNA polymerase.
Figure 2 is a picture of an electrophoresis gel (1% agarose), showing the
results of
low-temperature amplification with a moderately thermostable DNA polymerase in
40%
glycerol.
Figure 3A is a picture of an electrophoresis gel (1% agarose), showing the
results
of low-temperature cycle primer extension in 35% glycerol (lane 1) and in 15%
glycerol
(lane 2) with amplified products having a length of 250 base pairs. Figure 3B
is a picture
of an electrophoresis gel (1% agarose), showing the results of low-temperature
cycle
primer extension in 35% glycerol (lane 1) and in 15% glycerol (lane 2) with
amplified
products having a length of 400 base pairs. Figure 3C is a picture of an
electrophoresis
gel (1% agarose), showing the results of low-temperature cycle primer
extension in 35%
glycerol (lane 1) and in 15% glycerol (lane 2) with amplified products having
a length of
1 kilobase. Figure 3D is a picture of an electrophoresis gel (1% agarose),
showing the
results of low-temperature cycle primer extension in 35% glycerol (lane 1) and
in 15%
glycerol (lane 2) with amplified products having a length of 2 kilobases.
Figure 4 is a picture of an electrophoresis gel (1% agarose), showing the
results of
low-temperature cycle extension reaction of l7mer and 30mer primers with
moderately
thermostable DNA polymerases and I~lenow fragment. The reaction products with
l7mer primers are A1 (I~lenow fragment using the Iakobashvili and Lapidot
system), A2
(Klenow fragment with the Bst system), A3 (Bst-I polymerase with the Bst
system), A4
(Bst-II polymerase with the Bst system), and AS (Bca polymerase with the Bst
system).
The reaction products with 30mer primers are B1 (Klenow fragment using the
Iakobashvili and Lapidot system), B2 (I~lenow fragment with the Bst system),
B3 (Bst-I
polymerase with the Bst system), B4 (Bst-II polymerase with the Bst system),
and BS
(Bca polymerase with the Bst system).
Figures SA and SB represent two automated fluorescent DNA sequencing tracings
of a GC-rich segment, comparing the performance of AmpliTaqTM in the ABI
PrismTM
BigDyeTM Terminator cycle sequencing kit (5A) with that of the Bst-II cycle
sequencing
system (5B).
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Figure 6 is a picture of an electrophoresis gel (1% agarose), showing the
results of
cycle primer extension reactions conducted at various temperature steps, using
a
moderately thermostable DNA polymerase (Bst-II), with no glycerol and with 15%
glycerol.
Additional details about Figures 1-6 are included in the description and
examples
that follow.
DETAILED DESCRIPTION OF THE INVENTION
As noted above, this invention entails a unique combination of a moderately
thermostable DNA polymerase (such as Bacillus stea~othe~~cophilus, Bacillus
caldote~ax
or Bacillus caldolyticus ) in the presence of a low concentration of an agent
selected from
the group consisting of glycerol, ethylene glycol and mixtures of these, to
provide a way
to extend a primer (or pair of primers) in cycle DNA amplification for
automated cycle
sequencing and PCR at temperatures below about 80°C. The inventors
discovered that
both glycerol and ethylene glycol at low concentrations increase the sequence-
specific
DNA polymerization activity of the moderately thermostable DNA polymerases in
vitro.
At higher concentrations, for example greater than 35%, both glycerol and
ethylene
glycol exhibit a detrimental inhibitory effect on the DNA polymerization
activity of these
enzymes. However, the inventors achieved a reaction mixture with an optimum
concentration of glycerol or ethylene glycol, in which double-stranded DNA
templates
are denatured at 70°C while the polymerization activity of the
moderately thermostable
DNA polymerases can be preserved during low temperature sequence-specific
cycle
primer extension.
For instance, the inventors first observed that at the optimum enzymatic
reaction
temperature of 65°C, a fnal concentration of glycerol of up to about
20% increased the
5'-3' polymerization activity of the moderately thermostable DNA polymerases
(for
instance, see Figure 1 ). However, when the concentrations of glycerol was
increased to
greater than about 35% it invariably suppressed this enzymatic activity. When
the
glycerol concentration increases to 40% (v/v), this group of DNA polymerases
usually
lost more than two thirds (2/3) of the original polymerization activity. It
was found that
low-temperature cycle extensions with moderately thermostable DNA polymerases
in a
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solution containing 40% of glycerol generates poorly defined non-specific
amplified
products of varying fragment sizes (for instance, see Figure 2).
The inventors found that in the presence of 35% glycerol, low temperature
cycle
primer extension reactions catalyzed by moderately thermostable DNA
polymerises,
such as a Bst mutant or Bca, resulted in no amplification at all for a target
product of 250
by and 400 by long. When longer segments of DNA-for example, 1 kb and 2 kb in
length-are the amplification target, there is evidence of amplification; but
the reaction
products are non-specific. (For instance, see Figure 3).
It was further found that with a low concentration of glycerol, for example
15%
(v/v), and a Bst mutant or Bca DNA polymerise, sequence-specific amplification
products of less than 250 by to more than 2 kb in length can be generated
(see, for
example, Figure 3).
Thus, a low concentration of glycerol or ethylene glycol, for example between
about 10% and about 20% v/v, preferably 15%, can be used to lower the DNA
melting
temperature for cycle primer extension in conjunction with a moderately
thermostable
DNA polymerise to generate sequence-specific amplification products.
In this low-temperature cycle extension system, DNA fragments of a wide range
in length, including those having less than 30 base pairs, even shorter than
20 base pairs
in length can be used as the primers for sequence-specific extensions (f~r
instance, see
Figure 4). The thermolabile DNA polymerises, such as the I~lenow fragment,
fail to
generate any significant amount of amplification products useful for further
analysis (see,
for instance, Figure 4). ThermoSequenaseT"" and AmpliTaqT"", both being
modified forms
of the heat-resistant Taq DNA polymerise, cannot generate sequence-specific
products
useful for further analysis in the low-temperature cycle extension system.
However,
under their optimum high-temperature (melting at 95° C) cycle extension
conditions,
these two enzymes may extend the primers annealed to most DNA templates until
a GC-
rich segment is encountered. Compared with the results of using a moderately
thermostable DNA polymerise for low-temperature cycle extension, the low
processivity
of the heat-resistant DNA polymerises under high temperature cycle extension
becomes
evident when they are used for automated cycle sequencing of known GC-rich
templates.
The heat-resistant DNA polymerises generate no sequence-specific ddNTP
terminations
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down-stream to the GC-rich segment of the template whereas a moderately
thermostable
enzyme, for example the mutated Bst-II, can successfully overcome the GC-rich
obstacle
during DNA polymerase cycle extensions (see, for instance, Figure 5).
As indicated above, the use of moderately thermostable DNA polymerases is
quite critical to the methods of this invention. By "moderately thermostable"
it is meant
that these polymerases have an optimum reaction temperature at 65°C,
but are rapidly
inactivated above 70°C. To that end, the invention contemplates DNA
polyrnerases
obtained or derived from one or more of Bacillus stearothe~mophilus (Bst),
Bacillus
caldote~ax (Bca) or Bacillus caldolyticus (Bcy). All three of these organisms
are
classified as mesophilic microbes because, although their DNA polymerases axe
referred
to as thermostable (most active at 65°C), they are inactivated at
70°C or above. This is
contrasted with other enzymes, such as Taq, which are truly thermophilic-that
is, the
Taq DNA polymerase tolerates and remains active at temperatures higher than
95°C.
These mesophilic bacillus strains, especially Bacillus stearothe~mophilus,
produce DNA
polymerases that are useful in DNA cycle sequencing and PCR applications.
In a preferred embodiment, a moderately thermostable (also sometimes referred
to
as mesophilic) DNA polymerase may have proofreading 3'-5' exonuclease activity
during
DNA primer extension over a template, such that the DNA polymerase functions
to
excise mismatched nucleotides from the 3' terminus of the DNA strand at a
faster rate
than the rate at which the DNA polymerase functions to remove nucleotides
matched
correctly with nucleotides of the template. Such DNA polymerases are also
described by
the inventors in U.S. Patent No. 5,834,253, U.S. Patent No. 5,747,298, and
U.S. Patent
No. 6,165,765 (the contents of all of which are incorporated herein by
reference in their
entirety). One strain of Bacillus stearothermophilus (designated strain No.
320 for
identification purposes; described in U.S. Patent 5,747,298) produces a DNA
polymerase
(designated Bst 320) with a proof reading 3'-5' exonuclease activity which is
absent in
DNA polymerases isolated from other strains of Bacillus stearothermophilus.
(For this
invention, the term "proof reading" is intended to denote that the DNA
polymerase is
capable of removing mismatched nucleotides from the 3' terminus of a newly
formed
DNA strand at a faster rate than the rate at which nucleotides correctly
matched with the
nucleotides of the template are removed during DNA sequencing.) The strain Bst
320
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was deposited on October 30, 1995 in the American Type Culture Collection,
located at
12301 Parklawn Drive, Rockville, Maryland 20852, and has been given ATCC
Designation No. 55719. The DNA polymerase isolated from Bst 320 is composed of
587
amino acids as are the DNA polymerases of other known strains of Bacillus
stea~othermophilus, such as, for instance, the strains deposited by Riggs et
al (Genbank
Accession No. L42111 ) and by Phang et al. (Genbank Accession No. U23149).
However, the Bst 320 shares only 89.1% sequence identity at protein level with
the
Bacillus stearothermophilus DNA polymerase deposited by Riggs et al., and
shares only
87.4% sequence identity at protein level with the Bacillus stearothe~mophilus
DNA
polymerase deposited by Phang et al. For comparison, the above-referenced
enzyme
deposited by Riggs et al. and the enzyme deposited by Phang et al. share 96.9%
of their
amino acid sequence identity.
The inventors studied a thermostable DNA polymerase isolated from a different
species, Bacillus caldote~cax (Bca), which also has an optimum active
temperature at
65°C. The inventors discovered that the Bst 320 DNA polymerase shaxes
88.4% of the
amino acid sequence identity with Bca DNA polymerase (Uemori et al. J.
Biochem. 113:
401-410, 1993). Based on homology of the amino acid sequences, Bst 320 DNA
polymerase is as close to DNA polymerases isolated from Bacillus
stea~othermophilus as
to the DNA polymerase isolated from Bacillus caldoteaax, i.e. another species
of
bacillus. It was also discovered that both Bst 320 DNA polymerase and Bca DNA
polymerase functionally exhibit 3'-5' exonuclease activity, which is not
associated with
known amino acid sequence exonuclease motifs I, II and III as in the E. coli
DNA
polymerase I model, or other known Bacillus stea~-othe~mophilus polymerases.
One preferred Bst DNA polymerase is isolated from strain 320 with an amino
acid sequence as follows:
Amino acid sequence (SEQ ID N0:2):
AEGEKPLEEM EFAIVDVITE EMLADKAALV VEVMEENYHD
APIVGIALVNE HGRFFMRPE TALADSQFLA WLADETKKKS
MFDAKRAVVA LKWKGIELRG VAFDLLLAAY LLNPAQDAGD
IAAVAKMKQY EAVRSDEAVY GKGVKRSLPD EQTLAEHLVR
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KAAAIWALEQ PFMDDLRNNE QDQLLTKLEH ALAAILAEME
FTGVNVDTKR LEQMGSELAE QLRAIEQRIY ELAGQEFNIN
SPKQLGVILF EKLQLPVLKK TKTGYSTSAD VLEKLAPHHE
IVENILHYRQ LGKLQS.TYIE GLLKVVRPDT KVHTMFNQA
LTQTGRLSSA EPNLQNIPIR LEEGRKIRQA FVPSEPDWLI
FAADYSQIEL RVLAHIADDD NLIEAFQRDL DIHTKTAMDI
FQLSEEEVTA NMRRQAKAV NFGIVYGISDY GLAQNLNITR
KEAAEFIERY FASFPGVKQY MENIVQEAKQ KGYVTTLLHR
RRYLPDITSR NFNVRSFAER TAMNTPIQGS AADIIKKAMI
DLAARLKEEQ LQARLLLQVH DELILEAPKE EIERLCELVP
EVMEQAVTLR VPLKVDYHYG PTWYDAK
The characters represent the following amino acids:
where,
A: alanine (Ala) M: methionine (Met)
C: cysteine (Cys) N: asparagine (Asn)
D: aspartic acid (Asp) P: proline (Pro)
E: glutamic acid (Glu) Q: glutamine (Gln)
F: phenylanaline (Phe) R: arginine (Arg)
G: glycine (Gly) S: serine (Ser)
H: histidine (His) T: threonine (Thr)
I: isoleucine (Ile) V: valine (Val)
K: lysine (Lys) W: tryptophan (Trp)
L: leucine (Leu) Y: tyrosine (Tyr)
This Bst 320 DNA polymerase is characterized by possessing a proofreading 3'-
5'
exonuclease activity.
The nucleotide sequence encoding the Bst 320 DNA polymerase is indicated in
SEQ ID NO:1, below.
DNA sequence (isolated/purified):
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GCCGAAGGGG
AGAAACCGCT
TGAGGAGATG
GAGTTTGCCA
TCGTTGACGT CATTACCGAA GAGATGCTTG CCGACAAGGC
AGCGCTTGTC GTTGAGGTGA TGGAAGAAAA CTACCACGAT
GCCCCGATTG TCGGAATCGC ACTAGTGAAC GAGCATGGGC
GATTTTTTAT GCGCCCGGAG ACCGCGCTGG CTGATTCGCA
ATTTTTAGCA TGGCTTGCCG ATGAAACGAA GAAAA.AAAGC
ATGTTTGACG CCAAGCGGGC AGTCGTTGCC TTAAAGTGGA
AAGGAATTGA GCTTCGCGGC GTCGCCTTTG ATTTATTGCT
CGCTGCCTAT TTGCTCAATC CGGCTCAAGA TGCCGGCGAT
ATCGCTGCGG TGGCGAAAAT GAAACAATAT GAAGCGGTGC
GGTCGGATGA AGCGGTCTAT GGCAAAGGCG TCAAGCGGTC
GCTGCCGGAC GAACAGACGC TTGCTGAGCA TCTCGTTCGC
AAAGCGGCAG CCATTTGGGC GCTTGAGCAG CCGTTTATGG
ACGATTTGCG GAACAACGAA CAAGATCAAT TATTAACGAA
GCTTGAGCAC GCGCTGGCGG CGATTTTGGC TGAAATGGAA
TTCACTGGGG TGAACGTGGA TACAAAGCGG CTTGAACAGA
TGGGTTCGGA GCTCGCCGAA CAACTGCGTG CCATCGAGCA
GCGCATTTAC GAGCTAGCCG GCCAAGAGTT CAACATTAAC
TCACCAAAAC AGCTCGGAGT CATTTTATTT GAAAAGCTGC
AGCTACCGGT GCTGAAGAAG ACGAAAACAG GCTATTCGAC
TTCGGCTGAT
GTGCTTGAGA
AGCTTGCGCC
GCATCATGAA
ATCGTCGAAA ACATTTTGCA TTACCGCCAG CTTGGCAAAC
TGCAATCAAC GTATATTGAA GGATTGTTGA AAGTTGTGCG
CCCTGATACC
GGCAAAGTGC
ATACGATGTT
CAACCAAGCG
CTGACGCAAA CTGGGCGGCT CAGCTCGGCC GAGCCGAACT
TGCAAAACAT TCCGATTCGG CTCGAAGAGG GGCGGAAAAT
CCGCCAAGCG TTCGTCCCGT CAGAGCCGGA CTGGCTCATT
TTCGCCGCCG
ATTACTCACA
AATTGAATTG
CGCGTCCTCG
CCCATATCGC
CGATGACGAC
AATCTAATTG
AAGCGTTCCA
ACGCGATTTG
GATATTCACA
CAAAAACGGC
GATGGACATT
TTCCAGTTGA
GCGAAGAGGA
AGTCACGGCC
AACATGCGCC
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GCCAGGCAAA GGCCGTTAAC TTCGGTATCG TTTACGGAAT
TAGCGATTAC GGATTGGCGC AAAACTTGAA CATTACGCGC
AAAGAAGCTG CCGAATTTAT CGAACGTTAC TTCGCCAGCT
TTCCGGGCGT AAAGCAGTAT ATGGAAAACA TAGTGCAAGA
AGCGAAACAG AAAGGATATG TGACAACGCT GTTGCATCGG
CGCCGCTATT TGCCTGATAT TACAAGCCGC AATTTCAACG
TCCGCAGTTT TGCAGAGCGG ACGGCCATGA ACACGCCAAT
TCAAGGAAGC GCCGCTGACA TTATTAAA.AA AGCGATGATT
GATTTAGCGG CACGGCTGAA AGAAGAGCAG CTTCAGGCTC
GTCTTTTGCT GCAAGTGCAT GACGAGCTCA TTTTGGAAGC
GCCAAAAGAG GAAATTGAGC GATTATGTGA GCTTGTTCCG
GAAGTGATGG AGCAGGCCGT TACGCTCCGC GTGCCGCTGA
AAGTCGACTA CCATTACGGC CCAACATGGT ATGATGCCAA
ATAA (1764 nucleotides total)
The characters represent the following nucleotides:
A: Adenosine T: Thymidine C: Cytidine G: Guanosine
However, while quite useful with this invention, a disadvantage of the DNA
polymerases of the mesophilic strains Bacillus stea~othe~mophilus, Bacillus
caldotenax
or Bacillus caldolyticus, is that during DNA sequencing they all exhibit a
high degree of
selective discrimination against incorporation of certain particular members
of
fluorescent dye-labeled ddNTPs, namely the fluorescent dye-labeled ddCTP and
fluorescent dye-labeled ddATP, as terminators onto the 3' end of the extending
DNA
fragments during enzymatic reaction. This peculiar characteristic of selective
discrimination against incorporation of fluorescent dye-labeled ddCTP and
ddATP of the
natural DNA polymerases isolated from Bacillus stea~othermophilus and Bacillus
caldotev~ax was only recognized recently by the inventors. Such selective
discrimination
is apparently sequence-related, and cannot be corrected or compensated by mere
adjustment of the concentrations of the dNTPs.
Therefore, in another preferred embodiment the DNA polymerase used is a
mesophilic bacillus DNA polymerase (such as Bacillus stearothermophilus,
Bacillus
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caldotehax and Bacillus caldolyticus) which, during dye-labeled terminator
automated
DNA cycle sequencing, reduces the innate selective discrimination against the
incorporation of fluorescent dye-labeled ddCTP and fluorescent dye-labeled
ddATP,
without increasing the rate of incozporation of the other two dye-labeled
ddNTP
terminators (ddTTP and ddGTP) excessively. Such DNA polymerases are described
by
the inventors in LT.S. Patent No. 6,165,765 (the contents of all of which are
incorporated
herein by reference in their entirety).
For example, polymerases having this ability to reduce selective
discrimination
may be obtained or otherwise derived from a strain of Bacillus
stearothermophilus,
Bacillus caldote~ax and Bacillus caldolyticus, or made synthetically, where
the amino
acid sequences of the naturally-occurring DNA polymerase have leucine-
glutamate-
glutamate at positions corresponding respectively to positions 342-344 of Bst
320 DNA
polymerase and phenylalanine at a position corresponding to position 422 of
Bst 320
DNA polymerase. For instance, DNA polymerases derived from other strains of
Bacillus
stearothe~mophilus, Bacillus caldotehax and Bacillus caldolyticus, may be
easily
modified using conventional DNA modification techniques to include the amino
acid or
nucleotide substitutions identified above.
The following amino acid sequence represents the modified Bst 320 DNA
polymerase (also referred to herein as "Bst II" or "HiFi Bst II") as another
preferred
embodiment of this invention, modified from the naturally-occurring Bst 320
DNA
polymerase at positions 342-344 to substitute threonine, proline and leucine,
respectively,
for leucine, glutamate and glutamate, and at position 422 to substitute
tyrosine for
phenylalanine.
Amino acid sequence (SEQ ID:No 4):
MAEGEKPLEEMEFAIVDVITEEMLADKAAL V VEVMEENYHDAPIV GIAL
VNEHGRFFMRPETALADSQFLAWLADETKKKSMFDAKRAVVALKWKGIELRGV
AFDLLLAAYLLNPAQDAGDIAAVAKMKQYEAVRSDEAVYGKGVKRSLPDEQTL
AEHLVRKAAAIWALEQPFMDDLRNNEQDQLLTKLEHALAAILAEMEFTGVNVDT
KRT.EQMGSELAEQLRAIEQRIYELAGQEFNINSPKQLGVILFEKLQLPVLKKTKTG
YSTSAD VLEKLAPHHEIVENILHYRQLGKLQ STYIEGLLKV V RPDTGKVHTMFNQ
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ALTQTGRLSSAEPNLQNIPIRTPLGRKIRQAFVPSEPDWLIFAADYSQIELRVLAHIA
DDDNLIEAFQRDLDIHTKTAMDIFQLSEEEVTANMRRQAKAVNYGIVYGISDYGL
AQNLNITRKEAAEFIERYFASFPGVKQYMENIVQEAKQKGYVTTLLHRRRYLPDIT
SRNFNVRSFAERTAMNTPIQGSAADIIKKAMIDLAARLKEEQLQARLLLQVHDELI
LEAPKEEIERLCELVPEVMEQAVTLRVPLKVDYHYGPTWYDAK
The underlined amino acids are substituted amino acids produced by site-
directed
mutation of the naturally-occurring Bst 320 DNA polymerase.
The modified Bst 320 DNA polymerase is encoded by a DNA sequence such as
the following (SEQ ID N0:3):
ATG GCCGAAGGGG AGAAACCGCT TGAGGAGATG
GAGTTTGCCA TCGTTGACGT CATTACCGAA GAGATGCTTG
CCGACAAGGCAGCGCTTGTC GTTGAGGTGA TGGAAGAAAA
CTACCACGATGCCCCGATTG TCGGAATCGC ACTAGTGAAC
GAGCATGGGCGATTTTTTAT GCGCCCGGAG ACCGCGCTGG
CTGATTCGCAATTTTTAGCA TGGCTTGCCG ATGAAACGAA
GAAAAAAAGCATGTTTGACG CCAAGCGGGC AGTCGTTGCC
TTAAAGTGGAAAGGAATTGA GCTTCGCGGC GTCGCCTTTG
ATTTATTGCTCGCTGCCTAT TTGCTCAATC CGGCTCAAGA
TGCCGGCGATATCGCTGCGG TGGCGAAAAT GAAACAATAT
GAAGCGGTGCGGTCGGATGA AGCGGTCTAT GGCAAAGGCG
TCAAGCGGTCGCTGCCGGAC GAACAGACGC TTGCTGAGCA
TCTCGTTCGCAAAGCGGCAG CCATTTGGGC GCTTGAGCAG
CCGTTTATGGACGATTTGCG GAACAACGAA CAAGATCAAT
TATTAACGAAGCTTGAGCAC GCGCTGGCGG CGATTTTGGC
TGAAATGGAATTCACTGGGG TGAACGTGGA TACAAAGCGG
CTTGAACAGATGGGTTCGGA GCTCGCCGAA CAACTGCGTG
CCATCGAGCAGCGCATTTAC GAGCTAGCCG GCCAAGAGTT
CAACATTAACTCACCAAAAC AGCTCGGAGT CATTTTATTT
GAAAAGCTGCAGCTACCGGT GCTGAAGAAG ACGAAAACAG
GCTATTCGACTTCGGCTGAT GTGCTTGAGA AGCTTGCGCC
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GCATCATGAAATCGTCGAAA ACATTTTGCA TTACCGCCAG
CTTGGCAAACTGCAATCAAC GTATATTGAA GGATTGTTGA
AAGTTGTGCGCCCTGATACC GGCAAAGTGC ATACGATGTT
CAACCAAGCGCTGACGCAAA CTGGGCGGCT CAGCTCGGCC
GAGCCGAACTTGCAAAACAT TCCGATTCGG ACCCCACTGG
GGCGGAAAATCCGCCAAGCG TTCGTCCCGT CAGAGCCGGA
CTGGCTCATT TTCGCCGCCG ATTACTCACA AATTGAATTG
CGCGTCCTCGCCCATATCGC CGATGACGAC AATCTAATTG
AAGCGTTCCAACGCGATTTG GATATTCACA CAAAAACGGC
GATGGACATTTTCCAGTTGA GCGAAGAGGA AGTCACGGCC
AACATGCGCCGCCAGGCAAA GGCCGTTAAC TACGGTATCG
TTTACGGAATTAGCGATTAC GGATTGGCGC AAAACTTGAA
CATTACGCGCAAAGAAGCTG CCGAATTTAT CGAACGTTAC
TTCGCCAGCTTTCCGGGCGT AAAGCAGTAT ATGGAAAACA
TAGTGCAAGAAGCGAAACAG AAAGGATATG TGACAACGCT
GTTGCATCGGCGCCGCTATT TGCCTGATAT TACAAGCCGC
AATTTCAACGTCCGCAGTTT TGCAGAGCGG ACGGCCATGA
ACACGCCAATTCAAGGAAGC GCCGCTGACA TTATTAAAAA
AGCGATGATTGATTTAGCGG CACGGCTGAA AGAAGAGCAG
CTTCAGGCTCGTCTTTTGCT GCAAGTGCAT GACGAGCTCA
TTTTGGAAGCGCCAAAAGAG GAAATTGAGC GATTATGTGA
GCTTGTTCCGGAAGTGATGG AGCAGGCCGT TACGCTCCGC
GTGCCGCTGAAAGTCGACTA CCATTACGGC CCAACATGGT
ATGATGCCAAA
The characters represent the following nucleotides:
A: Adenosine T: Thymidine C: Cytidine G: Guanosine
The underlined nucleotides TAC are substituted nucleotides produced by site-
directed mutation of the naturally-occurring Bst 320 polymerase. (As would be
apparent
to someone skilled in this art, this DNA sequence does not indicate the
starting codon.)
1~
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The DNA polymerase may also be one that has a DNA sequence that is
complementary to Bst 320 or the modified Bst 320 DNA sequence, for instance,
DNA
sequences that would hybridize to one of the above DNA sequences of under
stringent
conditions. As would be understood by someone skilled in the art, the DNA
sequence
also contemplates those that encode a peptide having these characteristics and
properties
(including degenerate DNA code).
The DNA sequences and amino acid sequences contemplated include allelic
variations and mutations (for instance, adding or deleting nucleotide or amino
acids,
sequence recombination or replacement or alteration) which result in no
substantive
change in the function of the DNA polymerase or its characteristics. For
instance, the
DNA polymerases encompass non-critical substitutions of nucleotides or amino
acids that
would not change functionality (i.e., such as those changes caused by a
transformant host
cell). In addition, the invention is intended to include fusion proteins and
muteins of the
DNA polymerases.
The DNA sequences and amino acid sequences for the modified and ummodified
DNA polymerases are also obtainable by, for instance, isolating and purifying
DNA
polymerase from a Bacillus stea~othermophilus, or a bacterial strain otherwise
derived
from Bacillus stearothe~~nophilus, or other mesophilic bacillus strains such
as Bacillus
caldotehax or Bacillus caldolyticus. The DNA polymerases obtained from these
organisms may be easily modified using conventional DNA modification
techniques to
achieve the properties of high fidelity, high processivity, thermostability
and reduction in
fluorescent dye-labeled ddCTP and ddATP selective discrimination, as long as
the
unmodified amino acid sequences have leucine-glutamate-glutamate at positions
corresponding respectively to positions 342-344 of Bst 320 DNA polymerase and
phenylalanine at a position corresponding to position 422 of Bst 320 DNA
polymerase.
For instance, using the primers and methods of screening described herein,
someone
skilled in the art could isolate a DNA polymerase having the same properties
and
function from other strains.
In another preferred embodiment, a DNA polymerase is used which has highly
stable enzymatic activity for instance, stable enough to withstand drying-down
processes yet remain viable for DNA sequencing. Such DNA polymerases are
described
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by the inventors in U.S. Patent Application No. 09/735,677 (the contents of
which is
incorporated herein by reference in its entirety). These modified Bst DNA
polymerases have increased stability properties, such that they can be freeze-
dried or
dried-down in cold temperatures, or stored in ready-to-use liquid reaction
mixtures, for
extended lengths of time (e.g., at least eight weeks) at room temperature
without
significant loss of its quality as a DNA polymerase for accurate incorporation
of dNTPs
and ddNTPs, or their analogs, onto the 3' end of an extending primer upon
reconstitution
in solution. That is, upon reconstitution in solution and use in standard DNA
sequencing
there is no significant variability in the quality of sequences produced, when
compared to
control (e.g., non-freeze-dried or non-dried-down) DNA polymerase. Following
freeze-
drying or drying-down and subsequent reconstitution, these polymerases can be
used in
known DNA sequencing protocols to generate excellent quality DNA sequences.
These
DNA polymerases also demonstrate higher thermostability than the wild-type Bst
DNA
polymerases. For instance, these polymerases typically have a half life of
polymerase
activity at 65°C for about 16 minutes, which is roughly twice as long
as the wild-type Bst
DNA polymerase.
Throughout this disclosure, "HiFi Bst" or "Bst 320" DNA polymerase refers to
the
unmodified naturally occurring DNA polymerase having proofreading 3'-5'
exonuclease
activity, either isolated from the cells of a strain designated no. 320 of
Bacillus
stea~othermophilus or produced by overexpression of the gene encoding this
naturally
occurring DNA polymerase. (As noted above, this Bst strain no. 320 and DNA
polymerase are described in U.S. Patent 5,747,298 and U.S. Patent 5,834,253.)
"HiFi Bst-
II" refers to the modified form of "HiFi Bst" DNA polymerase which has the
ability to
reduce selective discrimination against fluorescent dye-labeled ddCTP and
ddATP. HiFi
Bst-II is an example of one preferred embodiment of this invention. (This Bst
strain and
DNA polymerase are described in U.S. Patent 6,165,765.) Bst-II also has
sufficient
stability to be dried-down or freeze-dried or stored in ready-to-use liquid
reaction
mixtures, at room temperature for an extended period of time (such as at least
eight
weeks), without significant loss of its quality as a DNA polymerase for
accurate
incorporation of dNTPs and ddNTPs, or their analogs, onto the 3' end of an
extending
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primer upon reconstitution in appropriate solution. (This Bst strain and DNA
polymerise
are described in copending U.S. Patent application 09/735,677.)
Thus, in one embodiment of the methods of the invention, the invention
contemplates a method for extending a primer (or a pair of primers) using an
enzymatic
cycle primer extension reaction at low cycling temperatures (that is,
temperatures below
about 80°C). The reaction mixture composition that comprises between
about 10% and
about 20% (and preferably about 15%) (v/v) glycerol, ethylene glycol, or a
mixture
thereof. The reaction is run in the presence of a moderately thermostable DNA
polymerise such as one of those described above. Ideally, the reaction is
carried out
under conditions that the cycle reaction temperature fluctuates between a
melting
temperature of about 70°C and a cooling (or annealing) temperature of
about 37°C, so
that the DNA polymerise repeatedly extends the primer or pair of primers. The
method
may include the further step of repeating the cycle primer extension reaction,
as many
times as is desired.
In another embodiment, a PCR or PCR-like reaction may be run at low
temperatures below 80°C. In this method, copies of a selected segment
of a double-
stranded DNA are amplified in the presence of a forward primer and a reverse
primer
(where both may be of various lengths) to the template by repeated heating and
cooling
(or annealing) cycles. The reaction mixture composition comprises between
about 10%
and about 20% (and preferably about 15%) (v/v) glycerol, ethylene glycol, or a
mixture
thereof, in the presence of one of the moderately thermostable DNA polymerises
described above. The reaction is preferably carried out under conditions that
the reaction
temperature fluctuates between a melting temperature of about 70°C and
a cooling (or
annealing) temperature of about 37°C, so that the DNA polymerise
repeatedly extends
the forward and reverse primers. The method may include the further step of
repeating
the reaction, as many times as is desired. .
In a further embodiment, molecules of a single primer of various lengths are
extended by a moderately thermostable DNA polymerise with specific nucleotide
terminations in the presence of ddNTPs or their analogs for low-temperature
cycle
sequencing below about 80°C. The ddNTP analogs may be fluorescent dye-
labeled so
that each members of the ddNTPs may emit different wavelengths, as those used
in
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automated dye-labeled terminator DNA cycle sequencing. Or instead, the
sequencing
primer will be labeled with four different dyes- to be used in pairing with
the
corresponding unlabeled member of the ddNTPs for a modified Sanger reaction as
in
fluorescent dye-labeled primer DNA cycle sequencing technology. Or
alternatively, the
low-temperature cycle primer extension termination reaction can be used in the
classic
Sanger protocol with radioactive isotope-labeled dATP for manual direct
sequencing of a
small amount of DNA template without prior PCR amplification.
Another embodiment contemplates a method for extending the molecules of a
single primer annealed to a single-stranded copy of the double-stranded DNA
product
amplified in vitro without prior isolation or purification for direct cycle
sequencing. For
instance, a diluted crude amplified reaction product (preferably generated
with a low-
temperature PCR reaction catalyzed by a moderately thermostable DNA polymerase
as
described herein) is used as template and mixed with an excess amount of a
sequencing
primer, the four standard ddNTP terminators (ddATP, ddGTP, ddTTP and ddCTP)
fluorescently labeled (or their corresponding analogs fluorescently labeled),
a moderately
thermostable DNA polymerase (preferably one with a reduced innate selective
discrimination against incorporation of a subset of dye-labeled ddNTPs), a
suitable
concentration of dNTPs (dATP, dGTP, dTTP and dCTP), and a composition
comprising
a buffer in a solution containing between about 10% and about 20% (preferably
15%)
(v/v) of glycerol, ethylene glycol, or mixture thereof. A standard cycle
primer extension
reactions) may then be run at a temperature below 80°C for a sufficient
number of times
to extend the sequencing primer molecules to desired varying lengths, which
extended
molecules will be terminated specifically by fluorescently labeled ddNTPs or
their
corresponding analogs. Preferably, the cycle reaction temperature fluctuates
between a
melting temperature of about 70°C and a coolinglannealing temperature
of about 37°C.
In one preferred embodiment, the method of sequencing a DNA strand may
comprise the steps of:
i) hybridizing a primer to a DNA template to be sequenced; and
ii)~ extending the primer using one of the above-described DNA polymerases, in
the presence of a solution containing between about 10% and about 20% (v/v)
(preferably about 15% (v/v)) glycerol, ethylene glycol, or a mixture thereof,
adequate
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amounts of the deoxynucleotide bases dATP, dGTP, dCTP and dTTP, and the four
dideoxynucleotide terminators, or their analogs, whereby the cycle reaction
temperature
fluctuates between a melting temperature of about 70°C arid a cooling
or annealing
temperature of about 37°C, and under such conditions that the DNA
strand is sequenced.
Preferably one of the deoxynucleotides is radioisotope-labeled, or the primer
molecules
are fluorescent dye-labeled, and more preferably all are fluorescent dye-
labeled.
In another embodiment, the invention entails a dry or liquid ready-to-use
reaction
mixture or kit suitable for use in a low-temperature cycle primer extension
reaction at
temperatures below about 80°C. This reaction mixture or kit comprises a
moderately
thermostable DNA polymerase (such as one of those described above) that is pre-
mixed
with at least one enzymatic DNA primer extension reaction component suitable
for use in
DNA amplification or for specific extension terminations with
dideoxyribonucleotide
analogs. The reaction mixture is preferably pre-distributed into
microcentrifuge tubes or
in multiple-well plates, such as, for instance, those that are suitable for
large-scale
automated PCR or for large-scale automated DNA sequencing. This ready-to-use
reaction mixture or kit can be stored at room temperature between about
22°C and about
25°C for at least eight weeks without losing its specific
polymerization activity for DNA
primer amplification or extension terminations.
As an example of the present.inventive methods, when a moderately thermostable
DNA polymerase is used for low-temperature cycle primer extension, both
annealing and
primer extension can take place simultaneously at 45°C. Alternatively,
37°C can be used
as the annealing temperature and 45-50°C the primer extension
temperature. (See Figure
6). Therefore, both the following protocol A and protocol B can be used for
the low-
temperature cycling steps with effective specific amplification:
(A) 70°C 30 seconds and 45°C 4 minutes for a total of 35 cycles;
or
(B) 70°C 30 seconds, 37°C 20 seconds and 50°C 3 minutes
for a total of 35 cycles.
However, protocol B is preferred when the enzymatic primer extension is to
generate
reaction products with fluorescent dye-labeled ddNTP terminations for
automated cycle
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sequencing. It is noted that the methods of this invention are not limited to
either
protocols A or B, but that these two protocols are exemplary of temperatures
and cycles
that work effectively with these methods. For instance, under certain
circumstances, the
extension time may be desired to be prolonged to about 11 minutes for long
target
segment amplification.
As another example, when the methods of this invention are used to generate
amplification products for DNA cycle sequencing, a single primer in excess can
be added
to a reaction mixture containing 15% glycerol and a moderately thermostable
DNA
polymerase. The single-stranded primer oligonucleotides can then be extended
to various
lengths with specific nucleotide terminations in the presence of ddNTPs or
their analogs,
which may be fluorescently labeled. The template used for the cycle sequencing
can be
any purified double-stranded or single-stranded DNA fragments containing the
target
sequence, or an aliquot of the diluted amplification products derived from the
low-
temperature cycle-extended primer strands of the double-stranded DNA template
described in this invention, without prior isolation and purification. Since
the
amplification products derived from the low-temperature cycle primer extension
using a
moderately thermostable DNA polymerase with high fidelity and high
processivity as
described in this invention are highly sequence-specific, prior isolation of
the PCR
product from the reaction mixture before being used as the template for DNA
cycle
sequencing is generally unnecessary.
The following non-limiting examples are illustrative of the invention.
EXAMPLES
Example 1: The effect of Glycerol on 5'- 3' polymerization activity of
moderately
thermostable DNA polymerases
The remaining activity of Bst-II DNA polymerase (produced according to US
patent No. 6,165,765) in the presence of different concentrations of Glycerol
was
determined as follows:
(a) In a series of numbered 0.5 ml microcentrifuge tubes were added the
following:
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Tube No. 1 2 3 4 5 6 7
~
x Reaction Buffer 5 5 5 5 5 5 5
(u1)
DNTPs (1 mM each)
(u1) 1 1 1 1 1 1 1
Calf Thymus DNA (u1)1 1 1 1 1 1 1
5 x Reaction Buffer (RB): 100mM Tris-Cl, pH 8.5 containing 100mM MgCl2.
Calf Thymus DNA: DNase I activated, l.Sug/ul.
The mixtures above were firstly evaporated by Speed-Vacuum, then varying final
concentrations of glycerol in each reaction mixture were achieved by adding an
appropriate amount of a glycerol stock solution to the above microcentrifuge
tubes as
indicated below.
Tube No. 1 2 3 4 5 6 ~
7
Final Glycerol Conc.0 10% 15% 20% 30% 40% 50%
(v/v)
a- "P-dATP (u1) 1 ' 1 1 1 1 1 1
Enzyme(0.36 ug/ul) 1 1 1 1 1 1 1
(u1)
Glycerol (80% stock)(ul)0 3.1 5.0 6.9 10.6 14.4 18.1
ddH20 (u1) 28 24.9 23.0 21.1 17.4 13.6 9.9
Total Volume(ul) 30 30 30 30 30 30 30
(a-32P-dATP : Amersham, 800Ci/mmol)
(b) All these tubes were incubated at 65°C for 30 minutes. Then each of
the
reaction mixtures was pipetted onto a DE-81 filter. After all of the fluid has
evaporated,
the amount of radioactivity on each filter was measured with scintillation and
recorded as
Xl. Thereafter, the filters were washed three times with 0.3 M Na2HP04
solution at room
temperature, 10 minutes each times, dried at room temperature and then the
amount of
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radioactivity on each filter was measured again and recorded as X2.
Incorporation ratio
=XalXi.
Remaining activity (%) _
Incorporation ratio of radioactivity in different glycerol Concentration
Incorporation ratio of radioactivity without glycerol
As shown in Figure 1, a low concentration of glycerol that did not exceed 20%
(v/v) increased the enzymatic activity of Bst-II DNA polymerase. However, at
higher
concentrations glycerol exhibited an inhibitory effect on the enzyme.
Example 2: The effect of 40% glycerol (v/v) on low-temperature cycle primer
extension
with moderately thermostable DNA polymerases
Bst-II DNA polymerase was used for the study.
Template: pBluescript(+)
Forward Primer: 5' GTAAAACGACGGCCAGT 3'
Reverse Primer: 5' AACAGCTATGACCATG 3'
Experimental Procedure:
(a) In a 0.2 ml microcentrifuge tube, were added the following
Template 1 u1 (16 nglul or 160 ng/ul)
Forward primer( 1 Opmol/ul) 2.5 u1
Reverse primer(lOpmol/ul) 2.5 u1
dNTPs(2.SmM each) 4 u1
SxRB 5 u1
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(b) The above mixture was firstly evaporated by Speed-Vacuum, then the
following
were added to the same microcentrifuge tube
ddHaO 11.75 u1
80% Glycerol 11.25 u1
Bst -II DNA polymerase (lUnit/ul) 2 u1
(c) The final mixture was subjected to the following temperature cycles.
70°C 30 sec
45°C 4 min
35 cycles
The reaction products were run on a 1% agarose gel for electrophoresis and
stained by ethidium bromide.
The results illustrated in Figure 2 show that in the presence of 40% glycerol
as the
reagent to lower the melting temperature of double-stranded DNA for cycle
primer
extension, non-specific amplification products of varying sizes were generated
during the
temperature cycling with either low or high concentration of template in the
reaction
mixture.
Example 3: The effect of reduced concentrations of glycerol on low-temperature
cycle primer extension with moderately thermostable DNA polymerases
This experiment was designed to demonstrate that reduction of the
concentration
of glycerol to about 15% is useful for lowering the DNA melting temperature
for specific
cycle primer extension with moderately thermostable DNA polymerases
Materials and Methods
Bst-II DNA polymerase was used for the study.
27
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Four different sets of templates and primers were selected representing
varying
lengths of the DNA segments to be amplified:
Template A. pBluescript(+) 10 ng/ul
Forward Primer: 5' GTAAAACGACGGCCAGT 3'
Reverse Primer: 5' AACAGCTATGACCATG 3'
Template B. A Rice genome BAC DNA 10 ng/ u1
Forward Primer: 5' CTTAATTTAAGGTTCCGTG 3'
Reverse Primer: 5' GCATTGGTAAGCAATGG 3'
Template C. A hybridization probe 50 ng/ul
Forward Primer: 5' ACAAAGCACTGAACCTG 3'
Reverse Primer: 5' TGGGACCTATCGTGTTG 3'
Template D. A subclone of BAC from rice genome 50 ng/ul
Forward Primer: 5' CGAATTCCTGCAGCC 3'
Reverse Primer: 5' GAACTAGTGGATCCCCC 3'
The low temperature cycle extension was carried out as follows:
(a) To a 0.2 ml microcentrifuge tube were added.
Template A, B, C or D 1 u1
Forward primer A, B, C or D (10 pmol/ul) 2.5 u1
Reverse primer A, B, C or D (10 pmol/ul) 2.5 u1
dNTP(2.SmM each) 4 u1
SxRB 5 u1
(b) The mixture above was firstly evaporated by Speed-Vacuum, then the
following .
were added to each microcentrifuge tube containing the evaporated reagents
with
different sets of template and primers to achieve a final concentration of 35%
glycerol
and 15% glycerol in the reaction mixture, respectively.
28
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1. with 35% Glycerol in mixture 2. with 15% GlKcerol in mixture
ddH20 12.1u1 18.3u1
80% Glycerol 10.9u1 4.7u1
Polymerase (lU/ul) 2u1 2u1
(c) All the microcentrifuge tubes with the reaction mixture were subjected to
low
temperature cycling as follows:
70°C 30 sec
45°C 4 min
35 cycles total.
The reaction products were run on a 1% agarose gel for electrophoresis and
stained by ethidium bromide. The reaction products from the mixture containing
35%
glycerol were loaded in lane 1, and the reaction products from the mixture
containing 15
glycerol were loaded in lane 2.
The results illustrated in Figure 3 show that, when a short segment of DNA of
2S0
by or 400 by long was the target product for cycle primer extension there were
no
amplification products produced at all during low temperature eycling, using
35%
glycerol as the reagent for lowering the DNA melting temperature (Figure 3 A l
and B 1 ).
When longer target products, for example, 1 Kb and 2 Kb in length, were to be
amplified
under the identical conditions, enzymatic cycle primer extension was achieved
with
generation of both specific and non-specific amplification products when 35%
of glycerol
was used to lowering the melting temperature (Figure 3 C 1 and D 1 ).
When a 15% glycerol was used as the reagent to lower the DNA melting
temperature, specific amplification products ranging from 250 by to 2 Kb in
length were
generated with a moderately thermostable DNA polymerase during low temperature
cycling (Figure 3 A2, B2, C2 and D2).
Based on the experimental results presented above, a low concentration of
glycerol, for example at about 15% of final, in the reaction mixture has been
adopted as
the preferred reagent for lowering the DNA melting temperature in specific
cycle primer
extension by moderately thermostable DNA polymerases.
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Example 4: Low-temperature cycle extension of DNA primers of different lengths
with
moderately thermostable DNA polymerases
In this example, the experiments were designed to demonstrate that the low-
temperature cycle extension system with moderately thermostable DNA
polymerases of
this invention can be used for sequence-specific extension of primers of up to
30 base
pairs in length.
The polymerases used were Bst-I (wild type produced according to US patent
5,834,254), Bst-II, and Bca (TaKaRa Co.). The I~lenow fragment (Sigma Chemical
Co.)
was used as a thermolabile DNA polymerase for comparison (Iakobashvili and
Lapidot).
The template used was rice genome BAC B414f7.
The two pairs of primers used were:
A: l7mer forward primer: 5'TAG CTA TCT AAC TTA AT3',
l7mer reverse primer: 5'TTG TTT CTC TGA TGC AT3',
B: 30mer forward primer: 5'TAG CTA TCT AAC TTA ATT TAA GGT TCC
GTG3',
30mer reverse primer: 5'TTG TTT CTC TGA TGC ATT GGT AAG CAA
TGG3' .
The following reaction system (referred to hereafter as the Bst system) was
used.
(a) In a 0.2 ml microcentrifuge
tube were added:
Template (5 ng/ul) 1 u1
Forward primer (15 pmol/ul) 2.5 u1
Reverse primer (15 pmol/ul) 2.5 u1
dNTPs (2.5 mM each) 4 u1
SxRB 5 u1
d~aG 3.3 u1
80% Glycerol 4.7 u1
DNA polymerase (4U/ul) 2 u1
(b) The microcentrifuge tubes were subjected to the following temperature
cycling.
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70°C 30 sec
37°C 20 sec
50°C 3 min
35 cycles total.
The reaction products were run on a 1% agarose gel for electrophoresis and
stained by ethidium bromide.
In addition, the method of using a reaction mixture containing 4.5 M proline
and
17% glycerol in Tris-HCl buffer as recommended by Iakobashvili and Lapidot was
also
adopted for the reactions with Klenow fragment as the DNA polymerase
(hereafter
referred to as the Iakobashvili and Lapidot system). The reaction products
were also run
parallel to those obtained with the Bst system, and illustrated as follows in
Figure 4.
In Figure 4, the following reaction products are shown in the respective
lanes.
A: Reaction products with 17mer primers:
A1: Klenow fragment using the Iakobashvili and Lapidot system.
A2: Klenow fragment with the Bst system.
A3: Bst-I polymerase with the Bst system.
A4: Bst-II polymerase with the Bst system.
A5: Bca polymerase with the Bst system.
B: Reaction products with 30mer primers:
B 1: Klenow fragment using the Iakobashvili and Lapidot system.
B2: Klenow fragment with the Bst system.
B3: Bst-I polymerase with the Bst system.
B4: Bst-II polymerase with the Bst system.
B5: Bca polymerase with the Bst system.
Molecular Ladders:
M1: ~, DNAIHz~d III.
M2: DL 2,000 (from TaKaRa Co., with the DNA fragment of 2000, 1000, 750, 500,
250 and 100 by respectively).
Figure 4 shows that the moderately thermostable DNA polymerases, namely the
natural form of Bst-I, the mutated Bst-II and Bca, all generated specific
amplification
31
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WO 02/101004 PCT/IB02/03341
products as a result of l7mer primer extension (A3-AS) and of 30mer primer
extension
(B3-BS) in the Bst system containing 15 % glycerol in the reaction mixture as
recommended for low temperature cycling. However, the thermolabile DNA
polymerase,
Klenow fragment, failed to produce a specific amplification product from l7mer
or
30mer primer extension either in the Iakobashvili and Lapidot system (A1 and
Bl) or in
the Bst system (A2 and B2).
Example 5: High fidelity low-temperature linear cycle sequencing with Bst-II
DNA
polymerase in stored ready-to-use reaction pre-mixture.
The current invention can be used to perform DNA sequencing with a genetically
modified moderately thermostable DNA polymerase, Bst-II, to extend the primer
over the
GC-rich segments of the template which the commonly used heat-stable DNA
polymerases with low processivity, such as ThermoSequenaseTM or AmpIiTaqTM,
are
unable to overcome. Furthermore, all pre-measured ingredients of the reaction
mixture
with or without the primer pre-added can be pre-mixed and stored in individual
microcentrifuge tubes or 96-well plates for at least eight (8) weeks at
temperatures
between 23°C and 25°C.
Bst-II Cycle Sequencing Experiment
Bst-II was used as the DNA polymerase.
Template: bg08. This was a GC-rich segment of a subclone of rice genome BAC
129.
Primer: 5'GAA TTG GAG CTC CAC CGC GG3'
Pre-mixed dye-ddNTPs: Optimized R6G-ddATP, ROX-ddCTP, TAMRA-ddUTP,
and Bodipy F1-14-ddGTP, purchased from NENTM Life Sciences Products.
(a) Into a 0.2 ml of microcentrifuge tube, the following ingredients were
added
dNTPs (2.5 mM each) 1 u1
SxRB 5 u1
Pre-mixed dye-ddNTPs 4
Bst-II DNA polymerase (l0U/ul) 1 u1
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ddH20 5.3 u1
80% Glycerol 4.7 u1
The reaction pre-mixture in microcentrifuge tubes was stored at temperatures
between 23°C and 25°C until use within eight (8) weeks.
(b) At the time of the experiment, 2.5 u1 of template (150 ng/ul) and 1.5 u1
of
primer were added into a microcentrifuge tube containing the above pre-
mixture.
(c) The contents in the microcentrifuge tube were mixed thoroughly and
subjected
to the .following linear low temperature cycling.
70°C for 30 sec,
37°C for 20 ec,
45°C for 3 min,
35 cycles total.
(d) Added 2.5 u1 3M NaOAc (pH5.2) and 55 u1 95% ethanol to each tube. The
tube was inverted several times and then placed at room temperature for 20 min
to
precipitate the extension products.
(e) The mixture was centrifuged at 12,OOOg for 20 min at room temperature.
(f) The supernatant was drawn off, and the pellet was rinsed with 120 u1 70%
ethanol.
(g) Inverted the tube several times, placed the tube at room temperature for
15
min, and centrifuged the tube for 10 min at 12,000 g.
(h) The pellet was dried at 45°C, and resuspended in 1.2 u1 loading
buffer (5:1 of
deionized formamide : 25 mM EDTA , pH8.0, with SOmglml Blue Dextran).
(i) The sample was denatured at 95°C for 3 min, then irnrnediately
placed on ice.
(j) All of each sample was loaded onto 4.5% (6M urea) sequencing gel and the
sequencing information was collected by an ABI PRISMTM 377 DNA Sequencer. The
data were analyzed with the corresponding instrument (matrix) file.
For comparison, DNA sequencing of the identical template with the same primer
was also performed, using two commercially available cycle sequencing kits,
namely the
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CA 02449560 2003-12-03
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DYEnamicTM ET terminator cycle sequencing kit with Thermo SequenaseTM
(Amersham)
and the ABI PrismTM BigDyeTM Terminator cycle sequencing kit with AmpliTaqTM
(ABI).
The cycle sequencing procedures were carried out by following the protocols
provided by
the respective companies.
The results of the DNA sequencing were presented in Figure 5 which shows the
ABI PrismTM BigDyeTM Terminator cycle sequencing kit with AmpliTaqTM (Figure 5
A)
failed to accomplish efficient specific fluorescent dye-labeled ddNTP
terminations during
cycle primer extension over the GC-rich segment of the DNA template. In
comparison,
the Bst-II Cycle Sequencing system, even after the Bst-II DNA polymerase had
been
stored in a pre-mixed form for eight (8) weeks at 23-25°C, successfully
overcame the
GC-rich barrier in the template and generated adequate specific dye.-labeled
ddNTP
terminations for DNA sequencing analyses (Figure 5 B). Similar to the ABI
AmpliTaqT""
kit, Thermo SequenaseTM used with the Amersham DYEnamicTM ET terminator cycle
sequencing kit also failed to overcome the GC-rich segment of the template
during the
cycle primer extension reaction for automated fluorescent DNA sequencing
(tracing not
shown here).
In conclusion, the Bst-II Cycle Sequencing system which remains stable in
ready-
to-use pre-mixture at room temperature for at least eight (8) weeks is most
suitable for
large-scale high fidelity automated fluorescent DNA sequencing, especially
when the
templates contain GC-rich segments.
Figure 5 shows DNA sequencing over a GC-rich segment, including a comparison
of the performance of AmpliTaqTM in the ABI PrismTM BigDyeTM Terminator cycle
sequencing kit (A) with that of the Bst-II Cycle Sequencing System (B). Figure
5 A and
B represent two automated fluorescent DNA sequencing tracings of a GC-rich
segment of
the same template using the same prime for cycle extension. Both sequences
were run in
an ABI 377 sequencer. The shadowed zone illustrated in A represents the region
out of
quality control evaluated and reported by the computer.
A = generated with the AmpliTaqTM BigDyeTM kit; B = generated with the Bst-II
Cycle
Sequencing system.
Example 6: Optimum temperature steps for cycle primer extension with
moderately
thermostable DNA polymerases
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This experiment was designed to determine the optimum temperature steps for
cycle primer extension with moderately thermostable DNA polymerases in a
reaction
mixture containing 15% glycerol as the agent to lower the DNA melting
temperature.
Bst-II was the DNA polymerase used.
Template: A rice genome BAC DNA
Forward Primer: 5' CTTAATTTAAGGTTCCGTG 3'
Reverse Primer: 5' GCATTGGTAAGCAATGG 3'
(a) To each 0.2 ml microcentrifuge tube were added:
Template (10 ng/ul) 1 u1
Forward primer (10 pmol/ul) 2.5 u1
Reverse primer ( 10 pmol/ul) 2.5 u1
dNTPs (2.5 mM each) 4 u1
SxRB 5 u1
(b) The following were added to the microcentrifuge tubes to achieve:
Final Concentration of Glycerol (v/v) 0% 15%
ddH20 8 u1 3.3 u1
80% Glycerol 4.7 u1
Bst-II DNA polymerase (1U/ul) 2 u1 2 u1
(c) The cycling temperature steps were as follows.
Steps 1 Steps 2 Steps 3 Steps Steps 5
4
70C 30s 70C 30s 70C 30s 70C 30s 70C 30s
37C 4min 37C 20s 37C 20s 37C 20s 45C 4min
45C 3min 50C 3min 60C 3mi
35 cycles 35 cycles 35 cycles 35 cycles35 cycles
The reaction products were run on a 1 % agarose . gel for electrophoresis and
CA 02449560 2003-12-03
WO 02/101004 PCT/IB02/03341
stained by ethidium bromide. The results are illustrated in Figure 6, in
which, the lanes
were loaded as follows:
1. No glycerol; 70°C 30s, 37°C 4min, 35 cycles.
2. No glycerol; 70°C 30s, 37°C 20s, 45°C 3min, 35 cycles.
3. No glycerol; 70°C 30s, 37°C 20s, 50°C 3min, 35 cycles.
4. No glycerol; 70°C 30s, 37°C 20s, 60°C 3min, 35 cycles.
5. 15% glycerol; 70°C 30s, 37°C 4min, 35 cycles.
6. 15% glycerol; 70°C 30s, 37°C 20s, 45°C 3min, 35
cycles.
7. 15% glycerol; 70°C 30s, 37°C 20s, 50°C 3min, 35
cycles.
8. 15% glycerol; 70°C 30s, 37°C 20s, 60°C 3min, 35
cycles.
9. 15% glycerol; 70°C 30s, 45°C 4min, 35 cycles.
The results in Figure 6 show that the most effective cycle primer extension
with
moderately thermostable DNA polymerases, such as Bst-II, is obtained with a
single
annealing and extension temperature at 45°C (Lane 9), or annealing at
37°C and
extension at 45°C-50°C in the presence of 15% glycerol used as
the melting-temperature-
lowering agent. Although both temperature cycling protocols of Steps 3 and
Steps 5 can
be used for specific primer extension in DNA amplification, the cycling
protocol of Steps
3 with 70°C 30s, 37°C 20s, 50°C 3min, 35 cycles is
preferred (Lane 7) when the
enzymatic primer extension is used to generate reaction products with
fluorescent dye-
labeled ddNTP terminators for automated cycle DNA sequencing.
Example 7: Direct low temperature cycle sequencing of amplified products
generated
by moderately thermostable DNA polymerases in stored ready-to-use reaction pre-
mixture.
This example demonstrated that all pre-measured ingredients of the reaction
mixture for low temperature primer extension, including a moderately
thermostable DNA
polymerase, with or without the primers pre-added can be pre-mixed and stored
in
individual microcentrifuge tubes or 96-well plates for at least eight (8)
weeks at
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CA 02449560 2003-12-03
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temperatures between 23°C and 25°C until use for the
amplification reaction. In addition,
the amplified reaction products can be used directly for automated DNA
sequencing
without prior purification.
Bst-II was used as the DNA polymerase.
Template: H525d9, a BAC of rice genome,
Forward primer: 5' TTT CAG GGT CCC TTA TAT CTC 3',
Reverse primer: 5'TCG CTT CTC CTC ATA ATC GAT 3'.
Pre-mixed dye-ddNTPs: Optimized R6G-ddATP, ROX-ddCTP, TAMRA-ddUTP, and
Bodipy F1-14-ddGTP, purchased from NENTM Life Sciences Products.
(a) Into a 0.2 ml of microcentrifuge tube, the following ingredients were
added:
Forward primer (10 pmol/ul) 2 u1
Reverse primer (10 pmol/ul) 2 u1
dNTPs (2.5 mM each) 2 u1
x RB 5 u1
Bst-II DNA polymerase (10 U/ul) 1 u1
ddHaO 4.3 u1
80% Glycerol 4.7 u1
The reaction pre-mixture in the microcentrifuge tube was stored at temperature
between
23°C and 25°C until use within eight (8) weeks.
(b) At the time of experiment, the following were added to the stored pre-
mixture:
Template (2.5 ng/ul) 1 u1
ddH20 3 u1
(c) The ingredients in the microcentrifuge tube were thoroughly mixed and
subjected to temperature cycling in the following protocol.
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70 °C for 30 sec,
37 °C for 20 sec,
45°C for 3 min,
35 cycles total.
(d) Cycle sequencing of the retrieved amplified products after purification.
(1) After the temperature cycling was completed, the reaction products were
loaded onto a 1 % low melting point agarose gel for electrophoresis.
(2) After electrophoresis, the target agarose blocks were cut out from the gel
and
weighed.
(3) To the cut-out agarose blocks, 0.04V of 25 x Conc. Buffer (Roche) was
added,
and the mixture was incubated at 65°C for 15 min to melt the gel.
(4) After additional incubation at 45°C for 5 min, an appropriate
amount of
agarase(lU/ul, Roche) was added at 1U/100 mg of agarose gel.
(5) After further digestion for 1 hour at 45°C, 1/10V of 3M NaOAc
(pH5.2 )
was added. The tube was placed on ice for I S min and then spun at 12,000 g
and at 4 °C
for 15 min.
(6) The supernatant was extracted with equal volume of phenol-chloroform
twice,
and of chloroform once. After each extraction, the mixture was centrifuged at
12,OOOg for
min to collect the top aqueous layer.
(7) A 3V of 95% ethanol was added to the extracted aqueous phase; then the
mixture was centrifuged at 12,000 g at 4°C for 15 min after chill on
ice for 15 min. The
pellet was washed in 250 u1 of 70% ethanol, dried and dissolved in 20 u1 of
ddH20.
(8) Sequencing the retrieved amplified products with the forward primer was
performed as described above in 5 (c) - (k), using the high fidelity low-
temperature linear
cycle sequencing with Bst-II DNA polymerase in stored ready-to-use reaction
pre-
mixture.
Alternatively, an aliquot of the amplified products in the reaction mixture
generated in step 7. (c) was sequenced with the Bst-II Sequencing System
directly
without retrieval and prior purification. The example of this direct cycle
sequencing
procedure is described as follows.
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(1) Into a 0.2 ml of microcentrifuge tube, the following ingredients were
added:
dNTPs (2.S mM each) 1 u1
SxRB 5 u1
Pre-mixed dye-ddNTPs (NENTM) 4 u1
Bst-II DNA polymerise (l0U/ul) 1 u1
ddHaO 5.3 u1
80% Glycerol 4.7 uI
The reaction pre-mixture in microcentrifuge tubes was stored at temperatures
between
23°C and 24°C until use within eight (8) weeks.
(2) At the time of experiment, the following were added into each
microcentrifuge
tube.
1120 diluted reaction product mixture [7. (c)] 1 u1
Forward primer (10 pmol/ul) 1.5 u1
ddH20 1.5 u1
(3) The contents in the microcentrifuge tube were mixed thoroughly and
subjected
to the following linear low temperature cycling.
70°C for 30 sec,
37°C for 20 sec,
45°C for 3 min,
35 cycles total.
The DNA in the reaction mixture was precipitated, washed and loaded onto
sequencing gel for electrophoresis and analyzed as described above under
Example 5
above. The sequencing tracings showed that the DNA sequences obtained by both
methods were identical. They indicate that low temperature cycle primer
extension with
moderately thermostable DNA polymerises may generate highly specific amplified
DNA
products which can be used for direct sequencing without further isolation.
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CA 02449560 2003-12-03
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References:
1. Sanger, F., Nicklen, S. & Coulson, A.R. Proc. Nat. Acad. Sci. USA 74: 5463-
5467.
1977
2. Linda G. Lee, Charles R. Connell, Sam L. Woo, et al. Nucleic Acids Res.
20(10):
2471-2483. 1992
3. Molly Craxton. Methods: A Companion to Methods in Enzymology 3(1): 20-26.
1991
4. Hanspeter Saluz, Jean-Pierre Jost. Proc. Nat. Acad. Sci. USA 86: 2602-2606.
1989
5. Ye, S. Y. 8i Hong, G.F., Scientia Sinica (Series B) 30: 503-506. 1987
6. Fuller, C. W.(1995) U.S. Patent No. 5432065
7. Robert Iakobashvili, Aviva Lapidot. Nucleic Acids Res. 27(6): 1566-1568.
1999
8. U.S. Patent No. 6,165,765, to Hong & Huang. DNA polymerase having high
stability and ability to reduce innate selective discrimination against
fluorescent dye-
labeled dideoxynucleotides.
All references are incorporated by reference herein in their entirety.
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SEQUENCE LIS TING
<110> FAN HONG, GUO
YANG, YONGJIE
ZHU, JIA
<120> LOW TEMPERATURE CYCLE EXTENSION OF DNA WITH HIGH
POLYMERIZATION SPECIFICITY
<130> LEE 113
<140> 09/878,131
<141> 2001-06-08
<160> 21
<170> PatentIn Ver. 2.1
<210> 1
<211> 1764
<212> DNA
<213> Bacillus stearothermophilus
<400> 1
gccgaagggg agaaaccgct tgaggagatg gagtttgcca tcgttgacgt cattaccgaa 60
gagatgcttg ccgacaaggc agcgcttgtc gtt gaggtga tggaagaaaa ctaccacgat 120
gccccgattg tcggaatcgc actagtgaac gagcatgggc gattttttat gcgcccggag 180
accgcgctgg ctgattcgca atttttagca tggcttgccg atgaaacgaa gaaaaaaagc 240
atgtttgacg ccaagcgggc agtcgttgcc ttaaagtgga aaggaattga gcttcgcggc 300
gtcgcctttg atttattgct cgctgcctat ttgctcaatc cggctcaaga tgccggcgat 360
atcgctgcgg tggcgaaaat gaaacaatat gaagcggtgc ggtcggatga agcggtctat 420
ggcaaaggcg tcaagcggtc gctgccggac gaacagacgc ttgctgagca tctcgttcgc 480
aaagcggcag ccatttgggc gcttgagcag ccgtttatgg acgatttgcg gaacaacgaa 540
caagatcaat tattaacgaa gcttgagcac gcgctggcgg cgattttggc tgaaatggaa 600
ttcactgggg tgaacgtgga tacaaagcgg ctt gaacaga tgggttcgga gctcgccgaa 660
caactgcgtg ccatcgagca gcgcatttac gagctagccg gccaagagtt caacattaac 720
tcaccaaaac agctcggagt cattttattt gaaaagctgc agctaccggt gctgaagaag 780
acgaaaacag gctattcgac ttcggctgat gtgcttgaga agcttgcgcc gcatcatgaa 840
atcgtcgaaa acattttgca ttaccgccag ctt ggcaaac tgcaatcaac gtatattgaa 900
ggattgttga aagttgtgcg ccctgatacc ggcaaagtgc atacgatgtt caaccaagcg 960
ctgacgcaaa ctgggcggct cagctcggcc gagccgaact t gcaaaacat tccgattcgg 1020
ctcgaagagg ggcggaaaat ccgccaagcg ttcgtcccgt cagagccgga ctggctcatt 1080
ttcgccgccg attactcaca aattgaattg cgcgtcctcg cccatatcgc cgatgacgac 1140
aatctaattg aagcgttcca acgcgatttg gat attcaca caaaaacggc gatggacatt 1200
ttccagttga gcgaagagga agtcacggcc aacatgcgcc gccaggcaaa ggccgttaac 1260
ttcggtatcg tttacggaat tagcgattac ggattggcgc a.aaacttgaa cattacgcgc 1320
aaagaagctg ccgaatttat cgaacgttac ttcgccagct -ttccgggcgt aaagcagtat 1380
atggaaaaca tagtgcaaga agcgaaacag aaa ggatatg -tgacaacgct gttgcatcgg 1440
cgccgctatt tgcctgatat tacaagccgc aatttcaacg -tccgcagttt tgcagagcgg 1500
acggccatga acacgccaat tcaaggaagc gccgctgaca -ttattaaaaa agcgatgatt 1560
gatttagcgg cacggctgaa agaagagcag ctt caggctc gtcttttgct gcaagtgcat 1620
gacgagctca ttttggaagc gccaaaagag gaaattgagc c~attatgtga gcttgttccg 1680
gaagtgatgg agcaggccgt tacgctccgc gtgccgctga aagtcgacta ccattacggc 1740
ccaacatggt atgatgccaa ataa 1764
<210> 2
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2/10
<211> 586
<212> PRT
<213> Bacillus stearothermophilus
<400> 2
Ala Glu Gly Glu Lys Pro Leu Glu Glu Met Glu Phe Ala Ile Val Asp
1 5 10 15
Val Ile Thr Glu Glu Met Leu Ala Asp Lys Ala Ala Leu Val Val Glu
20 25 30
Val Met Glu Glu Asn Tyr His Asp Ala Pro Ile Val Gly Ile Ala Leu
35 40 45
Val Asn Glu His Gly Arg Phe Phe Met Arg Pro Glu Thr Ala Leu Ala
50 55 60
Asp Ser Gln Phe Leu Ala Trp Leu Ala Asp Glu Thr Lys Lys Lys Ser
65 70 75 80
Met Phe Asp Ala Lys Arg Ala Val Val Ala Leu Lys Trp Lys Gly Ile
85 90 95
Glu Leu Arg Gly Val Ala Phe Asp Leu Leu Leu Ala Ala Tyr Leu Leu
100 105 110
Asn Pro Ala Gln Asp Ala Gly Asp Ile Ala Ala Val Ala Lys Met Lys
115 120 125
Gln Tyr Glu Ala Val Arg Ser Asp Glu Ala Val Tyr Gly Lys Gly Val
130 135 140
Lys Arg Ser Leu Pro Asp Glu Gln Thr Leu Ala Glu His Leu Val Arg
145 150 155 160
Lys Ala Ala Ala Ile Trp Ala Leu Glu Gln Pro Phe Met Asp Asp Leu
165 170 175
Arg Asn Asn Glu Gln Asp Gln Leu Leu Thr Lys Leu Glu His Ala Leu
180 185 190
Ala Ala Ile Leu Ala Glu Met Glu Phe Thr Gly Val Asn Val Asp Thr
195 200 205
Lys Arg Leu Glu Gln Met Gly Ser Glu Leu Ala Glu Gln Leu Arg Ala
210 215 220
Ile Glu Gln Arg Ile Tyr Glu Leu Ala Gly Gln Glu Phe Asn Ile Asn
225 230 235 240
Ser Pro Lys Gln Leu Gly Val Ile Leu Phe Glu Lys Leu Gln Leu Pro
245 250 255
Val Leu Lys Lys Thr Lys Thr Gly Tyr Ser Thr Ser Ala Asp Val Leu
260 265 270
Glu Lys Leu Ala Pro His His Glu Ile Val Glu Asn Ile Leu His Tyr
CA 02449560 2003-12-03
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275 280 285
Arg Gln Leu Gly Lys Leu Gln Ser Thr Tyr Ile Glu Gly Leu Leu Lys
290 295 300
Val Val Arg Pro Asp Thr Lys Val His Thr Met Phe Asn Gln Ala Leu
305 310 315 320
Thr Gln Thr Gly Arg Leu Ser Ser Ala Glu Pro Asn Leu Gln Asn Ile
325 330 335
Pro Ile Arg Leu Glu Glu Gly Arg Lys Ile Arg Gln Ala Phe Val Pro
340 345 350
Ser Glu Pro Asp Trp Leu Ile Phe Ala Ala Asp Tyr Ser Gln Ile Glu
355 360 365
Leu Arg Val Leu Ala His Ile Ala Asp Asp Asp Asn Leu Ile Glu Ala
370 375 380
Phe Gln Arg Asp Leu Asp Ile His Thr Lys Thr Ala Met Asp Ile Phe
385 390 395 400
Gln Leu Ser Glu Glu Glu Val Thr Ala Asn Met Arg Arg Gln Ala Lys
405 410 415
Ala Val Asn Phe Gly Ile Va1 Tyr Gly Ile Ser Asp Tyr Gly Leu Ala
420 425 430
Gln Asn Leu Asn Ile Thr Arg Lys Glu Ala Ala Glu Phe Ile Glu Arg
435 440 445
Tyr Phe Ala Ser Phe Pro Gly Val Lys Gln Tyr Met Glu Asn Ile Val
450 455 460
Gln Glu Ala Lys Gln Lys Gly Tyr Val Thr Thr Leu Leu His Arg Arg
465 470 475 480
Arg Tyr Leu Pro Asp Ile Thr Ser Arg Asn Phe Asn Va1 Arg Ser Phe
485 490 495
Ala Glu Arg Thr Ala Met Asn Thr Pro Ile Gln Gly Ser Ala Ala Asp
500 505 510
Ile Ile Lys Lys Ala Met Ile Asp Leu Ala Ala Arg Leu Lys Glu Glu
515 520 525
Gln Leu Gln Ala Arg Leu Leu Leu Gln Val His Asp Glu Leu Ile Leu
530 535 540
Glu Ala Pro Lys Glu Glu Ile Glu Arg Leu Cys Glu Leu Val Pro Glu
545 550 555 560
Val Met Glu Gln Ala Val Thr Leu Arg Val Pro Leu Lys Val Asp Tyr
565 570 575
His Tyr Gly Pro Thr Trp Tyr Asp Ala Lys
CA 02449560 2003-12-03
WO 02/101004 PCT/IB02/03341
4/10
580 585
<210> 3
<211> 1764
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Modified Bst
DNA sequence
<400> 3
atggccgaag gggagaaacc gcttgaggag atggagtttg ccatcgttga cgtcattacc 60
gaagagatgc ttgccgacaa ggcagcgctt gtcgttgagg tgatggaaga aaactaccac 120
gatgccccga ttgtcggaat cgcactagtg aacgagcatg ggcgattttt tatgcgcccg 180
gagaccgcgc tggctgattc gcaattttta gcatggcttg ccgatgaaac gaagaaaaaa 240
agcatgtttg acgccaagcg ggcagtcgtt gccttaaagt ggaaaggaat tgagcttcgc 300
ggcgtcgcct ttgatttatt gctcgctgcc tatttgctca atccggctca agatgccggc 360
gatatcgctg cggtggcgaa aatgaaacaa tatgaagcgg tgcggtcgga tgaagcggtc 420
tatggcaaag gcgtcaagcg gtcgctgccg gacgaacaga cgcttgctga gcatctcgtt 480
cgcaaagcgg cagccatttg ggcgcttgag cagccgttta tggacgattt gcggaacaac 540
gaacaagatc aattattaac gaagcttgag cacgcgctgg cggcgatttt ggctgaaatg 600
gaattcactg gggtgaacgt ggatacaaag cggcttgaac agatgggttc ggagctcgcc 660
gaacaactgc gtgccatcga gcagcgcatt tacgagctag ccggccaaga gttcaacatt 720
aactcaccaa aacagctcgg agtcatttta tttgaaaagc tgcagctacc ggtgctgaag 780
aagacgaaaa caggctattc gacttcggct gatgtgcttg agaagcttgc gccgcatcat 840
gaaatcgtcg aaaacatttt gcattaccgc cagcttggca aactgcaatc aacgtatatt 900
gaaggattgt tgaaagttgt gcgccctgat accggcaaag tgcatacgat gttcaaccaa 960
gcgctgacgc aaactgggcg gctcagctcg gccgagccga acttgcaaaa cattccgatt 1020
cggaccccac tggggcggaa aatccgccaa gcgttcgtcc cgtcagagcc ggactggctc 1080
attttcgccg ccgattactc acaaattgaa ttgcgcgtco tcgcccatat cgccgatgac 1140
gacaatctaa ttgaagcgtt ccaacgcgat ttggatattc acacaaaaac ggcgatggac 1200
attttccagt tgagcgaaga ggaagtcacg gccaacatgc gccgccaggc aaaggccgtt 1260
aactacggta tcgtttacgg aattagcgat tacggattgg cgcaaaactt gaacattacg 1320
cgcaaagaag ctgccgaatt tatcgaacgt tacttcgcca gctttccggg cgtaaagcag 1380
tatatggaaa acatagtgca agaagcgaaa cagaaaggat atgtgacaac gctgttgcat 1440
cggcgccgct atttgcctga tattacaagc cgcaatttca acgtccgcag ttttgcagag 1500
cggacggcca tgaacacgcc aattcaagga agcgccgctg acattattaa aaaagcgatg 1560
attgatttag cggcacggct gaaagaagag cagcttcagg ctcgtctttt gctgcaagtg 1620
catgacgagc tcattttgga agcgccaaaa gaggaaattg agcgattatg tgagcttgtt 1680
ccggaagtga tggagcaggc cgttacgctc cgcgtgccgc tgaaagtcga ctaccattac 1740
ggcccaacat ggtatgatgc caaa 1764
<210> 4
<211> 588
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Modified Bst
amino acid sequence
<400> 4
Met Ala Glu Gly Glu Lys Pro Zeu Glu Glu Met Glu Phe Ala Ile Val
1 5 10 15
CA 02449560 2003-12-03
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Asp Val Ile Thr Glu Glu Met Leu Ala Asp Lys Ala Ala Leu Val Val
20 25 30
Glu Val Met Glu Glu Asn Tyr His Asp Ala Pro Ile Val Gly Ile Ala
35 40 45
Leu Val Asn Glu His Gly Arg Phe Phe Met Arg Pro Glu Thr Ala Leu
50 55 60
Ala Asp Ser Gln Phe Leu Ala Trp Leu Ala Asp Glu Thr Lys Lys Lys
65 70 75 80
Ser Met Phe Asp Ala Lys Arg Ala Val Val Ala Leu Lys Trp Lys Gly
85 90 95
Ile Glu Leu Arg Gly Val Ala Phe Asp Leu Leu Leu Ala Ala Tyr Leu
100 105 110
Leu Asn Pro Ala Gln Asp Ala Gly Asp Ile Ala Ala Val Ala Lys Met
115 l20 125
Lys Gln Tyr Glu Ala Val Arg Ser Asp Glu Ala Val Tyr Gly Lys Gly
130 135 140
Val Lys Arg Ser Leu Pro Asp Glu Gln Thr Leu Ala Glu His Leu Val
145 150 155 160
Arg Lys Ala Ala Ala Ile Trp Ala Leu Glu Gln Pro Phe Met Asp Asp
165 170 175
Leu Arg Asn Asn Glu Gln Asp Gln Leu Leu Thr Lys Leu Glu His Ala
180 185 190
Leu Ala Ala Ile Leu Ala Glu Met Glu Phe Thr Gly Val Asn Val Asp
195 200 205
Thr Lys Arg Leu Glu Gln Met Gly Ser Glu Leu Ala Glu Gln Leu Arg
210 215 220
Ala Ile Glu Gln Arg Ile Tyr Glu Leu Ala Gly Gln Glu Phe Asn Ile
225 230 235 240
Asn Sex Pro Lys Gln Leu Gly Val Ile Leu Phe Glu Lys Leu Gln Leu
245 250 255
Pro Val Leu Lys Lys Thr Lys Thr Gly Tyr Ser Thr Ser Ala Asp Va1
260 265 270
Leu Glu Lys Leu Ala Pro His His Glu Ile Val Glu Asn Ile Leu His
275 280 285
Tyr Arg Gln Leu Gly Lys Leu Gln Ser Thr Tyr Ile Glu Gly Leu Leu
290 295 300
Lys Val Val Arg Pro Asp Thr Gly Lys Val His Thr Met Phe Asn Gln
305 310 3l5 320
CA 02449560 2003-12-03
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Ala Leu Thr Gln Thr Gly Arg Leu Ser Ser Ala Glu Pro Asn Leu Gln
325 330 335
Asn I1e Pro Ile Arg Thr Pro Leu Gly Arg Lys Ile Arg Gln Ala Phe
340 345 350
Val Pro Ser Glu Pro Asp Trp Leu Ile Phe Ala Ala Asp Tyr Ser Gln
355 360 365
Ile Glu Leu Arg Val Leu Ala His Ile A1a Asp Asp Asp Asn Leu Ile
370 375 380
Glu Ala Phe Gln Arg Asp Leu Asp Ile His Thr Lys Thr Ala Met Asp
385 390 395 400
Ile Phe Gln Leu Ser Glu Glu Glu Val Thr Ala Asn Met Arg Arg Gln
405 410 415
Ala Lys Ala Val Asn Tyr Gly Ile Val Tyr Gly Ile Ser Asp Tyr Gly
420 425 430
Leu Ala Gln Asn Leu Asn Ile Thr Arg Lys Glu Ala A1a Glu Phe Ile
435 440 445
Glu Arg Tyr Phe Ala Ser Phe Pro Gly Val Lys Gln Tyr Met Glu Asn
450 455 460
Ile Val Gln Glu Ala Lys Gln Lys Gly Tyr Val Thr Thr Leu Leu His
465 470 475 480
Arg Arg Arg Tyr Leu Pro Asp Ile Thr Ser Arg Asn Phe Asn Val Arg
485 490 495
Ser Phe Ala Glu Arg Thr Ala Met Asn Thr Pro Ile Gln Gly Ser Ala
500 505 510
Ala Asp Ile Ile Lys Lys Ala Met Ile Asp Leu Ala Ala Arg Leu Lys
515 520 525
G1u Glu Gln Leu Gln Ala Arg Leu Leu Leu Gln Val His Asp Glu Leu
530 535 540
Ile Leu Glu Ala Pro Lys Glu Glu Ile Glu Arg Leu Cys Glu Leu Val
545 550 555 560
Pro Glu Val Met Glu Gln Ala Val Thr Leu Arg Val Pro Leu Lys Val
565 570 575
Asp Tyr His Tyr Gly Pro Thr Trp Tyr Asp Ala Lys
580 585
<210> 5
<211> 17
<212> DNA
<213> Artificial Sequence
CA 02449560 2003-12-03
WO 02/101004 PCT/IB02/03341
7/10
<220>
<223> Description of Artificial Sequence: Primer
<400> 5
gtaaaacgac ggccagt 17
<210> 6
<211> 16
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 6
aacagctatg accatg 16
<210> 7
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 7
cttaatttaa ggttccgtg 19
<210> 8
<211> 17
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 8
gcattggtaa gcaatgg 17
<210> 9
<211> 17
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 9
acaaagcact gaacctg 17
<210> 10
CA 02449560 2003-12-03
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<211> 17
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 10
tgggacctat cgtgttg 17
<210> 11
<211> 15
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 11
cgaattcctg cagcc 15
<210> 12
<211> 17
<212> DNA
<2l3> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 12
gaactagtgg atccccc 17
<210> 13
<211> 17
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 13
tagctatcta acttaat 17
<210> 14
<211> 17
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 14
ttgtttetct gatgcat 17
CA 02449560 2003-12-03
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<210> 15
<2l1> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 15
tagctatcta acttaattta aggttccgtg 30
<210> 16
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> l6
ttgtttctct gatgcattgg taagcaatgg 30
<210> 17
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 17
gaattggagc tccaccgcgg 20
<210> 18
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 18
tttcagggtc ccttatatct c 21
<210> 19
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
CA 02449560 2003-12-03
WO 02/101004 PCT/IB02/03341
10/10
<400> 19
tcgcttctcc tcataatcga t 21
<210> 20
<211> 54
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Illustrative
oligonucleotide
<220>
<221> modified_base
<222> (37)
<223> a, t, c, g, other or unknown
<220>
<22l> modified_base
<222> (40)
<223> a, t, c, g, other or unknown
<220>
<221> modified_base
<222> (41)
<223> a, t, c, g, other or unknown
<220>
<221> modified_base
<222> (43)..(44)
<223> a, t, c, g, other or unknown
<220>
<221> modified_base
<222> (46..(47)
<223> a, t, c, g, other or unknown
<400> 20
ttgacacacg aacaaaacac agcccccccc cctcggnccn ntnntnnttt ctgt 54
<210> 21
<211> 53
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Illustrative
oligonucleotide
<400> 21
ttgacacacg aacaaaacac agcccccccc cctcgccccc cccccccccc tga 53
