U.S. patent application number 10/959015 was filed with the patent office on 2005-04-28 for dna polymerase having ability to reduce innate selective discrimination against fluorescent dye-labeled dideoxynucleotides.
Invention is credited to Hong, GuoFan, Huang, Wei-hua.
Application Number | 20050089910 10/959015 |
Document ID | / |
Family ID | 27388000 |
Filed Date | 2005-04-28 |
United States Patent
Application |
20050089910 |
Kind Code |
A1 |
Hong, GuoFan ; et
al. |
April 28, 2005 |
DNA polymerase having ability to reduce innate selective
discrimination against fluorescent dye-labeled
dideoxynucleotides
Abstract
The invention relates to genetical modification of DNA
polymerase to reduce its innate selective sequence-related
discrimination against incorporation of fluorescent dye-labeled
ddCTP and ddATP in the enzymatic reaction for preparation of
samples for automated florescent dye-labeled terminator DNA
sequencing. The modified DNA polymerases are more resistant to heat
inactivation and are more effective in dideoxynucleotide
incorporation than current DNA polymerases.
Inventors: |
Hong, GuoFan; (Shanghai,
CN) ; Huang, Wei-hua; (Zhejiang, CN) |
Correspondence
Address: |
Marlana Titus
Nash & Titus, LLC
6005 Riggs Road
Laytonsville
MD
20882
US
|
Family ID: |
27388000 |
Appl. No.: |
10/959015 |
Filed: |
October 5, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10959015 |
Oct 5, 2004 |
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09512019 |
Feb 24, 2000 |
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6818431 |
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09512019 |
Feb 24, 2000 |
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09157397 |
Sep 21, 1998 |
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6165765 |
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09157397 |
Sep 21, 1998 |
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08544643 |
Oct 18, 1995 |
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5747298 |
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09157397 |
Sep 21, 1998 |
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08642684 |
May 3, 1996 |
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5834253 |
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Current U.S.
Class: |
435/6.12 ;
435/199; 435/252.3; 435/252.31; 435/320.1; 435/69.1; 536/23.2 |
Current CPC
Class: |
C12Q 2563/107 20130101;
C12Q 2535/101 20130101; C12Q 1/6869 20130101; C12Q 2521/101
20130101; C12Q 2563/107 20130101; C12Q 2521/101 20130101; C12Q
2535/101 20130101; C12Q 1/6869 20130101; C12N 9/1252 20130101; C12Q
1/6869 20130101 |
Class at
Publication: |
435/006 ;
435/069.1; 435/199; 435/252.3; 435/320.1; 536/023.2;
435/252.31 |
International
Class: |
C12Q 001/68; C07H
021/04; C12N 009/22; C12N 001/21; C12N 015/74 |
Claims
1-23. (canceled)
24. A method for producing a modified form of a DNA polymerase
which during DNA sequencing selectively discriminates against
incorporation of fluorescent dye-labeled dideoxynucleotide
terminators ddCTP and ddATP but does not discriminate against
incorporation of fluorescent dye-labeled dideoxynucleotide
terminators ddTTP and ddGTP, comprising the step of modifying a DNA
polymerase which has an amino acid sequence that shares not less
than 95% homology of a DNA polymerase isolated from a strain of
Bacillus stearothermophilus, Bacillus caldotenax or Bacillus
caldolyticus, so that the modified DNA polymerase includes
threonine, praline and leucine at positions 342-344, respectively,
and tyrosine at position 422.
25. The method according to claim 24, wherein the DNA polymerase
has proofreading 3'-5' exonuclease activity during DNA sequencing
of a DNA strand from 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.
26. The method according to claim 24, wherein the DNA polymerase
has the amino acid sequence of SEQ ID NO:4.
27. The method according to claim 24, wherein the DNA polymerase is
encoded by a DNA segment having the nucleotide sequence of SEQ ID
NO:3.
28. A method for producing a modified form of a DNA polymerase
which during DNA sequencing selectively discriminates against
incorporation of fluorescent dye-labeled dideoxynucleotide
terminators ddCTP and ddATP but does not discriminate against
incorporation of fluorescent dye-labeled dideoxynucleotide
terminators ddTTP and ddGTP, comprising the step of modifying a
nucleotide sequence encoding a DNA polymerase which has an amino
acid sequence that shares not less than 95% homology of a DNA
polymerase isolated from a strain of Bacillus stearothermophilus,
Bacillus caldotenax or Bacillus caldolyticus, so that the modified
nucleotide sequence encodes threonine, praline and leucine at
positions 342-344, respectively, and tyrosine at position 422.
29-56. (canceled)
Description
[0001] This application is a continuation-in-part application of
Ser. No. 08/544,643 (now U.S. Pat. No. 5,747,298), filed Oct. 18,
1995, and Ser. No. 08/642,684, filed May 3, 1996, and the entire
contents of both applications are incorporated herein by
reference.
BACKGROUND OF THE INVENTION
[0002] 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.
[0003] 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.
[0004] 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 Sanger 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 dideoxynucleotide triphosphates (ddNTPs),
namely, ddATP, ddCTP, ddGTP and ddTTP, to terminate the extension
of the elongating deoxynucleotide polymers at various lengths.
[0005] In the classic one-step Sanger method, the sequence
determination is carried out in a set of four separate tubes, each
containing all four normal dNTPs, one of which is labeled with a
radioactive isotope, .sup.32P or .sup.35S, for autoradiographic
localization, a limiting amount of one of the four ddNTPs, a DNA
polymerase, a primer, and the DNA template to be sequenced. As a
result of the DNA polymerase activity, individual nucleotides or
nucleotide analogs are added to the new DNA chains, all starting
from the 3' end of the primer in a 5'-3' direction, and each linked
to adjacent ones with a phosphodiester bond in a base sequence
complementary to the DNA sequence of the template. Inasmuch as
there is a nucleotide analog in the reaction mixture, each tube
eventually contains numerous newly formed DNA strands of various
lengths, all ending in a particular ddNTP, referred to as A, C, G
or T terminator.
[0006] After resolving the four sets of reaction products by
high-resolution polyacrylamide/urea gel electrophoresis, the
populations of the newly formed DNA strands are separated and
grouped according to their molecular weight. An autoradiographic
image of the gel will show the relative positions of these DNA
strands as bands which differ from one another in distance measured
by one nucleotide in length, all sharing an identical primer and
terminating with a particular ddNTP (A, C, G or T). By reading the
relative positions of these bands in the "ladder" of the
autoradiograph, the DNA sequence of the template can be
deduced.
[0007] The DNA polymerase used in the reaction mixture plays a
pivotal role in DNA sequencing analysis. To be useful for DNA
sequencing, a DNA polymerase must possess certain essential
properties. For example, it must have its natural 5'-3' exonuclease
activity removed by mutagenesis or by posttranslational
modification, such as enzymatic digestion, and must be able to
incorporate dNTPs and ddNTPs, without undue discrimination against
ddNTP and with a sufficiently high processivity which refers to the
ability of the enzyme to polymerize nucleotides onto a DNA chain
continuously without being dislodged from the chain, and a
sufficiently high elongation rate. A 5'-3' exonuclease activity
associated with a DNA polymerase will remove nucleotides from the
primer, thus cause a heterogeneous 5' end for the newly formed DNA
strands, resulting in a false reading of the strand lengths on the
sequencing gel. A DNA polymerase with a low processivity and a low
elongation rate will cause many undesirable noise background bands
of radioactivity due to the presence of DNA strands which are
formed with improper lengths and improper terminations. Among the
more commonly used DNA polymerases, Sequenase.TM. has a higher
processivity and a higher elongation rate than others, such as the
Klenow fragment, Taq, and Vent polymerases (2), and is therefore
one of the most popular DNA polymerase selected for DNA sequencing
to-date.
[0008] However, even when a DNA polymerase has been endowed with
all the essential properties listed above, it may still generate
erroneous or misleading band patterns of radioactivity in the
sequencing gel. These artifactual patterns do not faithfully
reflect the true nucleotide sequence in the template being
sequenced. They may be caused by premature termination of the
elongating strands due to the presence of secondary structures
formed along the template, such as "hairpins" in the regions that
contain palindromic sequences or that are rich in G and C bases
(3); or, they may occur as a result of inadequate "proof-reading"
function of the DNA polymerase that will allow the removal of
misincorporated nucleotides at the 3' end of an elongating
strand.
[0009] Researchers in the field of DNA sequencing often have to use
several approaches to confirm their findings in order to avoid
being misled by these potentially erroneous sequence data. For
example, they sometimes rely on repeating the same sequencing
experiment with different DNA polymerases, or performing another
sequencing reaction with the template which is complementary to the
first single-stranded DNA template, and compare the results for
possible discrepancies.
[0010] Numerous investigators have tried to find an ideal DNA
polymerase for enzymatic sequencing, i.e. an enzyme that not only
has all the essential properties required for sequencing reaction,
but also is capable of resolving the secondary hairpin structures
and preventing the formation of strands containing nucleotides
noncomplementary to those of the template being sequenced.
[0011] The discovery by Ye and Hong (4) of the thermostable large
fragment of DNA polymerase isolated from Bacillus
stearothermophilus (Bst), an enzyme that is functional over the
temperature range between 25.degree. C. and 75.degree. C., but is
most active at 65.degree. C., and possesses all the essential
properties for DNA sequencing, has largely solved the problem
caused by secondary structures in the template since these
secondary structures are destabilized when the sequencing reaction
is carried out at 65.degree. C. In the past few years since this
enzyme was made commercially available under the name of Bst DNA
Polymerase (Bio-Rad Laboratories), independent reports have
confirmed that during sequencing reaction catalyzed by this enzyme
all four dNTPs, including dCTP) and other nucleotide analogs, such
as dITP and 7-deaza-dGTP, are incorporated equally effectively in
the chain elongation, thus eliminating the weak "C" band phenomena
often observed when other DNA polymerases are used, and producing a
very good band uniformity on the sequencing gel. It has been
further established that at this elevated temperature Bst DNA
polymerase system can be used both for the classic Sanger one-step
reaction as well as for the "labeling/termination" sequencing
reaction, double-stranded DNA sequencing, and the incorporation of
.sup.35S-labeled nucleotides, and .sup.32P-labeled nucleotides.
Since this system can be placed at room temperature for at least
two weeks without significant loss of its enzymatic activity, it
has been adapted for automation of DNA sequencing which requires a
stable DNA polymerase, using either fluorescent dye or radioactive
isotope labeling. (See also 9, 12, and 13.)
[0012] However, when this Bst enzyme is used for automated
fluorescent DNA sequencing, only partially satisfactory results
have been obtained with fluorescent dye-labeled primers (see 12 and
EG Bulletin 1771 of Bio-Rad Laboratories), and even less
satisfactory results are obtained with fluorescent dye-labeled
ddNTP terminators. Even when fluorescent dye-labeled primers are
used, a significant number of mismatched ddNTPs are incorporated
onto the 3' end of the extending nucleotides in the enzymatic
reaction, thus generating erroneous sequencing data (see Bio-Rad EG
Bulletin 1771). With this in mind, the inventors sought, and found,
a better DNA polymerase for DNA sequencing, especially for
automated fluorescent dye-labeled primer and fluorescent
dye-labeled terminator sequencing.
[0013] Another disadvantage of the Bst DNA polymerase currently
known in the art is its lack of 3'-5' exonuclease activity (5), and
specifically, proof-reading 3'-5' exonuclease activity. A survey of
the sequencing data collected from fourteen research centers which
have used this Bst DNA polymerase for their DNA sequencing work on
over 120 DNA clones showed that, statistically, base pair
mismatching occurs at a rate of about 1.5.times.10.sup.-5. That is,
approximately 1.5 errors can be expected in one hundred thousand
nucleotide incorporations during nucleotide polymerization
catalyzed by the enzyme.
[0014] It is generally known that the formation of incorrect DNA
sequences due to mismatching of base pairs between the template and
the growing nucleotide chain in DNA sequencing may be prevented by
a 3'-5' exonuclease activity which "proof-reads" the nucleotide
chain. However, even if a DNA polymerase exhibits 3'-5' exonuclease
activity in vitro, it is often the case that the polymerase will
not adequately "proof-read". Thus, the polymerase will not be
capable of removing mismatched nucleotides from a newly formed DNA
strand as efficiently as those nucleotides correctly matched with
the nucleotides of the template. In other words, a 3'-5'
exonuclease may excise the correctly matched nucleotides at a
faster rate than the mismatched ones from the 3' terminus, or
excise both the correctly matched and the mismatched nucleotides at
the same rate. Consequently, even where the DNA polymerase has
3'-5' exonuclease activity, it does not perform any useful
proof-reading function during DNA polymerization.
[0015] It is also known that a 3'-5' exonuclease activity
associated with a DNA polymerase, in the presence of low
concentrations of dNTPs, often counteracts the normal chain
elongation process catalyzed by the polymerase, induces cyclic
incorporation and degradation of nucleotides over the same segment
of template, or even operates more efficiently than the polymerase
activity per se, to the extent of causing degradation of the
primer. Consequently, removal of the 3'-5' exonuclease activity
along with the 5'-31 exonuclease activity from the native DNA
polymerases by chemical means or by genetic engineering techniques
has become a standard procedure in producing DNA polymerases for
sequencing. This is a common strategy to preserve the essential
properties of a DNA polymerase.
[0016] For example, among the major commercially available
sequencing enzymes (other than the native Taq (Thermus aquaticus)
DNA polymerase which lacks a 3'-5' exonuclease activity de novo)
the 3'-5' exonuclease activity has been removed from the native T7
DNA polymerase, which lacks a 5'-3' exonuclease, either by a
chemical reaction that oxidizes the amino acid residues essential
for the exonuclease activity (Sequenase.TM. Version 1) or
genetically by deleting 28 amino acids essential for the 3'-5'
exonuclease activity (Sequenase.TM. 2).
[0017] Vent.sub.R (exo.sup.-) DNA polymerase, which is recommended
as the preferred form of the Vent DNA polymerase for sequencing,
also has its 3'-5' exonuclease activity removed by genetic
modification. The native Vent DNA polymerase and the Klenow
fragment isolated from the native E. coli DNA polymerase I possess
a 3'-5' exonuclease; but these enzymes are no longer considered the
enzymes of choice for DNA sequencing.
[0018] The currently known Bst DNA polymerase (e.g., produced by
Bio-Rad Laboratories) isolated and purified from the cells of
Bacillus stearothermophilus for DNA sequencing is free of 3'-5'
exonuclease activity (5).
[0019] IsoTherm.TM. DNA Polymerase, a commercially available Bst
DNA polymerase for DNA sequencing, marketed by Epicentre
Technologies (1402 Emil Street, Madison, Wis. 53713), is also based
on a Bst DNA polymerase whose 3'-5' exonuclease activity has been
enzymatically removed (6).
[0020] Only the rBst DNA Polymerase produced from an
over-expressing recombinant clone in E. coli, which is the product
of the DNA pol I gene of Bacillus stearothermophilus, possesses a
3'-5' exonuclease activity in addition to a 5'-3' exonuclease
activity. However, due to the existence of an undesirable 5'-3'
exonuclease activity and a 3'-5' exonuclease activity of unknown
characteristics, the latter product is not recommended by the
company for DNA sequencing (6).
[0021] Over the past 10 years there has been a trend to develop and
improve the automated fluorescent DNA sequencing technology to
replace the classic radioactive isotope labeling manual method for
DNA sequencing because of the potential harmful effects of the
radioactive materials to humans and because of the need for
automated high throughput DNA sequencing systems. In using
fluorescent dyes as markers for labeling the DNA strands generated
in enzymatic reactions for sequencing, the dyes can be either
coupled with the primer, or coupled with the ddNTP terminators,
namely the dye-labeled ddATP, dye-labeled ddCTP, dye-labeled ddGTP
and dye-labeled ddTTP. Sequencing techniques based on these two
forms of labeling of the final enzymatic reaction products are
commonly referred to as "dye primer sequencing" and "dye terminator
sequencing", respectively.
[0022] In the dye primer sequencing, ddNTPs are employed as the
chain terminators, as in the original classic Sanger method which
uses radioactive isotope as the marker. The molecular structure of
ddNTPs are almost identical to that of dNTPs, the natural building
blocks of all DNA molecules. Therefore, any DNA polymerase which
has been used for radioactive isotope manual DNA sequencing can be
easily adapted for fluorescent dye primer DNA sequencing with
equally satisfactory results. The disadvantage in the dye primer
technology is that the primer for each template to be sequenced
must be labeled with four different fluorescent dyes and that the
enzymatic reaction must be performed in four separate test tubes
each containing only one of the ddNTPs, namely ddATP, ddCTP, ddGTP
or ddTTP, as in the classic Sanger radioisotope method.
[0023] In the dye terminator technology for DNA sequencing, the
fluorescent dye-labeled ddATP, dye-labeled ddCTP, dye-labeled ddGTP
and dye-labeled ddTTP are coupled with different fluorescent dyes,
each emitting a specific light spectrum, thus directly reporting
the type of ddNTP at the 3' terminus of the DNA fragment. Unlike
the situations in the dye primer technology in which four different
fluorescent dyes are coupled to a primer incorporated into all
newly formed DNA strands, these dye-labeled ddNTPs serve the dual
function of a specific base terminator and a "color marker". There
is no need to label the primer for each new template, and the
polymerase DNA extension reaction can be performed in a single test
tube to generate the required specifically terminated and
specifically dye-labeled DNA fragments of various sizes for DNA
sequencing.
[0024] The advantage of using fluorescent dye-labeled terminators
for DNA sequencing is obvious. However, there are certain
difficulties to overcome before an enzymatic reaction system
suitable for a radioisotope technique or suitable for a dye primer
technique can be adapted for a dye terminator technology. An
increase of the molecular weight from less than 500 for a ddNTP
terminator to about 800 or more for a fluorescent dye-labeled ddNTP
terminator may be associated with potential three-dimensional
structural changes. These molecular alterations may interfere with
the process of incorporation of the dye-labeled ddNTPs as chain
terminators by the DNA polymerase to the 3' end of an extending DNA
strand in terms of lowering the rate of incorporation, lowering the
processivity of the enzyme for this new substrate, reducing the
enzyme-terminator binding specificity and changing the
enzyme-terminator binding kinetics.
[0025] For example, both Taq DNA polymerase and Sequenase II.TM. (a
T7 DNA polymerase) have been used for radioisotope labeling DNA
sequencing with excellent results, and have been adapted for
fluorescent dye-labeled primer DNA sequencing. But neither can be
used for fluorescent dye-labeled terminator DNA sequencing
technologies. As reported in U.S. Pat. No. 5,614,365, when the Taq
DNA polymerase was used for fluorescent dye-labeled terminator
chemical reactions, the reaction products generated no readable
data on the DNA sequencer. Most of the fluorescence was either in
unincorporated dye-ddNTPs at the leading front of the test gel, or
in fragments greater than several hundred bases in length. Using a
Taq DNA polymerase mutant in which the amino acid, phenylalanine,
at position 667 of its amino acid sequence has been replaced by a
tyrosine and which has an increased ability to incorporate
dideoxynucleotides (6,000 times more efficient), to replace the
unmodified Taq DNA polymerase for the experiment, the results are
significantly improved. This F667Y mutant of Taq DNA polymerase is
now marketed by Amersham Life Science, Inc. under the trademark
ThermoSequenase.TM.. It is used for cycle-sequencing in which the
enzymatic reaction mixture is subjected to numerous cycles of
extension-termination, denaturing and annealing to ensure that
sufficient dye-terminator-labeled enzymatic reaction products are
generated for the DNA sequencing procedure. Because of the low
processivity of the parent Taq DNA polymerase, ThermoSequenase.TM.
is not recommended for direct DNA sequencing without precyclings.
Like Taq DNA polymerase, ThermoSequenase.TM. lacks a proof-reading
exonuclease activity.
[0026] Bacillus stearothermophilus, Bacillus caldotenax and
Bacillus caldolyticus are classified as mesophilic microbes;
although their DNA polymerases are referred to as thermostable
(most active at 65.degree. C.) they are inactivated at 70.degree.
C. or above. This is contrasted with other enzymes, such as Taq,
which are truly thermophilic--that is, their DNA polymerases
tolerate and remain active at temperatures higher that 95.degree.
C. These mesophilic bacillus strains, especially Bacillus
stearothermophilus, produce DNA polymerases that are useful in DNA
sequencing applications. However, a disadvantage of the DNA
polymerases of these strains 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 stearothermophilus
and Bacillus caldotenax was not previously recognized. Such
selective discrimination is apparently sequence-related, and cannot
be corrected or compensated by mere adjustment of the
concentrations of the dNTPs.
[0027] Thus, there is a need for a mesophilic bacillus DNA
polymerase that does not selectively discriminate against
incorporation of fluorescent dye-labeled ddCTP and ddATP, during
dye primer or dye terminator DNA sequencing.
SUMMARY OF THE INVENTION
[0028] This invention addresses the above-described problems
associated with mesophilic bacillus DNA polymerases by providing
novel DNA polymerases which, during direct DNA sequencing, reduce
the innate selective discrimination against the incorporation of
fluorescent dye-labeled ddCTP and fluorescent dye-labeled ddATP,
without increasing the rate of incorporation of the other two
dye-labeled ddNTP terminators (ddTTP and ddGTP) excessively. In
particular, this invention provides a novel genetic modification of
the amino acid sequence of a highly processive DNA polymerase (such
as isolated from Bacillus stearothermophilus, Bacillus caldotenax
or Bacillus caldolyticus) that, unmodified, selectively
discriminates against incorporation of fluorescent dye-labeled
dideoxynucleotide terminators ddATP and ddCTP (but does not
discriminate against incorporation of fluorescent dye-labeled
dideoxynucleotide terminators ddTTP and ddGTP). The modification
results in a reduction of the innate selective discrimination
against incorporation of fluorescent dye-labeled dideoxynucleotide
terminators ddATP and ddCTP, such that all four of the ddNTP
terminators are effectively incorporated into the DNA primer
elongated by the DNA polymerase. Thus, the modified DNA polymerase
of this invention is effective in reducing the innate selective
discrimination against incorporation of fluorescent dye-labeled
dideoxynucleotide terminators ddATP and ddCTP characteristic of the
DNA polymerase in its unmodified state.
[0029] In particular, the preferred DNA polymerase is a
modification of a DNA polymerase isolated from a strain of a
mesophilic bacterium, such as Bacillus stearothermophilus, Bacillus
caldotenax or Bacillus caldolyticus. The approach of modifying the
DNA polymerase described herein may be used to modify other DNA
polymerases which share a close amino acid homology of a DNA
polymerase isolated from a strain Bacillus stearothermophilus,
Bacillus caldotenax or Bacillus caldolyticus, as long as the
unmodified DNA polymerases have a selective discrimination against
incorporation of fluorescent dye-labeled dideoxynucleotide ddCTP
and/or ddATP as terminators in the enzymatic reaction for preparing
materials for automated fluroescent DNA sequencing. Consequently,
it is preferred that the modified DNA polymerase has an amino acid
sequence that shares not less than 95% homology of a DNA polymerase
isolated from a strain of Bacillus stearothermophilus, Bacillus
caldotenax or Bacillus caldolyticus.
[0030] The particularly preferred mesophilic species is Bacillus
stearothermophilus, which is highly heterogeneous. This is
indicated by the wide range of DNA base compositions as well as the
range of the phenotypic properties of strains assigned to this
species (see Bergey's Manual of Systemic Bacteriology, Eds. P. H.
A. Sneath, N. S. Mair, M. E. Sharpe and J. G. Holt, Williams &
Wilkins, 1986, Vol. 2, page 1135). Therefore, it is reasonable to
assume that the amino acid sequences of DNA polymerases isolated
from various strains would be heterogeneous with potential
functional differences. Although DNA polymerases isolated from the
known standard strains of Bacillus stearothermophilus have been
shown to lack a 3'-5' exonuclease activity, a questionable trace of
"contaminating" 3151 exonuclease has been observed in a purified
DNA polymerase preparations (see Kaboev et al., J. Bacteriology,
Vol. 145, page 21-26, 1981).
[0031] Consequently, the inventors began to address the
above-identified problems in the art by discovering a strain of
Bacillus stearothermophilus (designated strain No. 320 for
identification purposes; described in U.S. Pat. No. 5,747,298) that
produces a DNA polymerase (designated Bst 320) with a proof-reading
3'-51 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 was deposited on Oct. 30, 1995 in the American Type
Culture Collection, located at 12301 Parklawn Drive, Rockville, Md.
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
stearothermophilus, 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 stearothermophilus 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.
[0032] The inventors studied a thermostable DNA polymerase isolated
from a different species, Bacillus caldotenax (Bca), which also has
an optimum active temperature at 65.degree. C. The inventors
discovered that the Bst 320 DNA polymerase shares 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 stearothermophilus as to the DNA
polymerase isolated from Bacillus caldotenax, 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 stearothermophilus
polymerases.
[0033] The inventors has studied the DNA polymerases of three
different strains of Bacillus stearothermophilus (including DNA
polymerase obtained from Bst 320) and the DNA polymerase of
Bacillus caldotenax and found that 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 is especially the case when the preceding
3' end base of the extending DNA fragment is a dGMP (G) or a dAMP
(A). (By "dNTP" it is intended to denote the four commonly known
deoxynucleotide triphosphates, dATP, dTTP, dCTP, and dGTP.)
[0034] This selective discrimination causes missing peaks and
ambiguous peaks on a color plot generated by the automated
fluorescent DNA sequencer, and causes loss of sequencing data and
erroneous base callings. This is shown in FIGS. 6 and 8.
[0035] This disadvantage of the natural bacillus DNA polymerases in
fluorescent dye-labeled terminator DNA sequencing cannot be
corrected or compensated by mere adjustment of the concentrations
of the dNTPs and the fluorescent dye-labeled ddNTPs in the reaction
mixture. This selective discrimination against the specific
dye-labeled ddNTPs is also sequence-related as demonstrated with
respect to Bst in FIGS. 6 and 8, in which the missing or ambiguous
"C" peaks and "A" peaks tend to occur immediately following a
preceding "G", peak or a preceding "A" peak. Of particular interest
is the fact that the "C" and "A" peaks immediately following a
preceding "C" or a preceding "T" peak are quite strong and
resolvable in the same color plot analysis, indicating that the
concentrations of dNTPs and the fluorescent dye-labeled ddCTP and
the fluorescent dye-labeled ddATP were adequate for the termination
reaction.
[0036] According to the structural model studies carried out on E.
coli DNA polymerase I (Klenow fragment), certain amino acids in a
particular region or regions of a DNA polymerase appear to play
important roles in dNTP and ddNTP bindings and their final
incorporation, and affect discrimination between deoxy and
dideoxynucleotide substrates. For example, mutation of the amino
acids arginine, asparagine, lysine, tyrosine, phenylalanine,
aspartate, and glutamate in certain locations of amino acid
sequences of Klenow fragment may affect the binding of dNTP and
discrimination between deoxy and dideoxynucleotides. (See: Joyce,
C. M., Current Opinion in Structural Biology, 1:123-129, 1991.
Joyce and Steitz, Annu. Rev. Biochem., 63:777-822, 1993, page 800.
Carrol et al., Biochemistry 30:804-813, 1991).
[0037] The problem which faced the inventors was how to reduce the
selective discrimination against the incorporation of fluorescent
dye-labeled ddCTP and fluorescent dye-labeled ddATP by
site-directed mutagenesis of a DNA polymerase, without increasing
the rate of incorporation of the other two dye-labeled ddNTP
terminators excessively. In particular, the new mutant must be able
to incorporate more correctly base-matched dye-labeled ddCTP and/or
dye-labeled ddATP terminators to the dGMP (G) and dAMP (A) bases,
than to the dCMP (C) and dTMP (T) bases of the extending DNA
fragments during enzymatic reaction. A blanket increase in the
ability of an enzyme to incorporate all four dye-labeled ddNTPs to
the same proportion would serve no useful purpose for the group of
DNA polymerases isolated from mesophilic bacilli since, unlike the
Taq DNA polymerase, the unmodified natural enzymes of Bacillus
stearothermophilus and Bacillus caldotenax already possess a high
ability to incorporate fluorescent dye-labeled ddGTP and
fluorescent dye-labeled ddTTP, and even the fluorescent dye-labeled
ddCTP and dye-labeled ddATP provided at the immediately preceding
base at the 3' end of the extending DNA fragment is not a "G" or an
"A".
[0038] The inventors found that DNA polymerases isolated from
strains of Bacillus stearothermophilus and Bacillus caldotenax
possess the same amino acids at certain specific positions in their
amino acid sequence. For example, they all have
leucine-glutamate-glutamate at positions corresponding to positions
342-344 and phenylalanine at a position corresponding to position
422 of the amino acid sequence of the DNA polymerase isolated from
No 320 strain of Bacillus stearothermophilus. The inventors further
discovered that the most optimal modification to solve the problem
of selective discrimination in direct fluorescent DNA sequencing
for these DNA polymerases is to modify the four amino acids of the
natural DNA polymerases referenced above in such a form that
threonine-proline-leucine substitute respectively for
leucine-glutamate-glutamate at positions 342-344 and tyrosine
substitutes for phenylalanine at position 422 in their amino acid
sequences. Accordingly, the nucleotide sequence encoding the
natural forms of the DNA polymerases are modified at positions
1024-1032 from CTCGAAGAG to ACCCCACTG and at position 1265 from T
to A to encode for the DNA polymerases having the desired
properties. The combined effects of these amino acid modifications
reduce the selective discrimination against incorporation of
fluorescent dye-labeled ddCTP and dye-labeled ddATP of the
naturally-occurring mesophilic bacillus DNA polymerases during
enzymatic reaction for direct automated fluorescent DNA
sequencing.
[0039] Initially, the DNA polymerases used in the inventors'
research were obtained by overexpression of the genes encoding the
naturally-occurring enzymes of Bacillus stearothermophilus and
Bacillus caldotenax. Subsequently, modified DNA polymerases
obtained by overexpression of the site-directed mutated genes were
used. This invention provides both the nucleotide and amino acid
sequence for a modified DNA polymerase to illustrate the practice
of this new approach of modifying a special group of DNA
polymerases, as described below.
[0040] In one preferred embodiment, the Bst 320 DNA polymerase is
used for the unmodified, naturally-occurring DNA polymerase,
although DNA polymerases isolated from other strains of mesophilic
bacilli (for instance, Bacillus stearothermophilus and Bacillus
caldotenax) can be used as the starting enzymes for the genetic
modification. As noted above, the Bst 320 DNA polymerase is also
capable of proofreading 3'-5' exonuclease activity. In particular,
the invention provides the DNA and amino acid sequences for the
isolated and purified DNA polymerase having this function. These
sequences are also described below.
[0041] The invention also contemplates an isolated strain of
Bacillus stearothermophilus which produces a DNA polymerase having
an ability to reduce selective discrimination against incorporation
of fluorescent dye-labeled dideoxynucleotide terminators ddCTP and
ddATP, but not fluorescent dye-labeled dideoxynucleotide
terminators ddGTP and ddTTP, in the presence of dNTPs and the four
fluorescent dye-labeled dideoxynucleotide terminators. Preferably,
the Bst strain produces a DNA polymerase which also has
proofreading 3'-5' exonuclease activity during DNA sequencing of a
DNA strand from a template.
[0042] As mentioned above, the invention also contemplates DNA
polymerases obtained or otherwise derived from any bacillus strain,
or made synthetically, as long as the amino acid sequences of the
naturally-occurring DNA polymerases 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 example, DNA polymerases derived from other strains of Bacillus
stearothermophilus or Bacillus caldotenax or other mesophilic
bacilli may be easily modified using conventional DNA modification
techniques to include the amino acid or nucleotide substitutions
identified above.
[0043] The invention also provides a DNA construct comprising at
least one of the above-described DNA polymerase sequences and a
vector (such as a cloning vector or an expression vector), for
introducing the DNA construct into eucaryotic or procaryotic host
cells (such as an E. coli host cell). In addition, the invention
further provides a host cell stably transformed with the DNA
construct in a manner allowing production of the peptide encoded by
the DNA segment in the construct.
[0044] The invention also provides improved methods for replicating
DNA and sequencing DNA using the above-described DNA polymerases of
the invention. The DNA polymerases are useful in both direct dye
terminator DNA sequencing and dye-primer DNA sequencing.
[0045] Preferably, the method of sequencing a DNA strand may
comprise the steps of:
[0046] i) hybridizing a primer to a DNA template to be
sequenced;
[0047] ii) extending the primer using a DNA polymerase which has an
ability to reduce selective discrimination against incorporation of
fluorescent dye-labeled dideoxynucleotide terminators ddCTP and
ddATP, in the presence of adequate amounts of nucleotide bases
dATP, dGTP, dCTP and dTTP, or their analogs, and the four
fluorescent dye-labeled dideoxynucleotide terminators,
[0048] under such conditions that the DNA strand is sequenced.
[0049] Further objects and advantages of the invention will become
apparent from the description and examples below.
BRIEF DESCRIPTION OF THE DRAWINGS
[0050] In the Figures and 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 No. 320 strain of
Bacillus stearothermophilus or produced by overexpression of the
gene encoding this naturally occurring DNA polymerase. (This Bst
strain and DNA polymerase are described in U.S. Pat. No.
5,747,298.) "HiFi Bst-II" refers to the modified form of "HiFi Bst"
DNA polymerase which has an 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.
[0051] FIG. 1. This graph shows the thermostability at 65.degree.
C. of HiFI Bst-II and HiFI Bst.
[0052] Y: relative polymerase activity (%)
[0053] X: incubation time (minutes).
[0054] FIG. 2. This shows a autoradiograph of a DNA sequencing gel
obtained by using radiolabeled primer with HiFi Bst-II and HiFi
Bst, and shows the dideoxy-nucleotide incorporation of HiFi Bst-II
and HiFi Bst in a reaction mixture with a suboptimally low
ddNTP/dNTP ratios.
[0055] Template: single-stranded M13 mp18;
[0056] Primer: -20M13 forward primer.
[0057] FIG. 3. This shows a autoradiograph of a DNA sequencing gel
obtained by using radiolabeled dATP with HiFi Bst and HiFi Bst-II
in reaction mixtures with optimized ddNTP/dNTP ratios. The sequence
pattern with HiFi Bst-II is better than that with HiFi Bst.
[0058] Template: single-stranded M13 mp18;
[0059] Primer: -20M13 forward primer.
[0060] FIG. 4. This shows the results of dye-primer DNA sequencing
with HiFi Bst
[0061] Template: single-stranded pGEM-3Zf(+);
[0062] Primer: -21M13 forward DYEnamic Energy Transfer Dye
Primers.
[0063] FIG. 5. This shows the results of dye-primer DNA sequencing
with HiFi Bst-II.
[0064] Template: single-stranded M13mp18;
[0065] Primer: -21M13 forward DYEnamic Energy Transfer Dye
Primers.
[0066] FIG. 6. This shows the results of dye-terminator DNA
sequencing with HiFi Bst
[0067] Template: single-stranded pGEM-3Zf(+)
[0068] Primer: -20M13 forward primer.
[0069] FIG. 7. This shows the results of dye-terminator DNA
sequencing with HiFi Bst-II.
[0070] Template: single-stranded M13 mp18;
[0071] Primer: -20M13 forward primer.
[0072] FIG. 8. Like FIG. 6, this shows the results of four
fluorescent dye-labeled terminators DNA sequencing with HiFi Bst.
In FIG. 8 corrections of the missing or ambiguous bases, according
to the known pGEM sequence, are indicated below the letters "N" or
below the incorrect base letters.
[0073] Template: single-stranded pGEM-3Zf(+);
[0074] Primer: -20M13 forward primer.
DETAILED DESCRIPTION OF THE INVENTION
[0075] The DNA polymerases of the invention are capable of reducing
selective discrimination against incorporation of fluorescent
dye-labeled dideoxynucleotide terminators ddCTP and ddATP, (but not
ddGTP and ddTTP), in the presence of adequate amounts of dNTPs and
the four terminators.
[0076] The inventors discovered that certain modifications of the
amino acid sequence of DNA polymerases (i.e., modifying the amino
acids at positions 342-344 to substitute threonine, proline and
leucine, respectively, for leucine, glutamate and glutamate, and
modifying the amino acid at position 422 to substitute tyrosine for
phenylalanine, as corresponding to the amino acid sequence of Bst
320 DNA polymerase) result in a marked reduction of the innate
selective discrimination against incorporation of fluorescent
dye-labeled dideoxynucleotide ddCTP and ddATP, that is
characteristic of the unmodified DNA polymerase. This reduction of
selective discrimination can be demonstrated by direct automated
fluorescent terminator DNA sequencing as recovered missing or
ambiguous "C" peaks and/or "A" peaks in the automated sequencing
results, using a known template as standard.
[0077] The preferred source for the DNA polymerase is Bacillus
stearothermophilus because DNA polymerase isolated from this
mesophilic bacillus is highly processive, can be used and stored in
dried down form, has an optimum temperature at 65.degree. C., and
can be used for direct DNA sequencing without precycling. The
preferred Bst DNA polymerase is that isolated from strain 320 with
an amino acid sequence as follows:
[0078] Amino Acid Sequence (SEQ ID NO:2):
1 AEGEKPLEEM EFAIVDVITE EMLADKAALV VEVMEENYHD APIVGIALVN EHGRFFMRPE
TALADSQFLA WLADETKKKS MFDAKRAVVA LKWKGIELRG VAFDLLLAAY LLNPAQDAGD
IAAVAKMKQY EAVRSDEAVY GKGVKRSLPD EQTLAEHLVR KAAAIWALEQ PFMDDLRNNE
QDQLLTKLEH ALAAILAEME FTGVNVDTKR LEQMGSELAE QLRAIEQRIY ELAGQEFNIN
SPKQLGVILF EKLQLPVLKK TKTGYSTSAD VLEKIAPHHE IVENILHYRQ LGKLQSTYIE
GLLKVVRPDT GKVHTMFNQA LTQTGRLSSA EPNLQNIPIR LEEGRKIRQA FVPSEPDWLI
FAADYSQIEL RVLAHIADDD NLIEAFQRDL DIHTKTAMDI FQLSEEEVTA NMRRQAKAVN
FGIVYGISDY GLAQNLNITR KEAAEFIERY FASFPGVKQY MENIVQEAKQ KGYVTTLLHR
RRYLPDITSR NFNVRSFAER TAMNTPIQGS AADIIKKAMI DLAARLKEEQ LQARLLLQVH
DELILEAPKE EIERLCELVP EVMEQAVTLR VPLKVDYHYG PTWYDAK
[0079] The characters represent the following amino acids:
[0080] where,
2 A: alanine (Ala) C: cysteine (Cys) D: aspartic acid (Asp) E:
glutamic acid (Glu) F: phenylanaline (Phe) G: glycine (Gly) H:
histidine (His) I: isoleucine (Ile) K: lysine (Lys) L: leucine
(Leu) M: methionine (Met) N: asparagine (Asn) P: proline (Pro) Q:
glutamine (Gln) R: arginine (Arg) S: serine (Ser) T: threonine
(Thr) V: valine (Val) W: tryptophan (Trp) Y: tyrosine (Tyr)
[0081] The Bst 320 DNA polymerase is characterized by possessing a
proofreading 3'-51 exonuclease activity.
[0082] The nucleotide sequence encoding the unmodified Bst 320 DNA
polymerase is indicated in SEQ ID NO:1, in Example 2 below.
[0083] The following amino acid sequence represents the modified
Bst 320 DNA polymerase as the 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.
[0084] Amino Acid Sequence (SEQ ID:No 4):
3 MAEGEKPLEEMEFAIVDVITEEMLADKAALVVEVMEENYHDAPIV
GIALVNEHGRFFMRPETALADSQFLAWLADETKKKSMFDAKRAVV
ALKWKGIELRGVAFDLLLAAYLLNPAQDAGDIAAVAKMKQYEAVR
SDEAVYGKGVKRSLPDEQTLAEHLVRKAAAIWALEQPFMDDLRNN
EQDQLLTKLEHALAAILAEMEFTGVNVDTKRLEQMGSELAEQLRA
IEQRIYELAGQEFNINSPKQLGVILFEKLQLPVLKKTKTGYSTSA
DVLEKLAPHHEIVENILHYRQLGKLQSTYIEGLLKVVRPDTGKVH
TMFNQALTQTGRLSSAEPNLQNIPIRTPLGRKIRQAFVPSEPDWL
IFAADYSQIELRVLAHIADDDNLIEAFQRDLDIHTKTAMDIFQLS
EEEVTANMRRQAKAVNYGIVYGISDYGLAQNLNITRKEAAEFIER
YFASFPGVKQYMENIVQEAKQKGYVTTLLHRRRYLPDITSRNFNV
RSFAERTAMNTPIQGSAADIIKKAMIDLAARLKEEQLQARLLLQV
HDELILEAPKEEIERLCELVPEVMEQAVTLRVPLKVDYHYGPTWY DAK
[0085] The underlined amino acids are substituted amino acids
produced by site-directed mutation of the naturally-occurring Bst
320 DNA polymerase.
[0086] The modified Bst 320 DNA polymerase is encoded by a DNA
sequence such as the following (SEQ ID NO:3):
4 ATG GCCGAAGGGG AGAAACCGCT TGAGGAGATG GAGTTTGCCA TCGTTGACGT
CATTACCGAA GAGATGCTTG CCGACAAGGC AGCGCTTGTC GTTGAGGTGA TGGAAGAAAA
CTACCACGAT GCCCCGATTG TCGGAATCGC ACTAGTGAAC GAGCATGGGC GATTTTTTAT
GCGCCCGGAG ACCGCGCTGG CTGATTCGCA ATTTTTAGCA TGGCTTGCCG ATGAAACGAA
GAAAAAAAGC 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 ACCCCACTGG GGCGGAAAAT CCGCCAAGCG TTCGTCCCGT CAGAGCCGGA
CTGGCTCATT TTCGCCGCCG ATTACTCACA AATTGAATTG CGCGTCCTCG CCCATATCGC
CGATGACGAC AATCTAATTG AAGCGTTCCA ACGCGATTTG GATATTCACA CAAAAACGGC
GATGGACATT TTCCAGTTGA GCGAAGAGGA AGTCACGGCC AACATGCGCC GCCAGGCAAA
GGCCGTTAAC TACGGTATCG 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 TTATTAAAAA
AGCGATGATT GATTTAGCGG CACGGCTGAA AGAAGAGCAG CTTCAGGCTC GTCTTTTGCT
GCAAGTGCAT GACGAGCTCA TTTTGGAAGC GCCAAAAGAG GAAATTGAGC GATTATGTGA
GCTTGTTCCG GAAGTGATGG AGCAGGCCGT TACGCTCCGC GTGCCGCTGA AAGTCGACTA
CCATTACGGC CCAACATGGT ATGATGCCAA A
[0087] The characters represent the following nucleotides:
5 A: Adenosine T: Thymidine C: Cytidine G: Guanosine
[0088] The underlined nucleotides 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.)
[0089] The invention also contemplates any DNA sequence that is
complementary to the modified Bst 320 DNA sequence, for instance,
DNA sequences that would hybridize to the above DNA sequence of the
modified DNA polymerase under stringent conditions. As would be
understood by someone skilled in the art, the invention also
contemplates any DNA sequence that encodes a peptide having these
characteristics and properties (including degenerate DNA code).
[0090] This invention also contemplates 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
unique DNA polymerases of this invention.
[0091] The DNA sequences and amino acid sequences for the modified
DNA polymerase of this invention are also obtainable by, for
instance, isolating and purifying DNA polymerase from a Bacillus
stearothermophilus, or a bacterial strain otherwise derived from
Bacillus stearothermophilus, or other mesophilic bacillus strains
such as Bacillus caldotenax or Bacillus caldolyticus. The DNA
polymerases obtained from these organisms may be easily modified
using conventional DNA modification techniques to achieve the
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.
[0092] In the DNA polymerases currently used in conventional DNA
sequencing protocols, it is preferred that the enzymes have low or
no exonuclease activity. However, in this invention, it is
preferred that the DNA polymerases have a function of high fidelity
("HiFi") nucleotide incorporation. Therefore, in one preferred
embodiment the invention entails modification of a
naturally-occurring Bst DNA polymerase having a proofreading 3'-5'
exonuclease activity. This preferred modified DNA polymerase (e.g.,
"HiFi Bst-II") has a nucleotide sequence indicated in SEQ ID:NO 3
and an amino sequence indicated in SEQ ID:NO 4. To initially obtain
a Bst DNA polymerase having proofreading 3'-5' activity, strains of
Bacillus stearothermophilus can be segregated into different groups
according to the proof-reading exonuclease activity of their
respective DNA polymerases.
[0093] The invention also provides a DNA construct comprising at
least one of the DNA sequences of the modified DNA polymerase and a
vector (such as a cloning vector or an expression vector), for
introducing the DNA construct into host cells. An example of a
suitable vector is pYZ34/LF, described below.
[0094] The host cells need only be capable of being stably
transformed with the DNA construct in a manner allowing production
of the peptide encoded by the DNA segment in the construct
(preferably in large quantity). The host cells may be of eucaryotic
or procaryotic origin (such as a E. coli host cell). For instance,
the host cell may be a mesophilic organism, although this is not a
necessary requirement in order that a host cell be effective.
[0095] The invention also provides improved methods for DNA
sequencing using the above-described novel DNA polymerases. The
methods entail sequencing a DNA strand by conventional protocols
with the following modifications:
[0096] i) hybridizing a primer to a DNA template to be
sequenced;
[0097] ii) extending the primer using a DNA polymerase described
above, in the presence of radiolabeled dATP, nucleotides dGTP, dCTP
and dTTP, or their analogs, and ddNTP chain terminators; and
[0098] iii) allowing a DNA strand to be sequenced.
[0099] All four dNTPs, including dCTP, are incorporated equally
effectively in the chain elongation during sequencing reaction
catalyzed by the DNA polymerases of the invention with a high
processivity and a high elongating rate.
[0100] Preferably the nucleotide premix concentrations of modified
Bst DNA polymerase used in radiolabeled DNA sequencing are as
following:
[0101] A mix: dATP 0.8 .mu.M, dCTP 80M, dGTP 80 .mu.M, dTTP 80
.mu.M, ddATP 25 .mu.M;
[0102] C mix: dATP 0.8 .mu.M, dCTP 8 .mu.M, dGTP 80M, dTTP 80M,
ddCTP 20 .mu.M;
[0103] G mix: dATP 0.8 .mu.M, dCTP 80 .mu.M, dGTP 8 .mu.M, dTTP 80
.mu.M, ddGTP 50 .mu.M;
[0104] T mix: dATP 0.8 .mu.M, dCTP 80 .mu.M, dGTP 80 .mu.M, dTTP 8
.mu.M, ddTTP 50 .mu.M. (This mixture is useful for the particular
modified Bst 320 DNA polymerase set forth above, as well as for
other modified Bst DNA polymerases.)
[0105] In addition, the invention contemplates other uses of the
modified DNA polymerases. For instance, the DNA polymerase can also
be use in (1) filling-in 5' overhangs of DNA fragments; (2)
synthesis of DNA probes by random primers labeling methodology; and
(3) site-directed mutagenesis.
[0106] The following non-limiting examples are illustrative of the
invention.
EXAMPLE 1
Screening for Bst Polymerases
[0107] This invention also involves a method to measure the
proof-reading 3'-5' exonuclease activity of purified DNA
polymerases. The method is useful to screen a large number of
bacterial strains, such as Bacillus stearothermophilus and other
mesophilic bacterial strains, to select a strain which produces a
DNA polymerase with a high proof-reading 3'-5' exonuclease
activity. For instance, the method to test the proof-reading 3'-5'
exonuclease activity of DNA polymerase was carried out as
follows.
[0108] A DNA primer and two DNA templates with following sequences
were synthesized chemically, using a DNA synthesizer.
6 17-base primer 5' CATTTTGCTGCCGGTCA 3' (SEQ ID NO: 5) 1 mg/ml
Template (a) 3'------GTAAAACGACGGCCAGT- CTT------5' (SEQ ID NO: 6)
10 mg/ml Template (b) 3'-----GTAAAACGACGGCCAGTCGG-----5' (SEQ ID
NO: 7) 10 mg/ml
[0109] To produce the radiolabeled primer, 1 .mu.l (1 .mu.g) of
primer, 5 .mu.l (50 .mu.g) of template (a), 1 .mu.l of
[.alpha.-.sup.32P] dATP (800 Ci/mmole), 1 .mu.l of dGTP (0.5 mM), 1
.mu.l of Taq DNA polymerase. (1 unit), and 1 .mu.l of buffer
consisting of 500 mM Tris-Cl, pH 9.0, and 150 mM MgCl.sub.2, were
mixed in a test tube and incubated in a 65.degree. C. water bath
for 5 minutes. The mixture was subject to alkaline denaturing gel
electrophoresis. The radioactive band containing the 20-base
nucleotide was isolated and dissolved in 12 .mu.l of 10 mM Tris-Cl
buffer, containing 1 mM EDTA, pH 8.0. The final product represents
the following labeled 20-base primer.
7 5' CATTTTGCTGCCGGTCAGA*A* 3' (SEQ ID NO: 8) (* = 32P labeled)
[0110] To produce radiolabeled primer-template complexes, 5 .mu.l
of the labeled primer was mixed with 10 .mu.l of template (a) or
template (b) respectively to form the following:
8 Complex (a) 5'CATTTTGCTGCCGGTCAGA*A* 3' (same as SEQ ID NO: 8)
3'GTAAAACGACGGCCAGTCT T 5' (same as SEQ ID NO: 6) Complex (b) 5'
CATTTTGCTGCCGGTCAGA*A* 3' (same as SEQ ID NO: 8) 3'
GTAAAACGACGGCCAGTCG G 5' (same as SEQ ID NO: 7)
[0111] The free radiolabeled primer was removed through a G-50
Sephadex column.
[0112] An aliquot of complex (a) which had two correctly matched
radiolabeled A*s at the 3' terminus of the primer, and an aliquot
of complex (b) which had two mismatched A*s at the 3' terminus of
the primer, were then pipetted into two individual vials of
scintillation fluid and their radioactivity was measured in a
scintillation counter, and both complexes were adjusted with buffer
to a concentration containing the same molarity of incorporated
[.alpha.-.sup.32P] dAMP.
[0113] To perform the proof-reading 3'-5' exonuclease activity, 20
.mu.l of complex (a) or complex (b), 8 .mu.l reaction buffer
consisting of 15 mM Tris-Cl and 15 mM MgCl.sub.21 pH 8.5, 4 units
of DNA polymerase, and enough water to make up a total volume of 40
.mu.l were pipetted into a test tube and mixed well. The mixture
was subdivided into aliquots of 3 .mu.l each in 0.5 ml
microcentrifuge tubes and was then covered with 3 .mu.l paraffin in
each tube. The microcentrifuge tubes were incubated in a 65.degree.
C. water bath. At 1, 2, 3, 5, 10, and 20 minutes, a pair of the
microcentrifuge tubes were taken out from the water bath and the
content of each tube was dotted onto a DE-81 Whatman filter paper.
One of each pair of the filter papers was put in scintillation
fluid directly and the radioactivity was counted in cpm value in a
scintillation counter; the other was washed three times in 0.3 M
sodium phosphate buffer, pH 6.8 before being put into the
scintillation fluid for counting.
[0114] The difference in radioactivity expressed in cpm value
between the washed filter paper and the unwashed filter paper in
each pair was interpreted as representing the relative quantity of
labeled nucleotides excised by the 3'-5' exonuclease activity from
the 3' terminus of the primer. A DNA polymerase that excised the
radiolabeled nucleotides A*s from complex (b) more efficiently than
from complex (a) possessed proofreading 3'-5' exonuclease activity.
A DNA polymerase that excised the radiolabeled nucleotides A*s from
complex (a) faster than from complex (b), or at nearly the same
rate, was interpreted as possessing a non-specific 3'-5'
exonuclease activity which is considered unsuitable for DNA
sequencing.
[0115] Using these methods, a strain of bacteria was isolated from
among the strains of Bacillus stearothermophilus from various
sources which is distinguished in its fast growth rate. This strain
reached an optimum exponential growth within 3 hours for DNA
polymerase production. The strain was also able to produce a DNA
polymerase with a proof-reading 3'-5' exonuclease activity. This
strain of Bacillus stearothermophilus was labeled Bst No. 320.
[0116] As persons skilled in this art would appreciate, the
bacterial strain, or even the strain of Bacillus
stearothermophilus, from which a mesophilic DNA polymerase of the
invention can be obtained may be derived using the above-described
methods or others known in the art from strains of Bacillus
stearothermophilus or other bacteria strains (especially mesophilic
strains), including wild strains or mutant strains acquired by
various means, including spontaneous mutation.
[0117] To prepare the preferred purified Bst DNA polymerase, the
cells of Bst No. 320 were grown at 55.degree. C. in a liquid medium
consisting of 1% polypeptone, 0.5% yeast extract and 0.5% NaCl,
pH7.0-7.2. The 3 hr old cells were collected after centrifugation
and suspended in 4 volumes of TME buffer (50 mM Tris-HCl, pH7.5, 10
mM .beta.-mercaptoethanol, and 2 mM EDTA), containing 100 mg
lysozyme and 23 mg phenylmethylsulphonyl-fluorid- e/ml. The cells
were broken by sonication in ice. The supernatant was pooled after
centrifugation at 28,000 rpm in a Spinco L 30 rotor.
[0118] The purified Bst DNA polymerase of the invention was
prepared according to Okazaki and Kornberg (7) with appropriate
slight modifications and the large fragment of the DNA polymerase
was obtained by partial digestion of the whole DNA polymerase with
the proteinase subtilisin (type Carlsberg) basically according to
Jacobsen et al. (8).
[0119] The procedure for purification of enzyme was followed as
described in Ye and Hong (4). This Bst DNA polymerase possessed a
proof-reading 31'-5' exonuclease activity.
[0120] The Bst polymerase was tested for proofreading and
non-specific 3'-5' exonuclease activities as described above. The
results showed that the polymerase excised the mismatched
incorporated nucleotides from the 3' terminus of a double-stranded
DNA at a high rate, reaching the plateau of hydrolysis in about 3
minutes, about 8 times more efficiently in the first 3 minutes of
reaction than those correctly matched with the nucleotides of the
template. This enzyme is referred to herein as HiFi Bst DNA
polymerase, and is distinguishable from Bst DNA polymerases
isolated from other strains of Bacillus stearothermophilus.
[0121] This prodedure of using HiFi Bst as the DNA polymerase in
the classic radiolabeling Sanger reaction for DNA sequencing and
its autoradiograph are illustrated in Example 8. The data obtained
by adapting this procedure to use HiFi Bst as the DNA polymerase in
dye-labeled primer automated fluorescent DNA sequencing are
illustrated in Example 9. These results indicate that HiFi Bst DNA
polymerase can be used for the classic Sanger manual sequencing and
the fluorescent dye-labeled primer sequencing with high
processivity and high fidelity.
[0122] However, when the protocol was modified for fluorescent
dye-labeled ddNTP terminator automated DNA sequencing, numerous "C"
peaks and "A" peaks were missing or appeared ambiguous in the
sequence plot, especially when the "C" peak was immediately after a
"G" peak or after an "A" peak, and when the "A" peak was
immediately after a "G" peak. This selective discrimination against
incorporation of dye-labeled ddCTP and dye-labeled ddATP is
sequence-related since many "C" and "A" peaks following an
immediate "C" peak or an immeciate "T" peak remained strong and
correctly resolved in the same color plot of sequence analysis.
(See Example 9) This phenomenon of selective discrimination against
incorporation of fluorescent dye-labeled ddCTP and dye-labeled
ddATP is observed with all DNA polymerases isolated from different
strains of Bacillus stearothermophilus and Bacillus caldotenax, and
appears to be characteristic of DNA polymerases of the mesophilic
bacilli.
EXAMPLE 2
Mutation of the Gene for Naturally-Occurring Bst DNA Polymerase
Having Proofreading 3'-5' Exonuclease Activity
[0123] The DNA fragment LF containing the gene initially isolated
from the wild Bst 320 has the following sequence (see SEQ ID
NO:1):
[0124] DNA Sequence (Isolated/Purified):
9 GCCGAAGGGG AGAAACCGCT TGAGGAGATG GAGTTTGCCA TCGTTGACGT CATTACCGAA
GAGATGCTTG CCGACAAGGC AGCGCTTGTC GTTGAGGTGA TGGAAGAAAA CTACCACGAT
GCCCCGATTG TCGGAATCGC ACTAGTGAAC GAGCATGGGC GATTTTTTAT GCGCCCGGAG
ACCGCGCTGG CTGATTCGCA ATTTTTAGCA TGGCTTGCCG ATGAAACGAA GAAAAAAAGC
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 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 TTATTAAAAA 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)
[0125] Site directed mutagenesis was performed as described by
Kunkel et al. (14) The DNA fragment (designated "LF") containing
the gene for Bst DNA polymerase having proofreading exonuclease
activity was cloned from the expression vector pYZ23/LF into
plasmid pUC119. The constructed plasmid pUC119/LF was then
transformed into E. coli CJ236, a mutant of E. coli that lacks the
enzymes dUTPase and uracil N-glycosylase. Therefore, when grown in
a medium supplemented with uridine, this mutant of E. coli as well
as the plasmids in the cells will incorporate deoxyuridine into the
DNA in place of thymidine and the uracils will not be removed
readily.
[0126] As the constructed plasmid grew in the cells of E. coli
CJ236 and in the presence of uracil and M13K07 helper phage, the
normal thymidine bases of the DNA in the newly produced
single-stranded pUC119/LF were replaced by uracils. These
uracil-containing DNAs were used as the template in vitro for the
production of a complementary oligonucleotide that contained the
desired DNA sequence alteration, but with only TMPs and not dUMP
residues.
[0127] In practice, the expression vector pYZ23/LF was digested
with restriction enzymes Eco RI and Bam HI, and the DNA fragment LF
was separated and cloned into plasmid pUC119 which had been
previously digested with the same restriction enzymes. The
constructed plasmid pUC119/LF was then transformed into E. coli
CJ236. For gaining the uracil-containing single-stranded pUC119/LF,
a colony of E. coli CJ236 containing pUC119/LF was selected and
inoculated into 2 ml of 2.times.YT medium which was supplemented
with 0.25 ug/ml of uridine and 2.times.10.sup.8 to 4.times.10.sup.8
pfu/ml of M13KO7 as helper phage. After incubation at 37.degree. C.
with strong agitation for 1 hour, a kanamycin solution (25 mg/ml in
H.sub.2O) was added to the culture to a final concentration of 70
ug/ml. The incubation was allowed to continue for another 14-18
hours at 37.degree. C. with strong agitation. Then 1.5 ml of the
infected culture was transferred to a microcentrifuge tube, and
centrifuged at 12,000.times.g for 5 minutes at 4.degree. C. The
uracil-containing single-stranded pUC119/LF was precipitated and
purified from the supernatant according to standard PEG/NaCl and
ethanol procedures.
[0128] After performing a series of experiments, the inventors
found that the combined effects of changing the amino acids
leucine-glutamate-glutam- ate (LEE) at the location 342-344, to
respectively threonine-proline-leuci- ne (TPL), and the amino acid
phenylalanine (F) at location 422, to tyrosine (Y) in the peptide
structure of HiFi Bst DNA polymerase markedly reduced its selective
discrimination against incorporation of fluorescent dye-labeled
ddCTP and dye-labeled ddATP to such a level that direct automated
fluorescent DNA sequence (although not cycle-sequencing) can be
performed with the dye-terminator technology when the mutated
enzyme of the current invention is used. It is of interest to note
that this modified HiFi Bst, now referred to as HiFi Bst-II DNA
polymerase, exhibits the function of preferentially incorporating
more fluorescent dye-labeled ddCTP and dye-labeled ddATP onto the
3' end dGMP and the DAMP bases of the extending DNA strands during
enzymatic reaction, than the unmodified naturally occurring HiFI
Bst polymerase.
[0129] The end result is the recovery of the "C" and "A" peaks
which otherwise would have been missing or ambiguous on the
sequence analysis color plot. At the same time, the modified enzyme
did not indiscriminately generate an excess amount of dye-labeled
"G" terminated or dye-labeled "T" terminated DNA fragments. Even
the "C" and "A" peaks were not uniformly raised in a blanket
manner, but only raised in the formerly depressed locations after a
"G" and/or an "A". (See Example 9). Thus, this genetic modification
of the HiFi Bst to HiFi Bst-II results in a DNA polymerase that
reduces the selective discrimination against incorporation of the
fluorescent dye-labeled ddCTP and dye-labeled ddATP, rather than
merely increases the ability of the parent enzyme to incorporate
these dye-labeled dideoxynucleotides.
[0130] HiFi Bst-II, and the other novel similar DNA polymerases of
this invention, can be used for the classic radiolabeling Sanger
method. (See Example 8.) HiFi Bst-II appears to generate a better
sequencing pattern than HiFi Bst and requires less ddNTPs to
terminate the extending reaction (FIG. 3). For instance, in the
optimized reaction mixture for the unmodified HiFi Bst DNA
polymerase, the ddNTP/dNTP ratios in the A, C, G and T mix were 40,
6.25, 18.25 and 18.72, respectively. In the optimized reaction
mixture for the modified HiFi Bst-II DNA polymerase, the
corresponding ddNTP/dNTP ratios in the A, C, G and T mix were 40,
2.5, 6.25 and 6.25, respectively. Therefore, there was an up-to
about three-fold reduction in the amount of ddNTPs used after
genetic modification of the naturally-occurring DNA polymerase.
[0131] For the radiolabeling classic Sanger method of DNA
sequencing, the optimized reaction mixtures for either HiFi Bst or
HiFi Bst-II must contain much more ddNTPs than dNTPs to generate a
ladder of DNA fragments for sequencing analysis because the DNA
polymerases of the mesophilic bacilli tend to incorporate dNTPs
more efficiently than ddNTPs. The above-described genetic
modification appears to increase the ability of the
naturally-occurring enzymes to incorporate ddNTP in the presence of
a corresponding competing dNTP to about three-fold at the
concentration ratios commonly used for DNA sequencing. However, if
much higher concentrations of the nucleotides were used for the
experiment, and the ddNTP/dNTP ratio was reduced to a level that is
suboptimal for DNA sequencing (for instance at a ratio of 1/3), the
increased ability for incorporating ddNTPs after modification of
the enzyme could be dramatized. (See Example 6, FIG. 2).
[0132] Similar to the results obtained with radiolabeling Sanger
method, both HiFi Bst and HiFi Bst-II can be adapted for
fluorescent dye-labeled primer automated DNA sequencing and produce
comparable results without selective suppression of any specific
fluorescent peaks in the sequencing plot (see Example 9) although
the peaks generated by HiFi Bst-II appear to be more even than
those by HiFi Bst.
[0133] To change amino acids leucine, glutamic acid and glutamic
acid (LEE) at positions 342-344, respectively in the Bst polymerase
into threonine, proline and leucine (TPL), respectively, Primer 1
was designed as following (see SEQ ID NO 10):
10 5'-CATTCCGATTCGGACCCCACTGGGGCGGAAAACCG-3
[0134] To change amino acid phenylalanine (F) at position 422 in
the Bst DNA polymerase into tyrosine (Y), Primer 2 was designed as
following (see SEQ ID NO: 9):
11 5'-GCCGTTAACTACGGTATCGTTTACGG-3'
[0135] After phosphorylation of the 5' ends of the oligonucleotides
by T4 polynucleotide kinase, the two primers designed above were
annealed to the single-stranded uracil-containing pUC119/LF
purified from above. In the presence of the usual dNTPs (dATP,
dCTP, dGTP and dTTP), T4 DNA polymerase was used to synthesize in
vitro the strands of DNA complementary to the uracil-containing
pUC119/LF template, and T4 ligase was used to ligate the
synthesized strands to form a complete double-stranded plasmid
which was composed of one single-stranded, not mutagenic,
uracil-containing pUC119/LF and one complementary single-stranded,
mutagenic, thymidine-containing DNA fragment that had been altered
by primer 1 and primer 2 described above. These newly formed
double-stranded plasmids were then transformed into E. coli JM109.
The template strand was rendered biologically inactive. The
transformed strain of E. coli JM109 whose plasmids contained the
mutated DNA, now referred to as pUC119/LF-M, was screened out with
DNA sequencing of its plasmids.
EXAMPLE 3
Cloning and Expression of the Modified Bst DNA Polymerase Having
Both Ability to Reduce Selective ddNTP Discrimination and
Proofreading 3'-5' Exonuclease Activity
[0136] The plasmid pUC119/LF-M was prepared from the strain of
Escherichia coli JM109 containing the mutated DNA. The mutated DNA
fragment (LF-M) containing the mutated gene for the Bst polymerase
was recombined back into the expression vector pYZ23. The
constructed plasmid pYZ23/LF-M was then transformed into
Escherichia coli JF1125. The mutation was further confirmed by
double-stranded dideoxy DNA sequencing of isolated plasmid.
[0137] The strain of Escherichia coli JF1125 containing pYZ23/LF-M
was inoculated into LB culture containing 100 .mu.g/ml ampicillin,
and was incubated overnight at 30.degree. C. The overnight culture
was inoculated into a large volume of fresh culture, and was
incubated at 30.degree. C. until the OD.sub.600 of the culture
reached 0.7. The culture was then heated at 41.degree. C. for 3
hours for induction. The SDS-PAGE analysis of the cell extract
showed that the cloned mutated gene for the modified Bst DNA
polymerase was overexpressed.
EXAMPLE 4
Isolation and Purification of the modified Bst DNA Polymerase
Having Both Ability to Reduce Selective ddNTP Discrimination and
Proofreading 3'-5' Exonuclease Activity
[0138] The expressed cells of Escherichia coli JF1125 containing
pYZ23/LF-M grown in condition as described above were thawed and
washed with buffer [10 mM Tris-HCl (pH7.5 at room temperature), 10
mM .beta.-Mercaptoethanol, 2 mM EDTA, 0.9% NaCl]. The pellets were
then suspended in buffer [50 mM Tris-HCl (pH7.5 at room
temperature), 10 mM .beta.-Mercaptoethanol, 2 mM EDTA, 100 .mu.g/ml
Lysozyme, 23 .mu.g/ml PMSF](4 ml/g pellet). After 20 min at room
temperature, the mixture was cooled on salt-ice and sonicated
briefly to complete lysis. The cell extract obtained by
centrifugation at 18,000 rpm at 4.degree. C. for 20 minutes, was
then treated step by step as follows:
[0139] (A) The cell extract was heated at 60.degree. C. for 30
minutes, and cooled to 4.degree. C., then centrifuged at 15,000 rpm
at 4.degree. C. for 20 minutes;
[0140] (B) 5% Polymin P was added into supernatant to 0.6%, and
mixed quickly for 30 minutes, then centrifuged;
[0141] (C) The pellet was resuspended in Buffer A [50 mM Tris-HCl
(pH7.5 at room temperature), 1 mM EDTA, 1 mM
.beta.-Mercaptoethanol] containing 800 mM NaCl and 5% Glycerol at
4.degree. C., and then centrifuged;
[0142] (D) Ammonium sulfate was added into the supernatant to 60%
saturation at 4.degree. C., and mixed for 30 minutes, then
centrifuged;
[0143] (E) The ammonium sulfate pellet was resuspended in 30 ml of
60% saturated ammonium sulfate at 4.degree. C., and then
recentrifuged;
[0144] (F) The pellet was suspended in Buffer A containing 100 mM
KCl and dialysed against the same buffer for hours at 4.degree. C.,
then centrifuged. The insoluble protein was discard;
[0145] (G) The supernatant was added to pass through a DE-52
column. The column was washed, and the peak DNA polymerase was
eluted using a 100-600 mM KCl linear gradient in Buffer A,
concentrated in Buffer A containing 50%(w/v) PEG-6000, dialyzed in
Buffer A containing 100 mM KCl;
[0146] (H) The solution was then applied to Heparin-Sepharose CL-4B
column. The peak DNA polymerase was eluted with a linear gradient
of 100-800 mM KCl in Buffer A, concentrated and finally dialyzed in
buffer A containg 50% glycerol.
[0147] The resulting modified Bst DNA polymerase has been proved to
be homogenous by polyacrylamide gel electrophoresis. And the enzyme
obtained was stored in -20.degree. C.
EXAMPLE 5
Determination of the Thermostability of Unmodified Bst DNA
Polymerase and Modified Bst DNA Polymerase
[0148] The DNA polymerases of Examples 1 and 5 were incubated at
65.degree. C. for 0, 5, 10, 20, 30, 40, 50 minutes respectively,
and placed into ice-water immediately. The polymerase activity of
these DNA polymerases was determined at 60.degree. C.
[0149] The polymerase activity of DNA polymerase was determined as
follows:
[0150] 5.times. Reaction Solution:
12 1M Tris-HCl (pH7.6) 16.75 ml 1M MgCl.sub.2 1.675 ml 1M
.beta.-Mercaptoethanol 0.25 ml ddH.sub.2O adjusted to 50 ml
[0151] Reaction Storage:
13 5 .times. Reaction Solution 60 .mu.l dNTPs (1 mM each) 10 .mu.l
1.5 .mu.g/.mu.l DNase I activated 10 .mu.l calf thymus DNA
ddH.sub.2O 10 .mu.l .alpha.-.sup.32P-dATP appropriate amt.
[0152] Reaction Mixture:
14 Reaction Storage 30 .mu.l Sample 5 .mu.l ddH.sub.2O 65 .mu.l
[0153] The reaction mixtures were prepared as per the recipe above,
and incubated at 60.degree. C. for 30 minutes. Then the reaction
mixtures were pipetted onto DE-81 filters respectively. After all
of the fluid has evaporated, the amount of radioactivity on each
filter was measured with scintillation (X.sub.1). The filters were
washed three times with 0.3M Na.sub.2HPO.sub.4 at room temperature,
10 minutes each times, dried at room temperature and then the
amount of radioactivity on each filter was measured again
(X.sub.2).
[0154] The polymerase activity of sample 1 ( u / ml ) = X 2 X 1 - X
20 X 10 .times. 266
[0155] (X.sub.10 and X.sub.20 are the amount of radioactivity
measured with water as control sample)
[0156] Unit definition of polymerase activity: One unit is the
amount of DNA polymerase required to incorporate 10 nanomoles of
dNTPs into DNA in 30 minutes at 60.degree. C.
[0157] The thermostability of DNA polymerase is expressed with the
half life of polymerase activity at 65.degree. C. FIG. 1 shows the
comparison of thermostabilty of HiFi Bst and HiFi Bst-II. The half
life of HiFi Bst at 65.degree. C. was 8.5 minutes, and that of HiFi
Bst-II was 16 minutes. HiFi Bst-II was more thermostable than HiFi
Bst.
EXAMPLE 6
Demonstration of Increased ddNTP Incorporation by Modified Bst DNA
Polymerase in Suboptimal Sequencing Conditions
[0158] The following procedure was followed:
[0159] 1. The -20M13 forward primer was radiolabelled using
.gamma.-.sup.32P-ATP and T4 Polynucleotide kinase;
[0160] 2. The following components were combined in a
microcentrifuge tube:
15 5 .times. Reaction Buffer 2.0 .mu.l radiolabeled primer 1.0
.mu.l (2.5 ng) Template 7.0 .mu.l (1 .mu.g Ml3mp18 ssDNA)
[0161] The final volume was 10 .mu.l. The contents were mixed and
spun for 2-3 seconds;
[0162] 3. The tube were placed in a 75.degree. C. water bath for 5
minutes. Then the tube was allowed to cool slowly to ambient
temperature over a course of 10 minutes;
[0163] 4. 1.0 .mu.l of modified Bst DNA polymerase (of Example 5)
(1 u/.mu.l) was added. The mixture was mixed gently and spun for
2-3 seconds;
[0164] 5. 4 tubes were labelled "A", "C", "G", "T", respectively
and 2 .mu.l of each premixed nucleotide solution and 2.5 .mu.l of
main mixture (from step 3) was added to the respective reaction
tube;
[0165] 6. The tubes were incubated at 65.degree. C. for 15
minutes;
[0166] 7. The reactions were stopped by adding 4.0 .mu.l of Stop
Solution (95% deionized formamide, 10 mM EDTA, 0.05% xylene cyanol
FF, 0.05% bromophenol blue) to each tube;
[0167] 8. The samples were denatured at 90.degree. C. for 2
minutes, and immediately placed on ice;
[0168] 9. 4-5 .mu.l of samples were loaded onto each lane of 6% (8M
urea) sequencing gel, and electrophoresis was carried out.
[0169] Note: The Components of the Premixed Nucleotide
Solutions:
[0170] A mix: dNTPs 120 .mu.M, ddATP 40 .mu.M
[0171] C mix: dNTPs 120 .mu.M, ddCTP 40 .mu.M
[0172] G mix: dNTPs 120 .mu.M, ddGTP 40 .mu.M
[0173] T mix: dNTPs 120 .mu.M, ddTTP 40 .mu.M
[0174] FIG. 2 shows the comparison of ddNTP incorporation of HiFi
Bst-II DNA polymerase and HiFi Bst DNA polymerase. In this
radiolabeling DNA sequencing experiment, high concentrations of
nucleotides were used in the reaction mixture and the ddNTP/dNTP
ratio was reduced to a level (1/3) that is lower than the optimal
range for DNA sequencing. HiFi Bst-II is shown to have more
effective ddNTP incorporation. The DNA synthesis was often
terminated by ddNTP incorporation in the HiFi Bst-II mixture, and
the result showed uniform bands with synthesized small or large DNA
fragments. As a contrast, HiFi Bst had a lower ddNTP incorporation.
The DNA synthesis by HiFi Bst was less terminated, and most of the
synthesized products were the larger DNA fragments.
EXAMPLE 7
Preparation of Denatured Double-Stranded DNA Template
[0175] The following procedure was carried out.
[0176] 1. Double-stranded DNA (about 3-5 .mu.g) was adjusted to a
final volume of 10 .mu.l with TE (10 mM Tris-HCl, 1 mM EDTA,
pH8.0);
[0177] 2. 10 .mu.l of 0.4N NaOH, 0.4 mM EDTA, was added;
[0178] 3. The mixture was incubated at 65.degree. C. for 15
minutes;
[0179] 4. 2 .mu.l of 2M sodium acetate, pH4.5, and 55 .mu.l cold
ethanol was added, and the mixture was placed in ice-water bath for
5 minutes;
[0180] 5. The mixture was spun in a microcentrifuge at 4.degree.
C., 12500 rpm for 5 minutes;
[0181] 6. The supernatant was drawn off and the pellet was washed
with 200 .mu.l of 70% ethanol;
[0182] 7. The pellet was dried under vacuum for 2-3 minutes, and
the DNA was dissolved in appropriate solution.
EXAMPLE 8
DNA Sequencing Using Unmodified Bst DNA Polymerase/Modified Bst DNA
Polymerase with Radiolabeled dATP for Single- or Denatured
Double-Stranded DNA Template
[0183] The following procedure was carried out.
[0184] 1. The following components were combined in a labeled
microcentrifuge tube:
16 5 .times. Reaction Buffer 2.0 .mu.l Primer 1.0 .mu.l (2.5-5.0
ng) Template 7.0 .mu.l (250-500 ng ss DNA or 1-3 .mu.g denatured ds
DNA)
[0185] The final volume was 10 .mu.l. The contents were mixed and
spun for 2-3 seconds;
[0186] 2. The tube were placed in a 75.degree. C. water bath for 5
minutes. Then the tubes were allowed to cool slowly to ambient
temperature over a course of 10 minutes;
[0187] (Note: Step 2 is optional for single-stranded template, and
may be omitted at appropriate.)
[0188] 3. 1.0 .mu.l of HiFi Bst/HiFi Bst-II (1 u/.mu.l) and 1.0
.mu.l of [.alpha.-.sup.32P]dATP was added, and the mixture was
mixed gently and spun for 2-3 seconds;
[0189] 4. 4 tubes "A", "C", "G", "T" were labelled, and 2 .mu.l of
each premixed nucleotide solution and 2.5 .mu.l of main mixture
(from step 3) was added to the respective reaction tube;
[0190] 5. The tubes were incubated at 65.degree. C. for 2
minutes;
[0191] 6. 2.0 .mu.l of 0.5 mM dNTPs was added to each tube, and the
tubes were mixed gently, spun for 2-3 seconds, and incubated at
65.degree. C. for 2 minutes;
[0192] 7. The reactions were stopped by adding 4.01 of Stop
Solution (95% deionized formamide, 10 mM EDTA, 0.05% xylene cyanol
FF, 0.05% bromophenol blue) to each tube;
[0193] 8. The samples were denatured at 90.degree. C. for 2
minutes, and immediately placed on ice;
[0194] 9. 2-3 .mu.l of the samples were loaded onto each lane of 6%
(8M urea) sequencing gel, and electrophoresis was carried out.
[0195] Note: The Components of the Premixed Nucleotide Solutions
for HiFi Bst:
[0196] A mix: dATP 0.62 .mu.M, dCTP 62 .mu.M, dGTP 62 .mu.M, dTTP
62 .mu.M, ddATP 25 .mu.M;
[0197] C mix: dATP 0.8 .mu.M, dCTP 8 .mu.M, dGTP 80 .mu.M, dTTP 80
.mu.M, ddCTP 50 .mu.M;
[0198] G mix: dATP 0.8 .mu.M, dCTP 80 .mu.M, dGTP 4 .mu.M, dTTP 80
.mu.M, ddGTP 75 .mu.M;
[0199] T mix: dATP 0.8 .mu.M, dCTP 80 .mu.M, dGTP 80 .mu.M, dTTP 8
.mu.M, ddTTP 150 .mu.M.
[0200] The Components of the Premixed Nucleotide Solutions for HiFi
Bst-II:
[0201] A mix: dATP 0.8 .mu.M, dCTP 80 .mu.M, dGTP 80 .mu.M, dTTP 80
.mu.M, ddATP 25 .mu.M;
[0202] C mix: dATP 0.8 .mu.M, dCTP 8 .mu.M, dGTP 80 .mu.M, dTTP 80
.mu.M, ddCTP 20 .mu.M;
[0203] G mix: dATP 0.8 .mu.M, dCTP 80 .mu.M, dGTP 8 .mu.M, dTTP 80
.mu.M, ddGTP 50 .mu.M;
[0204] T mix: dATP 0.8 .mu.M, dCTP 80 .mu.M, dGTP 80 .mu.M, dTTP 8
.mu.M, ddTTP 50 .mu.M.
[0205] FIG. 3 shows the comparison of radiolabeled DNA sequencing
with HiFi Bst and HiFi Bst-II in their respective optimized
reaction mixtures. The bands on a DNA sequencing gel with HiFi
Bst-II were more uniform than those with HiFi Bst. The sequence
pattern using HiFi Bst-II was better than that using HiFi Bst. This
made the gel with HiFi Bst-II even easier to read. Moreover, the
ratio of dideoxy-nucleotide concentration to deoxy-nucleotide
concentration in Premixed Nucleotide Solutions for HiFi Bst-II was
lower than that for HiFi Bst. For instance, the ddATP/dATP,
ddCTP/dCTP, ddGTP/dGTP and ddTTP/dTTP ratios were 40, 6.25, 18.75
and 18.75, respectively, for HiFi Bst. The corresponding ratios for
HiFi Bst-II were 40, 2.5, 6.25 and 6.25, respectively. Therefore,
the concentrations of ddNTPs used in the termination reaction are
reduced to about three-folds after HiFi Bst has been genetically
modified.
EXAMPLE 9
DNA Sequencing Using Unmodified Bst DNA Polymerase/Modified Bst DNA
Polymerase with Dye-Primers for Single- or Denatured
Double-Stranded DNA Template
[0206] The following procedure was carried out.
[0207] 1. The following was combined: 4.0 .mu.l DNA template
(300-600 ng for ssDNA or 1-2 .mu.g denatured ds DNA) with 5.0 .mu.l
5.times. Reaction Buffer. The mixture was mixed and spun for 2-3
seconds in a microcentrifuge;
[0208] 2. 4 tubes were labeled "A", "C", "G", "T" and the pre-mixed
dATP, dCTP, dGTP, dTTP reagents were added to each reaction
tube:
17 Reaction tube A C G T A-REG primer (0.2 uM) 1.0 ul C-FAM primer
(0.2 uM) 1.0 ul G-TMR primer (0.4 uM) 1.0 ul T-ROX primer (0.4 uM)
1.0 ul A terminator mix 2.0 ul C terminator mix 2.0 ul G terminator
mix 2.0 ul T terminator mix 2.0 ul 5 .times. Reaction buffer 2.0 ul
2.0 ul 2.0 ul 2.0 ul with DNA template Total volume 5.0 ul 5.0 ul
5.0 ul 5.0 ul
[0209] 3. The tubes were placed in 75.degree. C. bath for 5
minutes, and allowed to cool slowly to ambient temperature over the
course of 10 minutes;
[0210] (Note: Step 3 is optional for single-stranded template, and
may be omitted as appropriate.)
[0211] 4. 1 .mu.l of HiFi Bst/HiFi Bst-II (0.5 u/.mu.l) was added
to each tube, and the tubes were spun for 2-3 seconds;
[0212] 5. The tubes were incubated at 65.degree. C. for 5
minutes;
[0213] 6. The contents of the "A", "C", "G" and "T" tubes were
pooled, and 1.5 .mu.l of 7.5M ammonium acetate and 55 .mu.l of
ethanol was added. The mixtured was mixed in a vortex and then
placed on ice for 20 minutes;
[0214] 7. The mixture was centrifuged at 12,500 rpm for 20 minutes
at 4.degree. C.;
[0215] 8. The supernatant was drawn off, and the pellet was washed
with 200 .mu.l of 70% ethanol;
[0216] 9. The pellet was vacuum dried for 2-3 minutes, and
resuspended in 4 .mu.l of loading buffer (5:1 deionized
formamide:25 mM EDTA with 50 mg/ml Blue Dextran);
[0217] 10. The sample was heated at 75.degree. C. for 2-3 minutes,
and immediately placed on ice;
[0218] 11. 2-3 .mu.l of sample was loaded onto a lane of the 4% (6
M urea) sequencing gel, and ABI PRISM.TM. 377 DNA Sequencer (from
Perkin Elmer) was used to collect data.
[0219] Note: Dye primer: DYEnamic Energy Transfer Dye Primers (from
Amersham):
18 -21 M13 forward: 5'-FAM-S.sup.TSSSSSTGT*AAAACGACGGCC (SEQ ID NO:
11) AGT-3'
[0220] TS=1'2'-dideoxyribose
[0221] T*=T attached with Dye 2 (A-REG, C-FAM, G-TMR, T-ROX)
[0222] FIG. 4 and FIG. 5 show the results of dye-primer DNA
sequencing with HiFi Bst and HiFi Bst-II. Both DNA polymerases
generated similar sequencing results although the peaks on the
color plot by HiFi Bst II appear to be more even in height.
EXAMPLE 10
DNA Sequencing Using Unmodified Bst DNA Polymerase/Modified Bst DNA
Polymerase with Dye-Terminators for Single- or Denatured
Double-Stranded DNA Template
[0223] The following procedure was carried out.
[0224] 1. The following components were combined in a labeled
microcentrifuge tube:
19 5 .times. Reaction Buffer 4.0 .mu.l Template 8.0 .mu.l (2-3
.mu.g ss DNA or 4-6 .mu.g denatured ds DNA) Primer 2.0 .mu.l (5-10
ng)
[0225] The final volume was 14 .mu.l. The contents were mixed and
spun for 2-3 seconds;
[0226] 2. The tube was placed in a 75.degree. C. water bath for 5
minutes;
[0227] 3. The tube was allowed to cool slowly to ambient
temperature over a course of 10 minutes; (Note: Steps 2 and 3 are
optional for single-stranded template, and may be omitted as
appropriate.)
[0228] 4. 1.0 .mu.l of HiFi Bst/HiFi Bst-II (1-2 u/.mu.l), 5 .mu.l
of nucleotides premix (containing Perkin Elmer-ABI fluorescent
dye-labeled nucleotide terminators), were added and the tube was
spun for 2-3 seconds;
[0229] 5. The mixture was incubated at 65.degree. C. for 10
minutes;
[0230] 6. 80 .mu.l of H.sub.2O was added to the reaction mix, and
the dye terminators were extracted with 1001 of
phenol:H.sub.2O:chloroform (68:18:14) reagent twice. The sample was
vortexed and centrifuged, and the aqueous upper layer was
transferred to a clean tube;
[0231] 7. To the tube was added 15 .mu.l of 2M sodium acetate, pH
4.5, and 300 .mu.l of ethanol, and the tube was vortexed and placed
in ice-water bath for 20 minutes;
[0232] 8. The tube was centrifuged with 12,500 rpm for 20 minutes
at 4.degree. C.;
[0233] 9. The supernatant was drawn off, and the pellet was washed
with 200 .mu.l of 70% ethanol;
[0234] 10. The pellet was vacuum dried for 2-3 minutes, and
resuspended in 4 .mu.l of loading buffer (5:1 deionized
formamide:25 mM EDTA with 50 mg/ml Blue Dextran);
[0235] 11. The sampled was heated at 90.degree. C. for 2-3 minutes,
and immediately placed on ice;
[0236] 12. 2-3 .mu.l of sample was loaded onto a lane of the 4% (6M
urea) sequencing gel, and ABI PRISM.TM. 377 DNA Sequencer (from
Perkin Elmer) was employed to collect data, using appropriate
amounts of nucleotide pre-mixed reagents.
[0237] FIG. 6 and FIG. 7 show the results of dye-terminator DNA
sequencing with HiFi Bst and HiFi Bst-II. There was data lost in
dye-terminator DNA sequencing with HiFi Bst, especially the "C"
after "G" or "A" and "A" after "G". In FIG. 8, corrections of the
missing or ambiguous bases, according to the known pGEM sequence,
have been indicated below the letters "N" or below the incorrect
base letters. This problem caused ambiguity in DNA sequencing. But
it was resolved in dye-terminator DNA sequencing with the modified
Bst DNA polymerase of this invention.
REFERENCES
[0238] 1. Sanger, F., Nicklen, S. & Coulson, A. R. Proc. Nat.
Acad. Sci., USA 74: 5463-5467. 1977.
[0239] 2. CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausubel, F. M.
et. Al. (Editors) Vol. I., John Wiley & Sons, Inc. 1995. pp
7.4.17-7.4.24.
[0240] 3. Ibid p. 7.4.31.
[0241] 4. Ye, S. Y. & Hong, G. F., Scientia Sinica (Series B)
30: 503-506. 1987.
[0242] 5. In Ref. 2, p. 7.4.18 Table 7.4.2.
[0243] 6. EPICENTRE TECHNOLOGIES CATALOG, 1994/95 Products for
Molecular & Cellular Biology, Page 1, "What's new in this
catalog?"
[0244] 7. Okazaki, T. & Kornberg, A. J. Biol. Chem. 239:
259-268. 1964.
[0245] 8. Jacobsen, H., Klenow, H. & Overgard-Hansen, K. Eur.
J. Biochem. 45: 623-627. 1974.
[0246] 9. McClary, J., Ye, S. Y., Hong, G. F. & Witney, F. DNA
Sequence 1: 173-180. 1991.
[0247] 10. Mead, D. A., McClary, J. A., Luckey, J. A., et Al.
BioTechniques 11: 76-87. 1991.
[0248] 11. Earley, J. J., Kuivaniemi, H. Prockop, D. J. &
Tromp, G. BioTechniques 17: 156-165,1994.
[0249] 12. Mardis, E. R. & Bruce, A. R. BioTechniques 7:
840-850. 1989.
[0250] 13. Chissoe, S. L. et al., Methods: A Companion to Methods
in Enzymology, 3, 555-65, 1991
[0251] 14. Kunkel, T. A. et al., Methods Enzymol. 154:367-382,
1987
[0252] All references mentioned herein are incorporated in their
entirety by reference.
Sequence CWU 1
1
16 1 1764 DNA Bacillus stearothermophilus 1 gccgaagggg agaaaccgct
tgaggagatg gagtttgcca tcgttgacgt cattaccgaa 60 gagatgcttg
ccgacaaggc agcgcttgtc gttgaggtga 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 cttgaacaga
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 cttggcaaac tgcaatcaac gtatattgaa
900 ggattgttga aagttgtgcg ccctgatacc ggcaaagtgc atacgatgtt
caaccaagcg 960 ctgacgcaaa ctgggcggct cagctcggcc gagccgaact
tgcaaaacat tccgattcgg 1020 ctcgaagagg ggcggaaaat ccgccaagcg
ttcgtcccgt cagagccgga ctggctcatt 1080 ttcgccgccg attactcaca
aattgaattg cgcgtcctcg cccatatcgc cgatgacgac 1140 aatctaattg
aagcgttcca acgcgatttg gatattcaca caaaaacggc gatggacatt 1200
ttccagttga gcgaagagga agtcacggcc aacatgcgcc gccaggcaaa ggccgttaac
1260 ttcggtatcg tttacggaat tagcgattac ggattggcgc aaaacttgaa
cattacgcgc 1320 aaagaagctg ccgaatttat cgaacgttac ttcgccagct
ttccgggcgt aaagcagtat 1380 atggaaaaca tagtgcaaga agcgaaacag
aaaggatatg tgacaacgct gttgcatcgg 1440 cgccgctatt tgcctgatat
tacaagccgc aatttcaacg tccgcagttt tgcagagcgg 1500 acggccatga
acacgccaat tcaaggaagc gccgctgaca ttattaaaaa agcgatgatt 1560
gatttagcgg cacggctgaa agaagagcag cttcaggctc gtcttttgct gcaagtgcat
1620 gacgagctca ttttggaagc gccaaaagag gaaattgagc gattatgtga
gcttgttccg 1680 gaagtgatgg agcaggccgt tacgctccgc gtgccgctga
aagtcgacta ccattacggc 1740 ccaacatggt atgatgccaa ataa 1764 2 587
PRT Bacillus stearothermophilus 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 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 Gly Lys Val His Thr Met Phe Asn Gln Ala 305 310
315 320 Leu Thr Gln Thr Gly Arg Leu Ser Ser Ala Glu Pro Asn Leu Gln
Asn 325 330 335 Ile Pro Ile Arg Leu Glu Glu Gly Arg Lys Ile Arg Gln
Ala Phe Val 340 345 350 Pro Ser Glu Pro Asp Trp Leu Ile Phe Ala Ala
Asp Tyr Ser Gln Ile 355 360 365 Glu Leu Arg Val Leu Ala His Ile Ala
Asp Asp Asp Asn Leu Ile Glu 370 375 380 Ala Phe Gln Arg Asp Leu Asp
Ile His Thr Lys Thr Ala Met Asp Ile 385 390 395 400 Phe Gln Leu Ser
Glu Glu Glu Val Thr Ala Asn Met Arg Arg Gln Ala 405 410 415 Lys Ala
Val Asn Phe Gly Ile Val Tyr Gly Ile Ser Asp Tyr Gly Leu 420 425 430
Ala Gln Asn Leu Asn Ile Thr Arg Lys Glu Ala Ala Glu Phe Ile Glu 435
440 445 Arg Tyr Phe Ala Ser Phe Pro Gly Val Lys Gln Tyr Met Glu Asn
Ile 450 455 460 Val Gln Glu Ala Lys Gln Lys Gly Tyr Val Thr Thr Leu
Leu His Arg 465 470 475 480 Arg Arg Tyr Leu Pro Asp Ile Thr Ser Arg
Asn Phe Asn Val Arg Ser 485 490 495 Phe Ala Glu Arg Thr Ala Met Asn
Thr Pro Ile Gln Gly Ser Ala Ala 500 505 510 Asp Ile Ile Lys Lys Ala
Met Ile Asp Leu Ala Ala Arg Leu Lys Glu 515 520 525 Glu Gln Leu Gln
Ala Arg Leu Leu Leu Gln Val His Asp Glu Leu Ile 530 535 540 Leu Glu
Ala Pro Lys Glu Glu Ile Glu Arg Leu Cys Glu Leu Val Pro 545 550 555
560 Glu Val Met Glu Gln Ala Val Thr Leu Arg Val Pro Leu Lys Val Asp
565 570 575 Tyr His Tyr Gly Pro Thr Trp Tyr Asp Ala Lys 580 585 3
1764 DNA Bacillus stearothermophilus 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 ttgcgcgtcc 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 4 588
PRT Bacillus stearothermophilus 4 Met Ala Glu Gly Glu Lys Pro Leu
Glu Glu Met Glu Phe Ala Ile Val 1 5 10 15 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 115 120 125 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 Ser 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 Val 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
315 320 Ala Leu Thr Gln Thr Gly Arg Leu Ser Ser Ala Glu Pro Asn Leu
Gln 325 330 335 Asn Ile 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
Ala 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 Ala 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 Glu 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
5 17 DNA Bacillus stearothermophilus 5 cattttgctg ccggtca 17 6 20
DNA Bacillus stearothermophilus 6 gtaaaacgac ggccagtctt 20 7 20 DNA
Bacillus stearothermophilus 7 gtaaaacgac ggccagtcgg 20 8 20 DNA
Bacillus stearothermophilus 8 cattttgctg ccggtcagaa 20 9 26 DNA
Bacillus stearothermophilus 9 gccgttaact acggtatcgt ttacgg 26 10 36
DNA Bacillus stearothermophilus 10 cattccgatt cggaccccac tggggcggaa
aatccg 36 11 24 DNA Bacillus stearothermophilus 11 sssssstgta
aaacgacggc cagt 24 12 743 DNA Unknown Organism Description of
Unknown Organism Template DNA sequence 12 ctcactatag ggcgaattcg
agctcggtac ccggggatcc tctagagtcg acctgcaggc 60 atgcaagctt
gagtattcta tagtgtcacc taaatagctt ggcgtaatca tggtcatagc 120
tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
180 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt
gcgttgcgct 240 cactgcccgc tttgcagtcg ggaaacctgt cgtgccagct
gcattaatga atcggccaac 300 gcgcggggag aggcggtttg cgtattgggc
gctcttccgc ttcctcgctc actgactcgc 360 tgcgctcggt cgttcggctg
cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt 420 tatccacaga
atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg 480
ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
540 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
ctataaagat 600 accaggcgtt tccccctgga agctccctcg tgcgctctcc
tgttccgaac cctgccgctt 660 accggatanc tgtccgcctt tttccttcgg
aaagcgtggc gtttctcata agctcacgtg 720 taggtattct cagttcggtt agc 743
13 657 DNA Unknown Organism Description of Unknown Organism
Template DNA sequence 13 atgcctgcag gtcgactcta gaggatcccc
gggtaccgag ctcgaattcg taatcatggt 60 catagctgtt tcctgtgtga
aattgttatc cgctcacaat tccacacaac atacgagccg 120 gaagcataaa
gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt 180
tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagcctgca ttaatgaatc
240 ggccaacgcg cggggagagg cggtttgcgt attgggcgcc agggtggttt
ttcttttcac 300 cagtgagacg ggcaacagct gattgccctt caccgcctgg
ccctgagaga gttgcagcaa 360 gcggtccacg ctggtttgcc ccagcaggcg
aaaatcctgt ttgatggtgg ttccgaaatc 420 ggcaaaatcc cttataaatc
aaaagaatag cccgagatag ggttgagtgt tgttccagtt 480 tggaacaaga
gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc 540
tatcagggcg atggccacta cgtgaaccat cacccaaatc aagttttttg gggtcgaggt
600 gccgtaaagc nctaaatngg anncctaaag ggagcccccg atttagagct tgacggg
657 14 672 DNA Unknown Organism Description of Unknown Organism
Template DNA sequence 14 ttgatatgnt catatagggg gtttcgngtc
ggtaccgggg ntcctctagn gtcgnctgna 60 ggnatgcaag nttgngtatt
ctatagtgtc anctaaatag nttggngtaa tcatggtcat 120 agntgtttcc
tgtgtgaaat tgttatccgn tcacaattcc acanaaaata cgngncggnn 180
gnataaagtg taaagcctgg ggtgnctaat gngtgngtta antcacatta attgngttgn
240 gntcaatgnc cgntttccag tcgggnaacc tgtcgtgnca gntgnattaa
tggttcggcc 300 aacgngcggg gngnggnggt ttgggtattg ggngntcttc
cgnttcctcg ntcantgatt 360 cgttgngntc ggtcgttcgg ntgnggngng
nggtatcaga tcantcaaag ggggtaatac 420 ggttatccac agaatcaggg
ggtaanggag gtaaggacat gtggggnaaa agggcagcaa 480 aagggcaggn
accgtaaaaa ggccggttgg ttggggtttt tccatagggt ccgcccccct 540
gggggggatc aaaaaaaatc cgnggccaag tcaaggggtg gggggacccn ccagggntta
600 taaaggtacc aggggttccc cctgggagtc cctccgtggg tctcctgtcc
gccctgcccg 660 ttacccggta ct 672 15 686 DNA Unknown Organism
Description of Unknown Organism Template DNA sequence 15 acgagctcga
attcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc 60
acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgccaatgag
120 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
ggaaacctgt 180 cgtgccagct gcattaatga atcggccaac gcgcggggag
aggcggtttg cgtattgggc 240 gccagggtgg tttttctttt caccagtgag
acgggcaaca gctggattgc ccttcaccgc 300 ctggccctga gagagttgca
gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc 360 ctgtttgatg
gtggttccga aatcggcaaa atcccttata aatcaaaaga ataggccgag 420
atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc
480 aacgtcaaag ggcgaaaaac cgtctatcag ggcgatgcca ctacgtgaac
catcacccaa 540 atcaagtttt ttggggtcga ngttgccgta aagcattaaa
tcgggaacct aaagggagcc 600 ccgatttaga gcttgagggg gaaagccggc
gaacgtgggc gagaaaaagg aggggnagaa 660 accgaaagga gcggcctnan gncgng
686 16 673 DNA Unknown Organism Description of Unknown Organism
Template DNA sequence 16 ttgatatgnt catatagggg gtttcgngtc
ggtaccgggg ntcctctaga gtcgacctgc 60 aggcatgcaa gcttgagtat
tctatagtgt cacctaaata gcttggcgta atcatggtca 120 tagctgtttc
ctgtgtgaaa ttgttatccg
ctcacaattc cacacaacat acgagccgga 180 agcataaagt gtaaagcctg
gggtgcctaa tgagtgagct aactcacatt aattgcgttg 240 cgctcactgc
ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc 300
caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
360 tcgctgcgct cggtcgttcg gctgcggcgc gcggtatcag ctcactcaaa
ggcggtaata 420 cggttatcca cagaatcagg ggataacgga ggtaaggaca
tgtggggnaa aagggcagca 480 aaagggcagg naccgtaaaa aggccggttg
gttggggttt ttccataggg tccgcccccc 540 tgggggggat caaaaaaaat
ccgnggccaa gtcaaggggt ggggggaccc nccagggntt 600 ataaaggtac
caggggttcc ccctgggagt ccctccgtgg gtctcctgtc cgccctgccc 660
gttacccggt act 673
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