JPH09140376A - New bst-dna polymerase with proof reading 3'-5'-exonuclease activity - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject heat stable new enzyme derived from a new Bacillus sterothermophilus strain, having a proofreading 3'-5'-exonuclease activity and useful for determining a DNA sequence, etc.

SOLUTION: This new heat stable DNA polymerase can actuate proofreading 3'-5'-exonuclease on determining the sequence of a DNA chain from a template by functioning to cut a mismatched nucleotide from the 3'-terminal of the DNA chain with a speed faster than the speed with which a DNA polymerase separates a nucleotide correctly matched with the nucleotide of the template and at the same time does not exhibit 5'-3'-exonuclease activity. Thus, the DNA polymerase is useful for determining a DNA sequence, etc. Further, the DNA polymerase is produced by culturing a new Bacillus stearothermophilus strain (ATCC 55719) in a medium and subsequently extracting the DNA polymerase from the cells of the microbes.

COPYRIGHT: (C)1997,JPO

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a thermostable Bacillus.
us stearothermophilus DNA polymerase.
The present invention also relates to a Bacillus stearothermophilus strain capable of producing its thermostable DNA polymerase.

[0002]

2. Description of the Prior Art The genetic material of all known living organisms is
Deoxyribonucleic acid (DNA), except for certain viruses that use ribonucleic acid (RNA) as their genetic material. DN
A consists of a chain of individual deoxynucleotides that are chemically linked by a specific sequence. Each deoxynucleotide is one of four types of nitrogen-containing bases, which are adenine (A), cytosine (C), guanine (G) or thymine (T), and a hydroxyl group attached to its 3'position as a pentose and It contains deoxyribose with a phosphate group attached to its 5'position. Adjacent deoxynucleotides that form a DNA strand are linked to each other by a phosphodiester bond that connects the 5'position of one pentose ring to the 3'position of the next pentose ring. In this case, the starting point of the DNA molecule always has a phosphate group attached to the 5'carbon of deoxyribose. Last D
NA molecules are always OH on the 3'carbon of deoxyribose
It has a (hydroxyl) group.

DNA usually exists as a double-stranded molecule,
Two antiparallel DNA strands are held together by a hydrogen bond between each nucleotide base of the two DNA strands in a closely matched "A and T" and "C and G" pairing fashion ． It is the order or sequence of bases in the DNA chain that determines the gene and thus the type of protein synthesized. Therefore, accurate determination of the base sequence in the DNA chain that also constitutes the genetic code of the protein is fundamentally important for understanding the properties of the related proteins.

The method used to determine the sequence of bases in a DNA molecule is called DNA sequencing. DNA
Among the sequencing techniques, the enzymatic method developed by Sanger et al. (1) is the most common. This is DN
A polymerase uses primers that have been annealed to the DNA template to be sequenced to generate four normal deoxynucleotide triphosphates (dNTPs), namely dATP, d.
Ability to extend in the presence of CTP, dGTP and dTTP, and the nucleotide analogue dideoxynucleotide triphosphate (ddNTP), ie ddAT
It is based on the ability of P, ddCTP, ddGTP and ddTTP to terminate the extension of extended deoxynucleotide polymers at various lengths.

In the classical one-step Sanger method, sequencing
Is done in a set of four separate test tubes, each of which
One containing all four normal dNTPs
Only for position determination by autoradiography
Radioisotope³²P or ³⁵Labeled with an S
In addition, one restriction amount in four kinds of ddNTPs, DNA poly
Melase, primer and DNA casting for sequence determination
Contains the mold. Depending on the activity of DNA polymerase,
Of new nucleotides or nucleotide analogs
Added to the A chain, in this case all 3'ends of the primer
Starting in the 5'-3 'direction from the edge, each of the D
Nucleotide sequence complementary to phosphodiester bond
It connects to more adjacent nucleotides. Nuku in the reaction mixture
As long as the reotide analog is present, each tube will eventually
All called A, C, G or T terminators
Various newly formed lengths that terminate in specific ddNTPs
Contains a large number of DNA strands.

After the four sets of reaction products are separated by high resolution polyacrylamide / urea gel electrophoresis, the newly formed DNA chain population is separated and grouped according to their molecular weights. The autoradiographic images of the gels all have the same primers, and the specific ddNTP
The relative position of these DNA strands stopped at (A, C, G or T) will be shown as bands that differ from each other at distances measured by the length of each nucleotide. By reading the relative positions of these bands in the "ladder" of the autoradiograph, the DNA sequence of the template can be deduced.

The DNA polymerase used in the reaction mixture plays a central role in the sequencing analysis of DNA. To be useful for DNA sequencing, the DNA
The polymerase must have certain essential properties. For example, it must have its natural 5'-3 'exonuclease activity removed by post-translational modifications such as mutagenesis or enzymatic digestion, and does not show excessive discrimination against ddNTPs and is removed from the DNA strand. DNTPs and dd with sufficiently high continuous mobility and a sufficiently high extension rate to dictate the ability of the enzyme to continuously polymerize nucleotides on its chain without being disturbed
It must be able to import NTP. The 5'-3 'exonuclease activity associated with DNA polymerase removes nucleotides from the primer and, thus, the heterologous 5'of the newly formed DNA strand.
This will cause the ends to give false readings of chain length on the sequencing gel. In DNA polymerases with low continuous mobility and slow extension rates, the presence of DNA strands formed with incorrect lengths and improper terminations results in the production of many unwanted background bands of radioactivity. Among the commonly used DNA polymerases, Sequenase has higher continuous mobility and higher extension rate than other, such as Klenow fragment, Taq, and Vent polymerases (2) and is therefore currently It is by far the most common DNA polymerase of choice for DNA sequencing.

[0008]

However, even if it possesses all the essential properties listed above,
Radioactive erroneous or confusing band patterns can occur in sequencing gels. The pattern of these artifacts does not faithfully reflect the true nucleotide sequence of the template to be sequenced. These may occur at premature termination of the extended strand due to the presence of secondary structures formed along the template, such as "hairpins" in regions containing palindromic sequences or regions rich in G and C bases. Yes (3), and can occur as a result of improper "proofreading" function of DNA polymerases that allows the removal of nucleotides incorrectly introduced at the 3'end of the stretch.

Researchers in the field of DNA sequencing often have to use some approaches to confirm their findings in order to avoid errors due to these possible incorrect sequence data. For example, by repeating the same sequencing experiment with another DNA polymerase or performing another sequencing reaction with a template complementary to the first single-stranded DNA template and comparing the results for inconsistencies. An ideal DNA polymerase for enzymatic sequencing, that is, it not only has all the essential properties required for a sequencing reaction, but also contains a nucleotide that is non-complementary to the template to be sequenced by eliminating the secondary hairpin structure. Many researchers have attempted to find a DNA polymerase capable of preventing the formation of a continuous chain.

Large thermostable fragment of DNA polymerase isolated from Bacillus stearothermophilus , 25
The discovery by Ye & Hong (3) of an enzyme that has functionality in the temperature range of ℃ to 75 ℃, but has the highest activity at 65 ℃ and has all the essential properties for DNA sequencing is
Since these secondary structures were destabilized when carried out at 65 ° C, most of the problems caused by the secondary structure in the template were solved. Since this enzyme was marketed under the trade name of Bst DNA polymerase (Bio-Rad Laboratories),
Over the course of a year, all four dNTPs, including dCTP, and other nucleotide analogs such as dITP and 7-deaza-in the sequencing reaction catalyzed by this enzyme.
dGTP was equally efficiently incorporated within the chain extension, thus eliminating the "C" band phenomenon often seen when other DNA polymerases were used, and very good band homogeneity on sequencing gels. It has been confirmed by multiple independent reports. Moreover, this high temperature Bst DNA polymerase system
Sanger one-step reaction, as well as "labeling / termination" sequencing reaction, double-stranded DNA sequencing, and ³⁵ S
It has also been established that it can be used for both introduction of labeled nucleotides and ³² P-labelled nucleotides. Because this system can be stored at room temperature for at least 2 weeks without significant loss of its enzymatic activity, fluorescent dyes or radioisotope labels have been used to produce DNA requiring a stable DNA polymerase.
It has been applied to automation of sequencing.

One problem with Bst DNA polymerase is that it lacks 3'-5 'exonuclease activity (5), especially proofreading 3'-5' exonuclease activity. Examination of sequencing data collected from 14 research centers that have used Bst DNA polymerase for DNA sequencing studies of over 120 DNA clones revealed that base pair mismatches were statistically about 1.5 × 10 ^-5 . It was revealed that it would happen. That is, when polymerizing nucleotides catalyzed by this enzyme, about 1.5 times errors are expected for 100,000 nucleotide introductions.

In determining DNA sequences, it is common that the formation of incorrect DNA sequences due to mismatches between the template and the growing DNA strands can be prevented by the 3'-5 'exonuclease activity which "proofreads" the nucleotide strands. Known to. However,
Even if a DNA polymerase exhibits 3'-5 'exonuclease activity in vitro, it often does not properly "proofread".
That is, the polymerase cannot remove the mismatched nucleotides from the newly formed DNA strand as efficiently as it did with the nucleotides that matched the template nucleotides correctly. In other words, the 3'-5 'exonuclease cleaves the correctly matched nucleotide from the 3'end at a faster rate than the mismatched nucleotide, or both the correctly matched and mismatched nucleotides at the same rate. Do.
As a result, even if the DNA polymerase has 3'-5 'exonuclease activity, the useful proofreading function at the time of DNA polymerization is not exerted.

3'-5 'related to DNA polymerase
Exonuclease activity often counteracts the normal chain extension process catalyzed by polymerases in the presence of low concentrations of dNTPs and induces circular uptake and nucleotide degradation on the same segment of the template. , Or even function more efficiently than the polymerase activity itself to the extent that it causes degradation of the primer. Therefore,
Removal of the 3'-5 'exonuclease activity along with the 5'-3' exonuclease activity from the native DNA polymerase by chemical or genetic engineering techniques is standard in the production of DNA polymerases for sequencing. It is an operation. This is a common strategy that preserves the essential properties of DNA polymerases.

For example, the commercially available major sequencing enzymes [native Taq (Thermus aquati, which lacks 3'-5 'exonuclease activity from the beginning).
cus) except for DNA polymerase], 5'-3 '
Native T7 DNA lacking exonuclease activity
The 3'-5 'exonuclease activity from the polymerase oxidizes the amino acid residues essential for exonuclease activity (sequenase type 1) or by the genetic manipulation of the 28 amino acids essential for 3'-5' exonuclease activity. (Sequenase 2) has been removed by the deletion of.

Vent _R (ex is recommended as the preferred form for sequencing Vent DNA polymerase.
o ^-) DNA polymerase also its 3 '-5' exonuclease activity has been removed by genetic modification. Klenow fragment isolated from native Vent DNA polymerase and native E. coli DNA polymerase I have 3'-5 'exonuclease activity, but these enzymes are already considered as the enzymes of choice for DNA sequencing There is no.

Bst DNA for DNA sequencing isolated and purified from Bacillus stearothermophilus cells
The polymerase has no 3'-5 'exonuclease activity (5).

[0017] Epicentre Technologies (1402 Emil Stre
etTerm, Madison, WI 53713), a commercially available Bst DNA polymerase for DNA sequencing, IsoTerm® is also commercially available Bst DNA with 3'-5 'exonuclease activity removed enzymatically.
It is a polymerase.

DNAp of Bacillus stearothermophilus
Only the rBst DNA polymerase, which is a product of the olI gene and is produced from an overexpressed recombinant clone in Escherichia coli, has 3′-5 ′ exonuclease activity in addition to 5′-3 ′ exonuclease activity. However, due to the presence of undesired 5'-3 'exonuclease activity and uncharacterized 3'-5' exonuclease activity, the latter product is not recommended by the company for DNA sequencing (6). ．

[0019]

The present invention addresses the above-mentioned problems in the art by providing a novel Bst DNA polymerase that operates with proofreading 3'-5 'exonuclease activity. In the present invention, the term "proofreading" means that the Bst DNA polymerase removes the mismatched nucleotides from the 3'end of the newly formed DNA strand at the time of DNA sequencing, and the nucleotides that correctly match the template nucleotides. It means that it can be removed at a speed faster than that.

The present invention also provides an improved method for sequencing DNA using the Bst DNA polymerase described above.

The present invention is further isolated from various sources.
Bs in different strains of Bacillus stearothermophilus
It provides a test for screening tDNA polymerase.

Other objects and advantages of the present invention will be apparent from the following description and examples.

[0023]

BEST MODE FOR CARRYING OUT THE INVENTION The DNA polymerase of the present invention is B
It is extracted from acillus stearothermophilus or derived from Bacillus stearothermophilus by other methods. The Bst DNA polymerase of the present invention is D
The NA polymerase functions to excise the mismatched nucleotides from the 3'end of the DNA strand at a rate faster than the nucleotides that correctly match the template nucleotide are removed, and the proofreading 3'-
The 5'exonuclease activity is operable and this D
NA polymerase does not show 5'-3 'exonuclease activity.

The present invention also provides an improved method of DNA sequencing using the Bst DNA polymerase described above. This method follows the conventional protocol with the following modifications: i) hybridizing the primer to the DNA template to be sequenced, and ii) the thermostable Bacillus stearothermophilus DN described above.
Using A polymerase, the nucleotide base dATP,
Sequencing of the DNA with modification by extending the primer in the presence of dCTP, dGTP and dTTP or analogs thereof, and the ddNTP chain terminator, and iii) allowing sequencing of the DNA strand.

The Bst DNA polymerase of the present invention is
Proofreading 3'-for DNA sequencing
Obtained using methods conventional in the art to screen and identify bacterial DNA polymerases having 5'exonuclease activity. This polymerase is preferably a strain of Bacillus stearothermophilus proofreading 3'-5 'of their respective DNA polymerases.
Obtained by separation according to exonuclease activity.

[0026] As an example, the present inventors have shown that the cells of the Bst strain (numbered Bst 320 for classification purposes) have had their 5'-3 'exonuclease activity removed by subtilisin digestion from a thermostable DNA polymerase. Was successfully purified. This Bst strain is 199 in accordance with the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the purposes of Patent Procedure.
ATCC (Maryland, USA, October 30, 5
It has been deposited at Rockville, Park Lawn Street 12301) and is assigned the deposit number ATCC 55719. The resulting enzyme has proofreading 3'-5 'exonuclease activity that removes mismatched nucleotides from the growing DNA strand at a faster rate than would be the case for nucleotides that matched the template correctly. This new enzyme is distinguishable from Bst DNA polymerase, which lacks 3'-5 'exonuclease activity.

Similar to the known Bst DNA polymerase (4) which has been well characterized by one of the present inventors, the Bst DNA polymerase (large fragment) of the present invention has an apparent molecular weight of about 75,000 daltons and a 5'-
It has no 3'exonuclease activity. It is 25 ℃ ~
It functions at a temperature of 75 ° C and is most active at 65 ° C. Thus, interference with secondary structure associated with single-stranded DNA templates during DNA sequencing can be overcome.

All four dNTPs including dCTP
Are likewise efficiently incorporated into chain extension during sequencing reactions catalyzed by the Bst DNA polymerases of the invention, which have high continuity of movement and high extension rates. There is no "thin C band" phenomenon often seen when using other DNA polymerases. In addition, this enzyme is a nucleotide analogue such as ddNTP, d
ITP and 7-deaza dGTP can be incorporated without undue discrimination, resulting in very good band homogeneity on sequencing gels.

The DNA polymerase of the present invention has a classical
Sanger one-step reaction, as well as "labeling / termination" sequencing reaction, can be used for both the introduction of double-stranded DNA sequencing and ³⁵ S-labeled nucleotides, and ³² P-labeled nucleotides. Since this enzyme can be stored at room temperature for at least 2 weeks without significant loss of its enzymatic activity, fluorescent dyes or radioisotope labels have been used to require a stable DNA polymerase.
It can be applied to the automation of NA sequencing by a robot.

The present invention includes a novel method for measuring the proofreading 3'-5 'exonuclease activity of purified DNA polymerase. This method is useful for screening large numbers of Bst strains to select for strains producing DNA polymerases with high proofreading 3'-5 'exonuclease activity. For example, a method for testing the proofreading 3'-5 'exonuclease activity of DNA polymerase is performed as follows.

A DNA primer having the following sequence and two kinds of DNA templates were chemically synthesized using a DNA synthesizer.

Embedded image

For the production of radiolabeled primer, 1 μl
(1 μg) primer, 5 μl (50 μg) template (a), 1 μl [α- ³² P] dATP (800 Ci / mmol
e), 1 μl of dGTP (0.5 mM), 1 μl of TaqD
NA polymerase (1 unit), and 1 μl of buffer solution (composition: 500 mM Tris-Cl, pH 9.0, and 1
50 mM MgCl ₂ ) was mixed in a test tube and incubated in a 65 ° C. water bath for 5 minutes. The mixture was subjected to alkaline denaturing gel electrophoresis. A radioactive band containing 20-base nucleotides was isolated and 10 mM containing 1 mM EDTA
It was dissolved in Tris-Cl, pH 8.0, 12 μl. The final product is the 20-base primer below.

Embedded image

For the preparation of radiolabeled primer-template complex, 5 μl of labeled primer was mixed with 10 μl of template (a) or template (b) respectively to form the following complex.

Embedded image

Free radiolabeled primer is G-50 Sephad
It was removed through an ex column. 2 at the 3'end of the primer
An aliquot of the complex (a) with two correctly matched radiolabels A * and an aliquot of the complex (b) with two mismatched radiolabels A * at the 3'end of the primer. The radioactivity was measured by pipetting into two separate vials containing ## STR4 ## in a scintillation counter and both complexes were buffered to a concentration containing the same molar introduced [[alpha]-< ³² > P] dATP.

Proofreading 3'-5 'Exon
To examine creatase activity, 20 μl of complex (a) or
Or (b), 15 mM Tris-Cl, and 15 mM Mg
Cl _Two8 μl of reaction buffer of pH 8.5 consisting of 4 units
Make the DNA polymerase a total volume of 40 μl with water and pipette.
Then, it was taken into a test tube and mixed well. Add 3 μl of each mixture to each well.
Divide into recoats and transfer to 0.5 ml microcentrifuge tubes.
Each tube was covered with 3 μl of paraffin. 65 microcentrifuge tubes
Incubated in a water bath at ° C. 1, 2, 3, 5, 10 and
And 20 minutes later, remove a pair of microcentrifuge tubes from the water bath.
Spot contents of microcentrifuge tube on DE-81 Whatmann filter paper
did. One of the pair of filter papers is as it is, the scintillation
Scintillation coun
Counter, the cpm value is counted, and the other is 0.3M phosphorus.
After washing 3 times in sodium phosphate buffer pH 6.8,
It was counted in a chelation solution.

The difference in radioactivity expressed in cpm between the washed and unwashed filter papers of each pair is the relative amount of labeled nucleotides cleaved from the 3'end of the primer by the 3'-5 'exonuclease activity. Was taken to mean. The DNA polymerase cleaving the radiolabeled nucleotide A * from the complex (b) more efficiently than from the complex (a) has proofreading 3'-5 'exonuclease activity. DNA polymerase cleaved the radiolabeled nucleotide A * more rapidly from the complex (a) than from the complex (b), or at almost the same rate from the both, shows a nonspecific 3′-5 ′ exonuclease activity. It is considered to be unsuitable for DNA sequencing.

Using these methods, B from various sources
A Bst strain excellent in its fast growth rate was isolated from the strains of acillus stearothermophilus . This strain reaches the optimum logarithmic growth phase for producing DNA polymerase within 3 hours. This strain is also novel in its ability to produce a DNA polymerase with 3'-5 'exonuclease activity. This Bacillus stearothermophilus strain contains B
Labeled st No. 320.

As will be appreciated by those skilled in the art,
Bacil that provides the thermostable DNA polymerase of the present invention
The lus stearothermophilus strain is not limited to Bst No. 320. On the contrary, the polymerase could be derived from Bacillus stearothermophilus strains including wild-type strains or mutant strains obtained by various means including spontaneous mutations.

For the production of DNA polymerase, Bst N
o.320 cells were grown at 55 ° C in liquid medium pH 7.0-7.2 consisting of 1% polypeptone, 0.5% yeast extract and 0.5% NaCl. After centrifuging, 3 hours old cells were collected and used in a TME buffer solution (composition: 50 mM Tris-HCl, pH 7.5, 10 mM) containing 100 ml of lysozyme and 23 mg / ml of phenylmethylsulfonyl fluoride.
β-mercaptoethanol, and 2 mM EDTA)
It was suspended in 4 volumes. The cells were sonicated in ice to destroy. After centrifugation at 28,000 rpm with a Spinco L 30 rotor, the supernatant was pooled.

DNA polymerase is Okazaki & Kornberg
A large fragment of the DNA polymerase was prepared by slightly modifying the method of (7), and the whole DNA polymerase was partially digested with the proteinase subtilisin (Carlsberg type) basically according to Jacobsen (8). ．

Details of the enzyme purification procedure were previously published (4). This new Bst DNA polymerase has proofreading 3'-5 'exonuclease activity and was named HiFi Bst.

HiFi Bst was tested for proofreading and nonspecific 3'-5 'exonuclease activity as described above. The result is HiFiBs
When t reaches the plateau for hydrolysis, the nucleotide incorporated by mismatch from the 3'end of the double-stranded DNA reaches the plateau of hydrolysis in about 3 minutes, and is about 8 times more efficient in the initial 3 minutes of the reaction than in the case where the template nucleotide is correctly matched. It was shown to cut at a high speed (Fig. 1).

[0043]

The following non-limiting examples are illustrative of the present invention.

D of Bst DNA polymerase of the present invention
Example of Use for NA Sequencing The following example is based on the standard Sanger protocol for single-stranded DNA sequencing and the enzyme Bst DN of the invention as the enzyme.
Use A polymerase.

1. Sequence 5'-GTA in a 1.5 ml centrifuge tube.
DNA 2.5 with AAACGACGGCCAGT-3 '
Universal DNA sequence primer containing ~ 5 ng 1.
SsDNA containing 0 μl, 250-500 ng of DNA 7.0 μ
l and 5 mM reaction buffer consisting of 100 mM Tris-Cl, pH 8.5, and 100 mM MgCl ₂ 2.0 μ
I took a pipette. 2. The mixture is placed in a water bath at 75 ° C for 5 minutes, then 5
It took a minute to gradually cool to room temperature. 3. In a test tube, add 1.5 μl of [α- ³⁵ S] dATP (1000
Ci / mmole) and 20 mM K ₂ HPO ₄ , pH 6.8
1.0 unit of enzyme, 10 mM β-mercaptoethanol, 50
Bst DNA polymerase of the invention containing% glycerol was added. The contents were mixed and subjected to microcentrifugation for 2-3 seconds. 4. An aliquot of 2.5 μl of the above mixture was added to one of the following mixed solutions preheated to 65 ° C. 1.5 mM Tris-Cl, 0.15 mM EDT
A, pH 8.0, 620 nM dATP, 62 μM dC
TP, 62 μM dGTP, 62 μM dTTP, 25 μM ddATP, C.I. 1.5 mM Tris-Cl, 0.15 mM EDT
A, pH 8.0, 800 nM dATP, 8 μM dC
TP, 80 μM dGTP, 80 μM dTTP, 50 μM ddCTP, G.I. 1.5 mM Tris-Cl, 0.15 mM EDTA,
800 nM dATP, 80 μM dCT in pH 8.0
P, 4 μM dGTP, 80 μM dTTP, 75 μM d
dGTP or T.I. 1.5 mM Tris-Cl, 0.15 mM EDTA,
800 nM dATP, 80 μM dCT in pH 8.0
P, 80 μM dGTP, 8 μM dTTP, 150 μM ddTTP, 2.0 μl were pipetted into each of the four test tubes. 5. After mixing and microcentrifuging for 2-3 seconds, all four tubes labeled A, C, G and T, respectively, were placed in a 65 ° C water bath for 2 minutes (extension-termination reaction). 6. In each of the 4 test tubes, 4 kinds of dNTPs (dATP, dCTP, dGTP and dTT) dissolved in water were added.
P) 2 μl of chasing solution containing 0.5 mM each was added. After mixing, the tubes were microcentrifuged, placed in a 65 ° C water bath and incubated for an additional 2 minutes. 7. All reactions in four tubes were stopped by the addition of 4 μl of stop solution containing 95% deionized formamide, 10 mM EDTA, 0.05% xylene cyanol FF, and 0.05% bromophenol blue. The mixture was subjected to microcentrifugation, loaded on a denaturing high resolution polyacrylamide gel and subjected to electrophoresis. 8. An autoradiographic image of the sequencing gel was obtained (Fig. 2).

Further testing of the Bst DNA polymerase of the present invention revealed that DN catalyzed by this enzyme was
The rate of incorporation of mismatch base pairs during A polymerization is
It was clarified that the order was 7.8 × 10 ^-7 . This is an almost 20-fold improvement in accuracy over the previously recorded error rate of about 1.5 × 10 ⁻⁵ for normal Bst DNA polymerase.

Furthermore, the Bst DNA polymerase of the present invention retains all the other advantages of normal Bst. It functions within a temperature range of 25 ° C to 75 ° C and is most active at 65 ° C. All four dNTs including dCTP
P, as well as other nucleotide analogs such as dIT
P and 7-deaza dGTP are similarly efficiently incorporated into chain elongation (hence no thin "C" band phenomenon) resulting in excellent band homogeneity on sequencing gels (Figure 2).

The DNA polymerase of the present invention has a classical
Sanger one-step reaction, as well as "labeling / termination" sequencing reaction, double-stranded DNA sequencing, and ³⁵ S
It can be used for both the introduction of labeled nucleotides and ³² P-labeled nucleotides (9). It should be used for rapid sequencing analysis using nanogram quantities of template (10) and for automation of DNA sequencing requiring stable DNA polymerases (11) with fluorescent dyes or radioisotope labels (12) Is possible.

All cited references cited here are incorporated by reference in their entirety. References 1. Sanger, F., Nicklen, S. & Coulson, AR, Proc.
Nat.Acad.Sci.USA 74: 5463-5467, 1977. 2. Current Protocols in Molecular Biology, Ausube
l, FM et al., Volume I, John Wiley & Sons, Inc. 1995,
Pages 7.4.17-7.4.24. 3. Ibid, page 7.4.31. 4. Ye, SY & Hong, GF, Scientia Sinica (Series
B) 30: 503-506, 1987. 5. Cited document 2, page 7.4.18, table 7.4.2. 6. Epicentre Technologies Catalog 1994/1995, Pr
oducts for Molecular & Cellular Biology, page 1, "Wha
t's new in this Catalog? ". 7. Okazaki, T. & Kornberg, AJ, J. Biol. Chem. 23
9: 259-268, 1964. 8. Jacobsen, H., Klenow, H. & Overgard-Hansen,
K., Eur. J. Biochem. 45: 623-627, 1974. 9. McClary, J., Ye, SY, Hong, GF & Witney,
F., DNA Sequence 1: 173-180, 1991. 10. Mead, DA, McClary, JA, Luckey, JA et al., Bi
oTechniques 11: 76-87, 1991. 11. Earley, JJ, Kuivaniemi, H., Prockop, DJ &
Tromp, G., BioTechniques 17: 156-165, 1994. 12. Mardis, ER & Bruce, AR, BioTechniques 7:84
0-850, 1989.

[Brief description of the drawings]

FIG. 1: HiFi Bst DNA of mismatched and correctly matched incorporated radiolabeled nucleotides.
Graph showing CPM (counts / minute) of excision from the 3'end by polymerase.

FIG. 2: Radioautograph of sequencing gel after electrophoresis on denaturing high resolution polyacrylamide gel obtained with Bst DNA polymerase of the present invention. The sequence pattern of the separated and uniformly labeled bands can be clearly seen.

Continuation of front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display part (C12N 1/20 C12R 1:07) (C12N 9/12 C12R 1:07) (C12N 15/09 ZNA C12R 1 : 07) (72) Inventor Feng Tsai, People's Republic of China Shanghai, Shanghai Institute of Biochemistry (no address)

Claims (6)

[Claims] 1. When sequencing a DNA strand from a template, D
The mismatched nucleotide from the 3'end of the NA chain is
The proofreading 3'- functions so that the DNA polymerase excises at a rate faster than the rate at which it removes nucleotides that match the template nucleotide correctly.
Bac capable of producing a thermostable DNA polymerase capable of activating 5'exonuclease activity and exhibiting no 5'-3 'exonuclease activity
illus stearothermophilus strain. 2. Bacillus stearothermo according to claim 1
philus strain thermostable DNA polymerase. 3. Bacil of claim 1 which is Bst 320
lus stearothermophilus strain. 4. When sequencing a DNA strand from a template, D
The mismatched nucleotide from the 3'end of the NA chain is
The proofreading 3'- functions so that the DNA polymerase excises at a rate faster than the rate at which it removes nucleotides that match the template nucleotide correctly.
A thermostable Bacillus stearothermophilus DNA polymerase capable of activating 5'exonuclease activity and exhibiting no 5'-3 'exonuclease activity. 5. The DNA polymerase of claim 4 having a molecular weight of about 75,000 daltons. 6. A method for determining the sequence of a DNA strand, comprising the steps of: i) hybridizing a primer to a DNA template; and ii) a nucleotide mismatched from the 3'end of the DNA strand is correctly matched with the template nucleotide by a DNA polymerase. Thermostable, capable of activating proofreading 3'-5 'exonuclease activity by acting to excise at a rate faster than the function of removing nucleotides, and showing no 5'-3' exonuclease activity The nucleotide bases dATP, d using the active Bacillus stearothermophilus DNA polymerase.
GTP, dCTP and dTTP or their analogs, and a step of extending a primer in the presence of a ddNTP chain terminator, and iii) performing a DNA strand sequencing.