JPH07313198A - Improved method for determination of base sequence of dna - Google Patents

Abstract

PURPOSE:To accurately determine the base sequence of a DNA with low noise by modifying and hybridizing a target DNA and a primer, treating the hybridized product with unlabeled and labeled modified nocleotide triphosphates and a nucleic acid synthetase and detecting the product. CONSTITUTION:A DNA for determining the sequence is thermally modified together with a primer to hybridize the DNA with the primer. The hybridized product is subjected to the chain extension reaction and a DNA reaction accompanying a non-specific chain-interruption reaction and a specific chain interruption reaction using an unmodified nocleotide triphosphate, a labeled modified nocleotide triphosphate derivative expressed by the formula (Q is 7-deazaadenine, 7-deazaguanine, cytosine or uracil; R<1> to R<4> each is H, sodium or lithium; sodium and lithium are not present at the same time; A is a label) and a nucleic acid synthetase. The DNA fragment formed by the specific chain-interruption reaction is non-electrically detected to determine the base sequence of the DNA with reduced noise.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to an improved non-RI labeled nucleotide sequencing method for DNA which does not use radioisotopes. More specifically, a novel DN in which a labeling substance necessary for detecting DNA is added to dideoxynucleotide triphosphate
This is a sequencing method using an A probe, which reduces noise in determining the base sequence of DNA, that is, improves accuracy.

[0002]

2. Description of the Related Art Nucleotide sequence analysis of DNA is a fundamental technique in molecular biology. There are two basic techniques as conventional DNA sequencing methods. One is the Maxam-Gilbert method (Methods En
zymology, Vol. 65, 1980, pp. 499-560), the other is the Sanger method (Proc. Natl. Acad. Sci., USA,
Vol.74, 1977, p.5463).

In recent years, most of the Sanger sequencing methods have been used for DNA sequencing, in which an oligonucleotide primer complementary to a portion of the DNA strand to be sequenced is used. Complementary copies of the DNA to be sequenced are synthesized by DNA polymerase in the presence of a mixture of four deoxynucleotide triphosphates and one dideoxynucleotide triphosphate. By incorporating the dideoxynucleotide triphosphate in newly synthesized DNA,
The terminal nucleotide hydroxyl groups of the extended nucleotide are gone, synthetic extension is no longer possible, and interruptions occur in sequence-specific synthesis. This extension generally performs separate reactions on all four possible mixtures of four deoxynucleotide triphosphates and one dideoxynucleotide triphosphate.

Subsequently, the length of the generated DNA fragment is separated by gel electrophoresis, and the separated DNA fragment is detected, whereby the DNA sequence can be directly read. To detect a DNA fragment, a part of an oligonucleotide primer, or at least one kind of deoxynucleotide, or four kinds of dideoxynucleotide is bound to a label, for example, a radioisotope, a fluorescent substance or an enzyme, and the label is detected. By doing.

At least the following steps are carried out in the most commonly used nucleotide sequencing method at present. First step: DNA to be sequenced and its complementary DNA are separated into single strands by leaving them at high temperature for several tens of seconds to several minutes. Second step: Then, the temperature is gradually lowered to allow the oligonucleotide primer to bind to the DNA to be sequenced (annealing). Third step: heating the DNA polymerase to an optimum temperature,
After extending the oligonucleotide primer along the template, a termination reaction is performed. Fourth step: Separate the DNA to be sequenced by leaving the extended oligonucleotide primer at high temperature for tens of seconds to several minutes. The oligonucleotide primer extended by electrophoresis is fractionated into sizes, and the nucleotide sequence is determined by detecting the labeling substance pre-labeled on the oligonucleotide primer.

When detecting a DNA fragment, it is not the best method to use a radioactive isotope in consideration of the influence on the human body and waste disposal. On the other hand, the fluorescent substance labeling method has an advantage that it can be detected safely and with high sensitivity by irradiating an appropriate excitation light (laser light).
However, all of these technologies require considerable investment in terms of facilities and equipment. Especially when processing a large number of samples, the processing capacity is limited depending on the scale of the equipment. There is a need for a DNA sequencing method that does not require special equipment or equipment, and several reports have been made (Nuclei
c Acids Research Vol.17, No.13, 1989, 5115-5123). One of these techniques is a method of determining a DNA sequence by the Sanger method using a biotin-labeled primer. Since biotin cannot be detected directly, it is detected indirectly. For example biotin-
It has also been reported that an avidin system indirectly labels a DNA fragment with an enzyme such as alkaline phosphatase or peroxidase (Anal. Biochem. V
ol.164, 512-520). By adding a substrate which is decomposed by these enzymes to develop color and emit light, such as 5-bromo-4-chloro-3-indolyl-phosphate and nitrobluetetrazolium, a case where a radioisotope or a fluorescent substance is used is used. It becomes possible to detect a DNA fragment with the same sensitivity.

[0007]

However, in the non-RI-labeled sequencing method using these biotin, dicoxigenin, enzyme or fluorescent dye, the non-RI-labeled sequencing method is apt to complicate the DNA labeling method and causes noise. There is a drawback that occurs. Therefore, the problem to be solved by the present invention is to solve the problem of DN of a labeling substance necessary for detection.
A novel method of adding to A is to reduce noise, that is, improve accuracy when determining the base sequence of DNA.

[0008]

That is, the present invention is an improved DNA nucleotide sequencing method comprising the following steps. a) a step of heat denaturing the DNA whose sequence is to be determined and a primer b) a step of hybridizing the above-mentioned primer to the DNA whose sequence is to be determined c) an unmodified nucleotide triphosphate, a labeling modification represented by the following chemical formula 2 Using a nucleotide triphosphate derivative and a nucleic acid synthase, a DNA synthesis step involving an elongation reaction, a non-specific chain-interruption reaction and a specific chain-interruption reaction d) Only a DNA fragment produced by the specific chain-interruption reaction is non-electrically Process to detect

[0009]

[Chemical 2] (In the formula, Q is a 7-deazaadenine, 7-deazaguanine, cytosine or uracil residue, and R ¹ , R ² ,
R ³ and R ⁴ each independently represent a hydrogen atom, a sodium atom or a lithium atom. However, sodium atom and lithium atom do not exist at the same time. A indicates a label. )

The DNA to be sequenced for use in the present invention includes, for example, M13 phage DNA-double-stranded DNA, pU
Double-stranded DN existing in a supercoiled form such as CDNA
Any DNA incorporated into A.

The primer used in the present invention is an oligonucleotide which is complementary to a part of the DNA strand to be sequenced. The number of oligonucleotides is generally 10-50.

There are two ways to use the primer.
One is a method of immobilizing a primer (deletion method). This is a method in which the sample DNA to be sequenced is ligated to a known DNA fragment, and the sample DNA is analyzed using a primer complementary to the fragment part of the known DNA. Therefore, some primers are labeled with biotin in advance. On the other hand, a primer complementary to a part of the sample DNA was used for analysis, and then a primer of a part several hundred bases apart was synthesized. This is a method in which another primer is synthesized and the analysis is sequentially advanced, that is, a primer complementary to the sample DNA is synthesized one after another (primer walking method).

The former requires less primers, but the size of the fragment that can be ligated is limited, so the sample is temporarily decomposed and reconstituted for analysis (necessary because the sample is decomposed at random). Requires labor. The latter requires a large number of primers, but the analysis results are always continuous because it is not necessary to decompose the sample finely. D
With the spread of NA automatic synthesizers and their high performance, the burden on primer synthesis is decreasing. Therefore, the disadvantages of using the primer walking method are decreasing.

The primer walking method for labeling a primer with biotin is a safe and reliable DNA analysis method, but sequential labeling of necessary primers with biotin is very wasteful and results in impairing the advantages of this method. Has become. In the present invention, such a defect is eliminated by using the labeled modified nucleotide triphosphate derivative represented by the above chemical formula 2.

The nucleic acid synthase used in the present invention includes
Tth DNA polymerase derived from Thermus thermophilus, Taq DNA polymerase derived from Thermus aquaticus, Pfu derived from hyperthermophilic Pyrococcus furiosus
Modification of thermostable DNA polymerase such as DNA polymerase, DNA polymerase lacking 5 '→ 3' exonuclease, DNA polymerase lacking 3 '→ 5' exonuclease, Klenow fragment of Escherichia coli polymerase I, T7 DNA polymerase Type DN
A polymerase etc. can be mentioned. Of these nucleic acid synthases, thermostable DNA polymerase is preferable, and thermostable DNA polymerase lacking 5 ′ → 3 ′ exonuclease and thermostable DNA polymerase lacking 3 ′ → 5 ′ exonuclease, for example, ΔTt
hDNA polymerase (manufactured by Toyobo) and ΔTaq DNA
Polymerase (United States Biochemical) is particularly preferable.

The labeled modified nucleotide triphosphate derivative represented by Chemical formula 2 used in the present invention is, for example, 7- (N-biotinyl- (3-amino-1-propynyl))-2'3'-.
Dideoxy-7-deazaguanosine-5'-triphosphate (the following chemical formula 3), 7- (N-biotinyl- (3-amino-
1-propynyl))-2'3'-dideoxy-7-deazaadenosine-5'-triphosphate (the following chemical formula 4), 5- (N
-Biotinyl- (3-amino-1-propynyl))-
2'3'-dideoxy-uridine-5'-triphosphate (the following chemical formula 5) or 5- (N-biotinyl- (3-amino-
1-propynyl))-2'3'-dideoxy-cytidine-5'-triphosphoric acid (Chemical Formula 6 below). These compounds were purchased from Organic Synthetic Chemical Industry Co., Ltd.

[0017]

[Chemical 3]

[0018]

[Chemical 4]

[0019]

[Chemical 5]

[0020]

[Chemical 6]

The label may be biotin, dicoxygenin, an enzyme or a fluorescent dye. The label forms a complex with a substance that produces a signal and produces a signal. When the label is biotin, the substances that give rise to signals are avidin and alkaline phosphatase, or avidin,
Such as biotin and alkaline phosphatase. When the label is a fluorescent substance, the substances that give rise to the signals include anti-fluorescent dyes and alkaline phosphatase. When the label is dicoxygenin, signal-giving substances include anti-dicoxygenin and alkaline phosphatase. When the label is alkaline phosphatase, the signal-producing substance is, for example, a derivative of 1,2 dioxetane. When the label is alkaline phosphatase, signal producing agents include 5-bromo-4-chloro-3-indolyl-phosphate and nitroblue tetrazolium.
The labeling method in the present invention is not limited to the use of the above compound.

There are three types of DNA labeling methods: the 5'end of the primer (end labeling method), the middle of the DNA fragment (internal labeling method), and the 3'end of the DNA fragment (terminator labeling method). .
In the present invention, the determination of the base sequence of DNA is achieved by generating a signal from any of the labeling substances and detecting the signal. The non-electrical detection in the present invention means
Rather than electrically amplifying and detecting the signal generated by the photon counter or liquid scintillation counter, it means to detect the signal optically by exposing it to a color or a film so that it can be visually confirmed. To do.

The accuracy of DNA sequencing is as follows:
Since a DNA fragment having a specific length is produced by a nucleic acid synthesizing enzyme, each molecule is accurately separated by a separation simple substance such as a denaturing polyacrylamide gel. The accuracy when using the end-label or internal-label method depends on the high substrate specificity of the nucleic acid synthase and the synthesized DNA.
Depends on the low decomposition activity of. In the present invention, 5'-3 '
And / or by using a nucleic acid synthase completely lacking the 3'-5 'degrading activity, the degrading activity of DNA can be eliminated. On the other hand, the substrate specificity is 100
A nucleic acid synthase having a% (so-called misreading) has not been discovered and causes noise. However, in the present invention, by labeling with dideoxynucleotide triphosphate, the frequency of this noise generation is maximized. Is decreasing. In other words, due to the misreading of deoxynucleotide triphosphate and noise due to the termination of the reaction due to the characteristics (purity / structure) of the DNA whose sequence should be determined, accurate synthesis was performed even when the enzyme was inaccurate and there were many errors. DN
Only A can be detected.

The terminator labeling method using biotin is particularly suitable for efficient and efficient DNA sequencing without special facilities and equipment. Furthermore, in order to reduce noise (improve accuracy), the biotin terminator method in which a labeling substance is added after a DNA fragment having a specific length is generated is suitable. More specifically, a sample DNA to be sequenced, a primer, a reaction buffer, four kinds of deoxyribonucleic acid and a DNA polymerase are mixed, and further, biotin-modified dideoxy-7-deazanucleotide-5'-3 'is added. Using phosphoric acid or the like, DNA synthesis involving elongation reaction, non-specific chain-interruption reaction and specific chain-interruption reaction is carried out to carry out the termination reaction. This termination reaction needs to be performed separately for all four types of dideoxyribonucleic acid labeled with biotin.

Dideoxynucleotide triphosphate is an analog in which deoxynucleotide triphosphate is hydrolyzed to reduce one oxygen atom. However, most DNA
Polymerases have poor uptake of this analog and are used at high concentrations during the reaction. In the case of dideoxyribonucleic acid labeled with biotin, uptake by DNA polymerase is particularly decreased due to an increase in molecular weight and a change in structure.

However, in the present invention, a DNA polymerase which is less susceptible to increase in molecular weight and change in structure is used, and the following steps are further carried out. a) a step of heat denaturing the DNA to be sequenced and the primer b) a step of hybridizing the primer to the DNA to be sequenced c) an unmodified nucleotide triphosphate, a labeling modification shown in the above chemical formula 2 Using a nucleotide triphosphate derivative and a nucleic acid synthase, a DNA synthesis step involving an elongation reaction, a non-specific chain-interruption reaction and a specific chain-interruption reaction d) Only a DNA fragment produced by the specific chain-interruption reaction is non-electrically Process to detect

Steps a) to c) are repeated several times, preferably 5 to
By repeating 60 times, it is possible to increase the sensitivity by increasing the chance that the labeled dideoxyribonucleic acid is taken up by the nucleic acid synthase.
Further, according to the method of the present invention, noise can be reduced because DNA for which the nucleic acid synthase has failed in the specific chain interruption reaction is not detected. Further, the detection does not involve electrical detection.

[0028]

INDUSTRIAL APPLICABILITY According to the present invention, by using a novel DNA probe in which a labeling substance necessary for detecting DNA is added to dideoxine nucleotide triphosphate,
Noise in the determination of the nucleotide sequence of, that is, the accuracy is improved. Further, the base sequence of DNA can be determined without using radioisotopes or special equipment. In the present invention, it is possible to reduce the decrease due to incorporation by DNA polymerase when using biotin as a label.

[0029]

EXAMPLES The present invention will be specifically described below with reference to Examples and Comparative Examples. Comparative Example 1 (Result 1) In the primer labeling method using T7 DNA polymerase
Sequencing reaction by M13mp18 DNA 50 fmol, biotin labeled universal primer (5'-Biotin-CGCC
GCCAGGGTTTTCCCAGTCACGAC-
3 ′) 1 pmole was added to the reaction buffer (40 mM Tri
s-HCl (pH 7.5), 20 mM MgCl ₂ , 5
It was dissolved in 10 μl of 0 mM NaCl). After heating at 65 ° C. for 2 minutes, the template and the primer were hybridized by returning to room temperature over 30 minutes. Then 0.1M
1 μl of dithiothreitol and 1UT7 DNA polymerase were added to make the total volume 15.5 μl. 3.5 μl of this mixed solution was dispensed into 4 tubes, and 80 μM each of deoxynucleotide triphosphate and 8 μM was placed in the first tube.
M 2′-dideoxyguanosine-5′-triphosphate 2.
5 μl, 80 μM each deoxynucleotide triphosphate, 8 μM 2′-dideoxyadenosine-5′-triphosphate 2.5 μl in the second tube, 80 μM each deoxynucleotide triphosphate, 8 μM 2 ′ in the third tube
2.5 μl of dideoxycytidine-5′-triphosphate, to the fourth tube 80 μM each deoxynucleotide triphosphate, 8 μM 2′-dideoxythymidine-5′-triphosphate 2.5 μl.

The reaction mixture was reacted at 37 ° C. for 5 minutes. 4 μl of a reaction stop solution (95% formamide, 20 mM sodium ethylenediaminetetraacetate, 0.05% bromphenol blue, 0.05% xylene cyanol) was added to each of the first, second, third, and fourth tubes. After heating at 80 ° C. for 2 minutes, it was rapidly cooled on ice.

Example 1 (Result 2) Terminator lab using Tth DNA polymerase
Sequencing reaction by the method of M13mp18DNA 50 fmol, universal primer (5'-CGCCGCCAGGGTTTTCC
CAGTCACGAC-3 ') 1 pmole, 200μ
M deoxynucleotide triphosphate 1 μl each, reaction buffer (10 mM Tris-HCl (pH 8.8), 10 m
3 μl of M KCl, 1.5 mM MgCl ₂ , 7 mM 2-mercaptoethanol, 5 μg / ml bovine serum albumin, and 4 units of Tth DNA polymerase (Toyobo) were mixed to make a volume of 17 μl. This mixed solution was dispensed into 4 tubes by 4 μl, and 1.5 mM 7- (N-biotinyl- (3-amino-1-propynyl))-2′3′-dideoxy-7 was placed in the first tube. −
2 μl of deazaguanosine-5′-triphosphate (Chemical Formula 3), second
Tube containing 1.5 mM 7- (N-biotinyl-
(3-Amino-1-propynyl))-2'3'-dideoxy-7-deazaadenosine-5'-triphosphate (Formula 4) 2
μl, 1.5 mM 5- (N-biotinyl- (3-amino-1-propynyl))-2′3 ′ in the third tube.
-Dideoxyuridine-5'-triphosphoric acid (Chemical formula 5) 2 μl,
To the fourth tube was added 2 μl of 1.5 mM 5- (N-biotinyl- (3-amino-1-propynyl))-2′3′-dideoxycytidine-5′-triphosphate (Chemical Formula 6).

The reaction mixture was coated with 20 μl of mineral oil,
Temperature programmable block (Thermal cycler PJ
-500, Perkin Elmer Cetus) at 95 ° C for 30 seconds; 60
30 ° C. for 30 seconds; and 72 ° C. for 1 minute
I went there. 4 μl of the reaction stop solution (95% formamide, 20 mM sodium ethylenediaminetetraacetate, 0.05% bromphenol blue, 0.05% xylene cyanol) was added to each of the first, second, third, and fourth tubes. After heating at 80 ° C. for 2 minutes, it was rapidly cooled on ice.

Example 1 and Comparative Example 1 Modified Polyacrylamide Gel Electrophoresis Modified acrylamide gel having a width of 20 cm, a length of 40 cm and a thickness of 0.5 mm (8% acrylamide, 7M urea, 8
9 mM Tris-HCl (pH 8.7), 89 mM
Boric acid, 2 mM sodium ethylenediaminetetraacetate, 1
0% formamide) in Example 1 and Comparative Example 1 above.
2 μl of each of the first, second, third, and fourth reaction solutions were sequentially added, and electrophoresis was performed at 25 W for 2 hours.

Transfer to nylon membrane and DNA
Fragment detection Peel off the denatured acrylamide gel after electrophoresis on one glass plate and use nylon membrane (Biodyne B, PA
LL) was adhered and a load of 2.5 g / cm 2 was applied overnight to transfer the DNA fragment in the gel to a nylon membrane. The transferred nylon membrane was used as a blocking buffer (25 mM phosphate buffer (pH 7.
2), 125 mM sodium chloride, 5% sodium dodecyl sulfate) at a rate of 0.5 ml / square centimeter for 5 minutes. Subsequently, 0.5 ml of blocking buffer containing 1 μg / ml of streptavidin was added.
The treatment was performed at a rate of / cm 2 for 5 minutes. Subsequently, the blocking buffer solution having a concentration of 1/10 was treated at a rate of 1 ml / cm 2 for 3 minutes, and this operation was repeated twice. Subsequently, with a blocking buffer containing 50 ng / ml biotin and alkaline phosphatase,
It was treated at a rate of 0.5 ml / square centimeter for 5 minutes. Then, 1m with 1/10 concentration of blocking buffer
The treatment was performed at a rate of 1 / cm 2 for 3 minutes, and this operation was repeated twice. Then, wash solution (10 mM Tr
It was treated with is-HCl (pH 9.6), 10 mM sodium chloride, 1 mM magnesium chloride at a rate of 1 ml / cm 2 for 3 minutes, and this operation was repeated twice. Then, the luminescent liquid (0.75M 2-amino-2-
Methyl-1-propanol (pH 9.6), 1 mM magnesium chloride, 1 mM cetyltrimethylammonium bromide bromide, 20 ng / ml PPD) 0.5 m
After treatment for 5 minutes at a rate of 1 / cm <2> to remove the luminescent solution, the mixture was left at room temperature for 30 minutes. Subsequently, the nylon membrane treated in a dark room was brought into close contact with the X-ray film, exposed for 30 minutes and developed. The nucleotide sequence of the DNA was determined by analyzing the obtained DNA fragment pattern.

The results are shown in FIG. Result 1 in Figure 1
Is Comparative Example 1 (primer label + T7 DNA polymerase), and Result 2 is Example 1 (biotin terminator +
2 is a photograph of electrophoresis showing the result of Tth DNA polymerase). As is clear from FIG. 1, no noise is observed in the present invention, but a large amount of noise is observed in the comparative example.

[Brief description of drawings]

FIG. 1 is an electrophoretic photograph showing the results of sequencing reactions in the present invention and comparative examples.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Yoshihisa Kawamura 10-24 Toyomachi, Tsuruga City, Fukui Prefecture Toyobo Co., Ltd.

Claims (18)

[Claims] 1. An improved DNA nucleotide sequencing method comprising the following steps. a) a step of heat denaturing the DNA to be sequenced and the primer b) a step of hybridizing the primer to the DNA to be sequenced c) an unmodified nucleotide triphosphate, a labeling modification represented by the following chemical formula 1 Using a nucleotide triphosphate derivative and a nucleic acid synthetase, a DNA synthesis step involving an elongation reaction, a non-specific chain-interruption reaction and a specific chain-interruption reaction. D) A DNA fragment produced by the specific chain-interruption reaction is non-electrically Detecting process [Chemical formula 1] (In the formula, Q is a 7-deazaadenine, 7-deazaguanine, cytosine or uracil residue, and R ¹ , R ² ,
R ³ and R ⁴ each independently represent a hydrogen atom, a sodium atom or a lithium atom. However, sodium atom and lithium atom do not exist at the same time. A indicates a label. ) 2. The improved DNA sequencing method according to claim 1, wherein the label is biotin, dicoxygenin, an enzyme or a fluorescent dye. 3. The improved DNA nucleotide sequencing method according to claim 1, wherein the nucleotide triphosphate derivative is dideoxy-7-deazanucleotide-5′-triphosphate. 4. The labeled modified nucleotide derivative is 7-
(N-biotinyl- (3-amino-1-propynyl))
-2'3'-dideoxy-7-deazaguanosine-
5'-triphosphate, 7- (N-biotinyl- (3-amino-
1-propynyl))-2'3'-dideoxy-7-deazaadenosine-5'-triphosphate, 5- (N-biotinyl- (3-amino-1-propynyl))-2'3'-die Deoxyuridine-5'-triphosphate or 5- (N-biotinyl- (3-amino-1-propynyl))-2'3 '
-Dideoxycytidine-5'-triphosphate. The improved DNA sequencing method according to claim 1, which is characterized in that it is dideoxycytidine-5'-triphosphate. 5. The improved DNA nucleotide sequencing method according to claim 1, wherein the label forms a signal by forming a complex with a substance that gives rise to a signal. 6. The improved DNA sequencing method according to claim 1, wherein the label is biotin and the signal-generating substance is avidin and alkaline phosphatase. 7. The improved DNA sequencing method according to claim 1, wherein the label is biotin and the substance that produces a signal is avidin, biotin and alkaline phosphatase. 8. The improved DNA sequencing method according to claim 1, wherein the label is dicoxygenin, and the substance that gives rise to a signal is anti-dicoxygenin and alkaline phosphatase. 9. The improved DNA sequencing method according to claim 1, wherein the label is a fluorescent substance, and the substance that produces a signal is an anti-fluorescent dye and alkaline phosphatase. 10. The improved DNA according to claim 1, wherein the label is alkaline phosphatase and the substance producing the signal is a 1,2-dioxetane derivative.
Nucleotide sequencing method. 11. The label is alkaline phosphatase, and the substance producing the signal is 5-bromo-4-chloro-3.
Improved DN according to claim 1, characterized in that it is indolyl-phosphoric acid and nitroblue tetrazolium.
A sequencing method of A. 12. The non-modified nucleotide triphosphate is a deoxynucleotide triphosphate.
The improved DNA sequencing method described. 13. A DNA polymerase lacking 5 ′ → 3 ′ exonuclease as a nucleic acid synthase.
The improved DNA sequencing method according to claim 1, wherein a polymerase or a DNA polymerase lacking a 3 '→ 5'exonuclease is used. 14. The DNA polymerase is Thermu
s thermophilus-derived Tth DNA polymerase, Thermus aquaticus-derived Taq DNA polymerase, Pyrococcus
14. The improved DNA sequencing method according to claim 13, which is at least one DNA polymerase derived from furiosus. 15. The improved DNA according to claim 1, wherein a DNA polymerase lacking 5 ′ → 3 ′ exonuclease and / or 3 ′ → 5 ′ exonuclease is used as the nucleic acid synthesizing enzyme. Nucleotide sequencing method. 16. The improved DNA sequencing method according to claim 1, wherein steps a) to c) are repeated a plurality of times. 17. The improved D according to claim 16, characterized in that the two-step or three-step reaction is carried out at different temperatures.
NA sequencing method. 18. The labeled modified nucleotide triphosphate derivative is 1/10 of the unmodified nucleotide triphosphate derivative.
The improved DNA sequencing method according to claim 1, wherein the concentration (v / v) is -50/1.