JP2002360298A - Method for determining base sequence of dna - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new method for determining the base sequence of DNA. SOLUTION: This method for determining the base sequence of DNA comprises reading a nucleic acid sequence by reacting an NTP derivative with FL-3'dNTP in the presence of an RNA polymerase and an DNA fragment containing a promoter sequence for the RNA polymerase to obtain a nucleic acid transcript and optically detecting the fluorescent labeling of the FL-3'dNTP taken in the nucleic acid transcript product. The FL-3'dNTP is one of the compounds represented by the general formula [Ia] to [Id]. Wherein, R is 3'-deoxy-1-ribofuranosyl-5'-triphosphate; and Q is a residue represented by the general formula [II].}. wherein, V is C≡C or C=C; n and m are each independently 2-4 integer; and R<0> is a fluorophore represented by the general formula [III].}. [wherein, R<1> and R2 are each independently H, CH3 , CH2 CH2 -COOH, Br, the formula (1), the formula (2), the formula (3), the formula (4), the formula (5), the formula (6) or the formula (7).].

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for determining a DNA base sequence. Among them, the present invention is a chain terminator method using a mutant RNA polymerase
DNA sequencing method, as a terminator,
The present invention relates to a DNA sequencing method using a 3′-deoxyribonucleotide derivative labeled with BODIPY, which is a fluorescent label.

[0002]

2. Description of the Related Art In genome analysis and gene diagnosis represented by today's human genome project, DNA sequencing is one of very important techniques. Currently, DN
A typical example of the A sequencing method is the Sanger method [Sanger, F., et al. Proc. Natl. Acad. Sci. USA, 7
4: 5463 (1977)]. This method is widely used in current DNA sequencing and is a general method.

As an application of the Sanger method, a cycle sequence method using a polymerase chain reaction (PCR) has become mainstream at present. In this method, the template DNA does not need to be single-stranded, and the reaction is performed by adding DNA, one kind of primer, dNTPs, ddNTPs, and a heat-resistant DNA polymerase. Incorporation of ddNTPs during the cycle sequencing reaction, which stops DNA chain extension and results in 5 '
DNs with a common terminal sequence but different chain lengths
A chain is synthesized. By subjecting them to electrophoresis, the nucleotide sequence of DNA is determined.

[0004] Furthermore, this cycle sequence method includes:
There are a dye primer method in which primers are fluorescently labeled, an internal label method in which dNTP is labeled, and a dye terminator method in which ddNTP is labeled. Above all, the dye terminator method allows sequencing using any unlabeled primer. Also, while the dye primer method needs to be performed in separate reaction solutions for A, G, C, and T, the DNA chain extension reaction can be performed in the same reaction solution, which is a relatively simple method. Widely used for DNA sequencing.

[0005] However, the above-mentioned sequencing method has several problems. In recent years, as an application of the above-mentioned Sanger method, a method in which a product amplified by a PCR method is directly used in a sequencing reaction without performing a cloning operation and sequenced has been performed. This method is called the direct sequencing method, and the base sequence of many samples can be obtained in a short time without performing complicated operations such as library creation and screening of the library that have been performed by conventional cloning. Can be determined [Corinne Wong et al. (1988) Natur
e, 330, 384-386]. However, in this method, the PCR
If the product is used directly in a sequencing reaction, primers or 2 'deoxyribonucleotide 5' triphosphates (hereinafter referred to as 2 'dNTPs) that were not incorporated during the PCR reaction
Remain during the sequence reaction and inhibit the sequence reaction. In order to solve this, conventionally, such PC
In order to remove excess primer and 2'dNTP generated during the R reaction before the sequencing reaction, complicated operations such as ethanol precipitation and gel filtration have been required [DoritR.L., Et al. (19)
91) Current Protocols in Molecular Biology, vol. 11,
John Wiley and Sons, New York, 15.2.1-15.2.11].

[0006] In addition, the time required for the sequence reaction in the above-mentioned sequence method takes 2-3 hours, and the reaction results vary depending on the reaction conditions. Therefore, it is necessary to set delicate conditions.

[0007] The sequencing method having the above two major drawbacks requires complicated operations, and can be a major obstacle to mass analysis and automation of DNA base sequence determination represented by the present human genome project.

As another problem, it is very difficult to analyze a base sequence having a high GC content by the above-mentioned sequencing method [Choi, JSet al.,
Exp. Mol. Med., 31:20 (1999)]. In fact, in the DNA nucleotide sequence analyzed by the Human Genome Project, the portion with a high GC content is a gap, and it is extremely difficult to decode the nucleotide sequence in the gap portion with current technology, and it takes more time and cost Is required [McMurray, A.
A., et al. Genome Res., 8 (5): 562 (1998)].

Therefore, in order to solve these problems, the present inventor did not remove the unreacted primers and 2 ′ deoxyribonucleotide 5 ′ triphosphate remaining in the PCR reaction solution, and did not remove the PCR reaction product. A DNA sequencing method using RNA polymerase was devised, which eliminates the need for denaturation of the product. This method uses an RNA polymerase such as T7 RNA polymerase and a 3 ′ deoxyribonucleotide (hereinafter, 3 ′ dN
Using TP as a terminator, a method for determining the DNA base sequence by performing an RNA transcription reaction (transcription sequence method) was devised [US Pat. No. 6,074,824].

[0010] By using this method, the complicated work required for the conventional sequencing reaction can be omitted, and the automation required for processing a large number of samples can be made possible. However, when the above method was studied in detail, further improvement was required to obtain longer and more accurate DNA sequence data.

One problem to be improved is the development of a fluorescently labeled terminator. That is, generally
Rhodamine, which is used as a fluorescent dye in DNA nucleotide sequencing, is labeled with 3 'deoxyribonucleotide 5' triphosphate used in the present method, and when a sequencing reaction is performed as a fluorescent label terminator, the effect of the reaction solution, particularly It is susceptible to the reaction solution such as pH, and shows variations in signal. And due to them, incorrect sequence results were shown, and only short DNA sequences could be read. Therefore, the transfer sequence method using the above terminator requires considerable skill.

[0012]

SUMMARY OF THE INVENTION Accordingly, an object of the present invention is to provide an accurate and longer chain by making the signal intensity less likely to be affected by reaction solution conditions such as pH.
An object of the present invention is to provide a transcription sequence method capable of determining a DNA base sequence.

The present inventors have found that a transcription sequence method using 3 ′ deoxyribonucleotide 5 ′ triphosphate to which a fluorescent dye BODIPY is added as a terminator solves the above-mentioned problems, and completed the present invention. did.

[0014]

SUMMARY OF THE INVENTION The present invention for solving the above-mentioned problems has been accomplished by the use of an RNA polymerase and a DNA fragment containing a promoter sequence for the RNA polymerase.
TP, GTP, CTP and UTP or their derivatives, and 1
Or 3 or more fluorescently labeled 3 'deoxyribonucleoside 5' triphosphates (hereinafter FL-3'dNTP
) To obtain a nucleic acid transcription product, and by optically detecting the fluorescent label of the FL-3'-dNTP incorporated in the nucleic acid transcription product, a DNA for reading a nucleic acid sequence. A method for determining a nucleotide sequence, wherein the FL-3′dNTP is represented by the following general formula:
The present invention relates to a method for determining a DNA base sequence, which is a compound represented by any one of [Ia] to [Id].

Embedded image [Wherein, R represents 3′-deoxy-1-ribofuranosyl-5′-triphosphate, and Q represents a residue represented by the following general formula [II]. ]

Embedded image[Wherein, V represents -C≡C- or -C = C-, and n and m are independent
Represents an integer of 2 to 4, R ⁰Is represented by the following general formula [III]
Fluorophore. ]

Embedded image Wherein, R1 and R2, H _{independently, CH 3 -, - CH 2} CH 2 -COOH, B
r,

Embedded image It is. ]

[0015] The present invention further provides a method for increasing the ability of an RNA polymerase to take up a 3 'dNTP derivative as compared to the ability of a corresponding wild-type RNA polymerase,
It is preferably a mutant RNA polymerase in which at least one amino acid of the wild type RNA polymerase has been substituted.

[0016]

BEST MODE FOR CARRYING OUT THE INVENTION [FL-3'dNTP] In the DNA sequencing method of the present invention, any of the compounds represented by the general formulas [Ia] to [Id] is used as FL-3'dNTP. FL-3 'dNTP represented by the general formula [Ia] ~ [Id], (a) 3'dNTP residues (b) a linker _{moiety: -V- (CH 2) n -NH} -CO- (CH 2) _m- and (c) BODIPY, which is a fluorescent dye (R ⁰ ), can be divided into three components. The 3 ′ dNTP residue is represented by the general formula [Ia] cytidine, the general formula [Ib] uridine, the general formula [Ic] 7-deazaguanidine, the general formula [I
d] is 7-deazaadenosine. The basic form of the fluorescent dye BODIPY represented by R ⁰ is represented by the general formula [III].

The group represented by the general formula [III] is a fluorescent dye group which can be detected by an apparatus capable of detecting luminescence by receiving energy such as an argon laser. 3′-deoxyribonucleotide residue and B represented by R ⁰
It is desirable that the combination with ODIPY has independent peaks without overlapping of the excitation wavelength peaks.

Specific 3'-deoxyribonucleotides
The combination of (A, G, C, U) and BODIPY represented by R ⁰ is
It is as follows. A… BODIPY R6G G… BODIPY FL C… BODIPY 581/591 U… BODIPY 564/570

BODIPY R6G is a compound represented by the general formula [III]
1 is H and R2 is

Embedded image (General formula [IV] below). In BODIPY FL, R1 in the general formula [III] is CH _3- and R2 is CH _3- (the following general formula:
[V]). In BODIPY 581/591, R1 in the general formula [III] is H, and R2 is

Embedded image (General formula [VI] below). BODIPY 564/570 has the general formula [I
II], R1 is H and R2 is

Embedded image (General formula [VII] below).

[0020]

Embedded image

In FL-3'dNTP, the 5 'side is H, PO ₃ H ₂ , P ₂ O
₆ H ₃ , P ₃ O0H ₄ , or a salt thereof, specific examples of the salt include sodium salts, potassium salts, alkali metal salts such as lithium salts, alkaline earth metal salts such as barium salts, ammonium salts, Organic amine salts such as triethylammonium salt and pyridine salt are exemplified.

The above linker portion: -V- (CH ₂ ) _n -NH-CO- (C
H ₂ ) _m -is a 3 ′ deoxyribonucleotide residue and a fluorescent dye R
It is a linker for binding to BODIPY of ⁰ . 3 '
Deoxyribonucleotide residues are purine nucleotides
(Guanidine (general formula [Ic]) and adenosine (general formula [Id]))
In position 7, a pyrimidine nucleotide (cytidine (general formula
[Ia]) and uridine (general formula [Ib])) bind to a linker at the 5-position. Further, the fluorescent dye (BODIPY dye) represented by the general formula [III] binds to a position opposite to the 3 ′ deoxyribonucleotide of the linker portion.

In the linker represented by -V- (CH ₂ ) _n -NH-CO- (CH ₂ ) _m- , n and m are each independently an integer of 2 to 4,
(CH ₂ ) _n- and-(CH ₂ ) _m -are each a methylene chain. This is because, after the sequencing reaction, when the terminator having a fluorescent dye and the polynucleotide bound to the 3 ′ side move by electrophoresis on a gel, the mutual distance between adjacent polynucleotides becomes uniform. This is for adjusting the number of methylene chains n corresponding to the linker of the terminator. By equalizing the mutual distances with respect to the mobility of each polynucleotide, when reading a long sequence, it is possible to read a longer DNA base sequence as a result without overlapping the fluorescent labels peaked on each polynucleotide. Will be possible. The specific number of methylene chains n is as follows. A n = 4 G n = 4 C n = 2 U n = 2

The FL-3'dNTP represented by the general formulas [Ia] to [Id]
Production Method FL-3'dNTP represented by general formulas [Ia] to [Id] can be produced, for example, by the following production method. General formula [I
FL-3'dNTP represented by [a] to [Id] is disclosed in JP-A-63-152.
The compound can be easily synthesized with reference to JP-A-364-364. In addition, FL-3′dNTP can be easily obtained by using a labeling method and a purification method recommended by Molecular Probes, Inc. for the fluorescent dye.

The fluorescent dye BODIPY represented by the general formula [III]
Is currently manufactured and sold by Molecular Probes. BODIPY having a heterocyclic ring is a dipyrromethene boron difluoride compound, which has been improved so that any substance can be easily fluorescently labeled. Furthermore, compared to conventional fluorescent dyes, it has a longer wavelength and a narrower bandwidth. Therefore, there is a characteristic that the signal intensity at the time of detecting the fluorescence wavelength is high, the signal intensity is sharp, and the peaks of the excitation wavelengths do not overlap with each other due to the narrow bandwidth, thereby reducing errors in the analysis by the computer. Therefore, when 3 ′ deoxyribonucleotide having BODIPY having such properties as a label is used as a terminator, a DNA base sequence determination result can be obtained as compared with the case where a terminator labeled with a conventional fluorescent dye is used. .

That is, in a DNA base sequence determination method by a transcription sequence method using BODIPY as a fluorescent dye (R) represented by the general formula [III] and using 3 'deoxyribonucleotide as a terminator, the sequence result is determined by the pH in the reaction solution. There is an advantage that the DNA base sequence is hardly affected by conditions and the like, the signal intensity is high, and a uniform and longer DNA base sequence can be determined.

[RNA polymerase] The RNA polymerase may be a wild-type RNA polymerase derived from T7 phage, T3 phage, SP6 phage or K11 phage. In addition, RNA polymerases may increase the ability to incorporate 3'dNTP derivatives, for example, as compared to the ability of the corresponding wild-type RNA polymerase as described in, for example, JP-A-11-75867 (Japanese Patent No. 3172710). Furthermore, a mutant RNA polymerase in which at least one amino acid of the wild type RNA polymerase has been substituted can also be used.

A method for enzymatically synthesizing a nucleic acid transcription product using an RNA polymerase with a DNA fragment containing a promoter sequence for RNA polymerase as a template, a method for separating a nucleic acid transcription product, and a method for separating the separated fractions In principle, any method for reading a nucleic acid sequence from is known. Accordingly, in these respects, basically, any known methods, conditions, devices, and the like can be appropriately used.

(1) Step of Obtaining Nucleic Acid Transcription Reaction Product The DNA fragment used as a template is not particularly limited except that it contains a promoter sequence for RNA polymerase.
For example, a DNA fragment containing a promoter sequence can be a DNA product amplified by the polymerase chain reaction. Further, without removing the primers and / or 2 ′ deoxyribonucleoside 5 ′ triphosphates and / or derivatives thereof used in the polymerase chain reaction from the amplified DNA product, the nucleic acid transcription production reaction in the method of the present invention can be performed. It can be carried out. As the polymerase chain reaction for DNA amplification, a method widely used as a PCR method can be used as it is. Further, the DNA fragment containing the promoter sequence may be a DNA fragment obtained by ligating the promoter sequence and the DNA fragment to be amplified and then cloning using an appropriate host. That is, in the present invention, there are no particular restrictions on the DNA sequence to be amplified, primers, conditions for amplification, and the like.

For example, as a reaction system of a polymerase chain reaction for amplifying a DNA fragment containing a promoter sequence, 10 to 50 ng of genomic DNA or 1 pg of cloned DNA, 10 μM of each primer,
As a DNA polymerase, for example, Taq polymerase or the like can be used in a 20 μl volume containing μM of each 2 ′ deoxyribonucleoside 5 ′ triphosphate (dATP, dGTP, dCTP, dTTP).

However, either one of the primers for the polymerase chain reaction or the amplified insert DN
A needs to include a promoter sequence for the RNA polymerase described below. The direct transcription sequence method is obtained by using a primer having a phage promoter sequence in one of two types of primers in a PCR method, or by having a phage promoter sequence in amplified insert DNA. PCR
The product can be subjected to in vitro transcription using an RNA polymerase driven by the promoter.

[0032] The promoter sequence for RNA polymerase can be appropriately selected according to the type of RNA polymerase to be used. However, by introducing two promoters derived from two phages, for example, two promoters specifically recognized by T7 and T3 phage to each strand of the template, any of the RNAs during the transcription sequence reaction
By simply selecting a polymerase, it is possible to analyze sequence data of both ends of the target DNA.

The method of the present invention includes a promoter sequence.
A nucleic acid transcript such as an RNA transcript is synthesized from a DNA fragment. D
Since the NA fragment contains a promoter sequence for the RNA polymerase, the promoter sequence activates the RNA polymerase to synthesize a nucleic acid transcript such as an RNA transcript.

For the synthesis of a nucleic acid transcript such as an RNA transcript, a substrate containing at least a fluorescent label 3′-deoxynucleotide (FL-3′dNTP) is used as a substrate for an RNA polymerase described later. More specifically, the substrate is ATP, G
Ribonucleoside 5 'triphosphates (NTPs) consisting of TP, CTP and UTP or derivatives thereof;
In addition, one or more FL-3'dNTPs are used. FL-3'dN
As TP, any of the compounds represented by the general formulas [Ia] to [Id] is used. As the ribonucleoside 5 ′ triphosphates (NTPs), at least four types of compounds having different bases are necessary for the synthesis of a transcript, including the case where some of them are derivatives such as ATP. However, two or more compounds containing the same base can be used.

By incorporating FL-3'dNTP into the 3 'end of RNA or nucleic acid which is a transcription product, the 3' hydroxy group is lost and synthesis of RNA or nucleic acid is inhibited.
As a result, RNs of various lengths whose 3 'end is FL-3'dNTP
A or a nucleic acid fragment is obtained. Such a ribonucleoside analog in which FL-3'dNTP having a different base and a different fluorescent label is incorporated at the 3 'end is obtained. This ribonucleoside
By preparing an analog having 4 bases at the 3 ′ end, it can be used for determination of RNA or nucleic acid sequence [Vladimir D. Axelred er al. (1985) Biochemist
ry Vol. 24, 5716-5723].

One or more FL-3'dNTPs can be used in one nucleic acid transcription reaction. Only one kind
When one nucleic acid transcription reaction is performed using FL-3'dNTP, the nucleic acid transcription reaction is performed four times, so that the base at the 3 'end (FL-3'dNT
Four different transcription products of P) are obtained. In one nucleic acid transcription reaction, a transcription product which is a mixture of various RNAs or nucleic acid fragments having the same FL-3'dNTP at the 3 'end and different molecular weights can be obtained. The resulting four transcription products can be independently subjected to separation and sequence reading described below. In addition, two or more of the four types of transcription products are mixed,
This mixture can also be subjected to separation and sequence reading.

In one nucleic acid transcription reaction, two or more types of FL-3'dNT
When P is used at the same time, two or more transcription products having different bases (FL-3'dNTP) at the 3 'end are contained in one reaction product. This can be subjected to separation and sequence reading described below. More than one kind of nucleic acid transcription reaction
The simultaneous use of FL-3'dNTP is preferable because the number of nucleic acid transcription reaction operations can be reduced. In the present invention, in particular, the transcription of nucleic acids such as RNA is performed by using four types of FL-
It is preferable to terminate by 3 ′ dNTP, separate it, and read the sequence of four bases at once (simultaneously).

(2) Step of separating nucleic acid transcription reaction product In the method of the present invention, RNA or nucleic acid transcription products are separated. This separation can be appropriately performed by a method capable of separating a plurality of product molecules having different molecular weights contained in the transcription product according to the molecular weight. An example of such a separation method is an electrophoresis method. In addition, HPLC and the like can be used.

The conditions of the electrophoresis method are not particularly limited.
It can be performed by a conventional method. An RNA or nucleic acid sequence can be read from a band (RNA or nucleic acid ladder) obtained by subjecting the transcription product to electrophoresis.

(3) Step of Reading Nucleic Acid Sequence RNA or nucleic acid ladder can be read by detecting a fluorescent label of FL-3'dNTP. Specifically, for example, the transcription product sequence can be read by subjecting the transcription product obtained using FL-3'dNTP to electrophoresis and detecting the fluorescence of a band obtained.
In particular, in the present invention, the above-mentioned general formulas [Ia] to FL-3′dNTP are used.
Since any FL-3'dNTP represented by [Id] is used, pH
It is hardly affected by reaction solution conditions such as the above, and the signal intensity becomes uniform, so that the base sequence of a longer-chain DNA can be determined accurately and accurately. The detection of a ladder that generates fluorescence can be performed, for example, by using an apparatus used for DNA sequencing or RNA sequencing as appropriate.

In addition, four types of F labeled with different fluorescence
It is obtained by subjecting a mixture of various transcription product fragments having any of the FL-3'dNTPs represented by the general formulas [Ia] to [Id] to an end by electrophoresis using L-3'dNTP. The sequence of RNA or nucleic acid can also be read by detecting the four types of fluorescence of the band. In this method, four types of FL-3'dNTPs are labeled with different fluorescence. By subjecting a mixture of four types of transcription products having different 3 'ends to electrophoresis, a band emitting fluorescence corresponding to four different types of FL-3' dNTPs at the 3 'end was obtained. , The sequence of four types of RNA or nucleic acid can be read at a time.

[0042]

According to the present invention, by using 3 ′ deoxyribonucleotide 5 ′ triphosphate to which a fluorescent dye BODIPY is added as a terminator, the signal intensity is less affected by reaction solution conditions such as pH. By providing uniformity, it is possible to provide a transcription sequence method capable of determining the base sequence of a longer-chain DNA accurately and accurately.

[0043]

The present invention will be described in more detail with reference to the following examples. [Example 1] Example of transcription sequence using BODIPY-terminator As BODIPY-terminator, BODIPY R6G-3'-dATP (n =
4), BODIPY FL-3'-dGTP (n = 4), BODIPY 581 / 591-3'-dCTP
(n = 2) and BODIPY 564 / 570-3′-dUTP (n = 2). In each case, BODIPY synthesized by the method described in JP-A-63-152364 was converted to 3′-dNT by a known method.
It is labeled P.

The in vitro transcription reaction was performed by Melton, DA
The method shown in [Nucleic Acids Res., 12: 7035-7056 (1984)] was used. Specifically, a circular DNA of a plasmid vector pBluescriptSK (+) having a T7 promoter was used as a template. The reaction was performed using BODIPYR6G-3'-dATP (n = 4), BODIPY
FL-3'-dGTP (n = 4), BODIPY 581 / 591-3'-dCTP (n = 2), BOD
IPY 564 / 570-3'-dUTP (n = 2) plus 500 μM ITP, UTP and 2
50 [mu] M ATP, mixed CTP, by performing dilution capable analysis of about 500 bases, to determine the condition, 8mMMgCl _2, 2m
M spermidine- (HCl) ₃ , 5 mM DTT, 40 mM Tris / HCl pH 8.0
Under the conditions described above, 50 units of mutant T7 RNA polymerase F644Y were added to make a total reaction volume of 10 μl and reacted at 37 ° C. for 1 hour.

Next, ethanol was added to remove unreacted die terminator remaining in the reaction product, the mixture was kept at -20 ° C. for 20 minutes, and centrifuged to obtain a precipitated nucleic acid. The supernatant was removed and dried using a centrifugal evaporator. According to ABI 377XL Sequencing System instruction manual Ver.1.0 of PerkinElmer Japan, formamide / EDTA / Blue dextra
n Dissolve in 6 μl loading buffer, and transfer 2 μl to 6 μl
M urea / 4% long ranger acrylamide solution (FMC
ABI 377 using denaturing gel for sequence analysis
Analysis was performed using an XL sequencer and an analysis program. The result is shown in FIG. As a result, this time
Very good sequence electrograms were obtained using the BODIPY-terminator.

[Brief description of the drawings]

FIG. 1 shows an analysis example using a transcription sequence using 3′-deoxynucleotide bound to a Bodipy dye as a terminator.

Continued on the front page (72) Inventor Masanori Watahiki 1-1-24 Arakawa, Toyama-shi, Toyama Nippon Genetech Toyama Research Laboratories Co., Ltd. 4B024 AA20 CA01 EA04 FA18 HA19 4B063 QA13 QQ42 QR32 QR62 QR63 QR66 QS03

Claims (2)

[Claims] 1. An RNA polymerase and said RNA polymerase
A in the presence of a DNA fragment containing a promoter sequence for
TP, GTP, CTP and UTP or their derivatives, and 1
Or 3 or more fluorescently labeled 3 'deoxyli
Bonnucleoside 5 'triphosphate (hereinafter FL-3'dNTP
) To obtain a nucleic acid transcription product,
Fluorescent label of the FL-3'-dNTP incorporated in the photoproduct
DNA salts that read nucleic acid sequences by optical detection
A method for determining a base sequence, wherein the FL-3'dNTP is represented by the following general formula:
The compound is one of the compounds represented by [Ia] to [Id].
Characteristic DNA sequencing. Embedded image[Wherein R is 3′-deoxy-1-ribofuranosyl-5′-trif
And Q is represented by the following general formula [II]:
Indicates the residue to be used. [Chemical formula 2][II] [wherein, V represents -C≡C- or -C = C-, and n and m are independent
Represents an integer of 2 to 4, R ⁰Is represented by the following general formula [III]
Fluorophore. [Chemical formula 3]Wherein R1 and R2 are independently H, CH_Three-, -CH_TwoCH_Two-COOH, B
r,It is. ] 2. A mutant in which at least one amino acid of a wild-type RNA polymerase has been substituted so that the RNA polymerase has an increased ability to incorporate a 3 ′ dNTP derivative as compared to the ability of the corresponding wild-type RNA polymerase. Type
The method according to claim 1, which is an RNA polymerase.