JPH06197798A - Determination of dna base sequence by fluorescent substrate - Google Patents

Abstract

PURPOSE:To determine the base sequence of a DNA in shortened time with simple operation. CONSTITUTION:The method for the determination of a DNA base sequence is composed of (1) a step to fractionate 4 kinds of DNA specimens individually synthesized by complemental strand synthesis and having different kinds of terminal bases by forcing the specimens to migrate through different migration channels, (2) a step to transfer the fractionated DNA specimen to a filter by suction, (3) a step to bond an enzyme to each DNA specimen and (4) a step to react the above enzyme with a fluorescent substrate consisting of a naphthol derivative or an anthracene derivative and detecting the emitting fluorescent light.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a DNA nucleotide sequence determination method, and more particularly to a DNA nucleotide sequence determination method using a fluorescent substrate.

[0002]

2. Description of the Related Art DNA sequencing methods have been performed so far using radioisotope (RI) labeling, but with the increasing demand for genomic analysis, fluorescence sequencers have recently been developed. There is an urgent need for RI. However, a system for exciting a fluorescently labeled DNA base sequence pattern with a laser beam and detecting the fluorescent signal is very expensive in view of its widespread use in general laboratories. This is because the laser is used as the excitation light source and the software for analyzing the fluorescence signal is expensive.

In order to solve such a problem, a DNA nucleotide sequencing method by chemiluminescence method was developed (Reference: Be.
ck et al., Nucleic Acids Res. , 1
7, 5115 (1989), Gillevet et al., N.
ATURE, 348, 657 (1990)). In this method, in the dideoxy method (Sanger method) which is widely used among DNA nucleotide sequencing methods, a sequencing primer is labeled with biotin, and then biotinylated alkaline phosphatase is bound via streptahidine to bind AMPPD or It is reacted with a chemiluminescent substrate such as CSPD. Specifically, the reaction of dideoxy method is performed using a biotin-labeled primer, and the individual band patterns are separated by electrophoresis, transferred to a nylon membrane filter, and then bound to alkaline phosphatase as described above for chemiluminescence. The DNA base sequence pattern is detected by reacting with a substrate and exposing a luminescent signal to an X-ray film.

[0004]

The problems of this method are as follows.
It is necessary to perform an exposure operation on the X-ray film in order to detect the luminescence signal. In order to obtain an optimum signal, it is usually necessary to carry out monitoring and expose for an appropriate time, but there is no suitable monitoring method for detection by chemiluminescence method, so there is no choice but to actually carry out the detection operation. So in many cases 2-3
Multiple exposure operations are required. After all, despite the advantage that the exposure time of one time is about 30 minutes, X
Since it takes about 20 minutes to develop a linear film,
Eventually it will take 3-4 hours to detect the signal. In addition, the complexity of the operation, which requires multiple exposure operations, poses a problem. Therefore, an object of the present invention is to make it possible to determine the base sequence of DNA in a shorter time and with a simple operation.

[0005]

[Constitution of the invention]

In order to solve the above-mentioned problems, the present inventors have (1) individually migrating four kinds of DNA samples having different kinds of terminal bases synthesized for complementary strands in different migration paths. And then fractionating, (2) transferring the fractionated DNA sample onto a filter by suction, (3) each DNA
A method for determining the nucleotide sequence of DNA was found by sequentially performing the steps of binding an enzyme to a sample, (4) reacting the fluorescent substrate with a naphthol derivative or anthracene derivative, which is a fluorescent substrate, and detecting the emitted fluorescence. It was More specifically, as a method for individually preparing four complementary DNA samples having different terminal base species by complementary strand synthesis, vector DNA can be used.
Prepare a single-stranded DNA sample whose nucleotide sequence to be incorporated into
Hybridizes to NA. 4 samples obtained in this way
After the division, it can be obtained by individually performing complementary chain synthesis so that the terminal base species are adenine, guanine, cytosine and thymine.

Further, as the naphthol derivative used as the fluorescent substrate, those represented by the following structural formula can be used.

[0007]

Embedded image 3-hydroxy-2-naphthalenecarboxylic acid-
2'-Phenylanilidenate 3-Hydroxy-N-2'-biphenyl-2
-Naphthalene carboxamide p
hosphate

[0008]

Embedded image 3-hydroxy-2-naphthalenecarboxylic acid-
3 ', 5'-dimethoxyaniliderinic acid body 3-Hydroxy-N-3', 5'-dimetho
xyphenyl-2-naphthalenecar
boxamide phosphate

[0009]

Embedded image 3-hydroxy-2-naphthalenecarboxylic acid-
2 ', 4'-dimethoxyanilidenate 3-Hydroxy-N-2', 4'-dimetho
xyphenyl-2-naphthalenecar
boxamide phosphate

[0010]

Embedded image 6-Bromo-3-hydroxy-2-naphthalenecarboxylic acid-2 ′, 4′-dimethylaniliderinic acid derivative 6-Bromo-3-Hydroxy-N-2 ′, 4 ′
-Dimethylphenyl-2-naphtha
lenecarboxamide phosphate

[0011]

Embedded image 3-hydroxy-2-naphthalenecarboxylic acid-
2'-methyl-4'-bromoanilidene acid body 3-Hydroxy-N-2'-methyl-4-B
romophenyl-2-naphthalenec
arboxamide phosphate

[0012]

Embedded image 3-hydroxy-2-naphthalenecarboxylic acid-
3 ', 5'-Dimethylanilidene acid body 3-Hydroxy-N-3', 5'-dimethyl
lphenyl-2-naphthalenecarb
oxamide phosphate

[0013]

Embedded image 3-hydroxy-2-naphthalenecarboxylic acid-
2'-Bromo-4'-methylanilidenate 3-Hydroxy-N-2'-Bromo-4'-m
Ethylphenyl-2-naphthalene
carboxamide phosphate

[0014]

Embedded image 3-hydroxy-2-naphthalenecarboxylic acid-
2'-Isopropylanilidenate 3-Hydroxy-N-2'-isopropylp
henyl-2-naphthalene carbox
amide β-D-galactopyranosi
de

[0015]

Embedded image 3-hydroxy-2-naphthalenecarboxylic acid-
2'-Phenylanilide β-D-galactopyranoside 3-Hydroxy-N-2'-biphenyl-2
-Naphthalene carboxamide β
-D-galactopyranoside

[0016]

Embedded image 3-Hydroxy-2-naphthalenecarboxylic acid-3 ′, 5′-dimethoxyanilide β-D-galactopyranoside 3-Hydroxy-N-3 ′, 5′-dimetho
xyphenyl-2-naphthalenecar
boxamide β-D-galactopyran
oside

[0017]

Embedded image 3-Hydroxy-2-naphthalenecarboxylic acid-2 ′, 4′-dimethoxyanilide β-D-galactopyranoside 3-Hydroxy-N-2 ′, 4′-dimetho
xyphenyl-2-naphthalenecar
boxamide β-D-galactopyran
oside

[0018]

Embedded image 6-Bromo-3-hydroxy-2-naphthalenecarboxylic acid-2 ′, 4′-dimethylanilide β-D-
Galactopyranoside 6-Bromo-3-Hydroxy-N-2 ′, 4 ′
-Dimethylphenyl-2-naphtha
lenecarboxamide phosphate

[0019]

Embedded image 3-Hydroxy-2-naphthalenecarboxylic acid-2′-methyl-4′-bromoanilide β-D-galactopyranoside 3-Hydroxy-N-2′-methyl-4-B
romophenyl-2-napphaleneca
rboxamide β-D-galactopyrra
noside

[0020]

Embedded image 3-Hydroxy-2-naphthalenecarboxylic acid-3 ′, 5′-dimethylanilide β-D-galactopyranoside 3-Hydroxy-N-3 ′, 5′-dimethy
lphenyl-2-naphthalenecarb
oxamide β-D-galactopyrano
side

[0021]

Embedded image 3-Hydroxy-2-naphthalenecarboxylic acid-2′-bromo-4′-methylanilide β-D-galactopyranoside 3-Hydroxy-N-2′-Bromo-4′-m
Ethylphenyl-2-naphthalene
carboxamide β-D-galactopy
ranside

[0022]

Embedded image 3-Hydroxy-2-naphthalenecarboxylic acid-2′-isopropylanilide β-D-galactopyranoside 3-Hydroxy-N-2′-isopropylp
henyl-2-naphthalene carbox
amide β-D-galactopyranosi
de

Further, as the anthracene derivative, one represented by the following structural formula can be used.

Embedded image 3-Hydroxy-2-anthracenecarboxylic acid-2 ′, 4′-dimethoxyaniridolinic acid derivative 3-Hydroxy-N-2 ′, 4′-dimetho
xyphenyl-2-anthracenecarb
oxamidephosphate

[0024]

Embedded image 3-Hydroxy-2-anthracenecarboxylic acid-2′-phenylanilideneic acid body 3-Hydroxy-N-2′biphenyl-2-
anthracene carboxamide phos
phate

[0025]

Embedded image 3-Hydroxy-2-anthracenecarboxylic acid-2 ′, 4′-dimethylaniliderinic acid derivative 3-Hydroxy-N-2 ′, 4′-dimethy
lphenyl-2-anthracenecarbo
xamidephosphate

[0026]

Embedded image 3-Hydroxy-2-anthracenecarboxylic acid-2-methoxyanilidene acid 3-Hydroxy-N-2′-methoxyphe
nyl-2-anthracene carboxami
dephosphonate

[0027]

Embedded image 3-Hydroxy-2-anthracenecarboxylic acid-2 ′, 4′-dimethoxyanilide β-D-galactopyranoside 3-Hydroxy-N-2 ′, 4′-dimetho
xyphenyl-2-anthracenecarb
oxamide β-D-galactopyrano
side

[0028]

Embedded image 3-Hydroxy-2-anthracenecarboxylic acid-2′-phenylanilide β-D-galactopyranoside 3-Hydroxy-N-2′biphenyl-2-
anthracene carboxamide β-D-
galactopyranoside

[0029]

Embedded image 3-Hydroxy-2-anthracenecarboxylic acid-2 ′, 4′-dimethylanilide β-D-galactopyranoside 3-Hydroxy-N-2 ′, 4′-dimethy
lphenyl-2-anthracenecarbo
xamide β-D-galactopyranosi
de

[0030]

Embedded image 3-Hydroxy-2-anthracenecarboxylic acid-2-methoxyanilide β-D-galactopyranoside 3-Hydroxy-N-2′-methoxyphe
nyl-2-anthracene carboxami
deβ-D-galactopyranoside

[0031]

In the present invention, the DNA transferred onto the filter
Since the fluorescent substrate is bound to, the emitted fluorescence can be directly photographed. For this reason, the exposure operation and the development operation, which are required in the conventional method using the X-ray film, are not necessary, and thus the operability is improved, and the time required for the exposure operation and the development operation is omitted, and the base is shortened in a short time. It becomes possible to determine the sequence.

[0032]

EXAMPLES Specific examples of the present invention will be described below. For reference, the steps of Example 1 and Example 2 described below are shown in FIG.

Example 1 A non-radioactive labeled DNA sequence kit (PlexT) commercially available from Millipore
The DNA nucleotide sequence was determined using M13 DNA as a template using the M luminescence kit. First, DNA is prepared by the dideoxy method described below. That is, the template DNA is prepared by incorporating the DNA whose nucleotide sequence is to be investigated into M13 DNA (vector DNA).
A, and a primer labeled with biotin is annealed. This is divided into four, and deoxynucleotides (dATP, dGTP, d) are individually added so that the terminal base species are adenine, guanine, cytosine and thymine.
CTP, dTTP) and dideoxynucleotide (d
1 of dATP, ddGTP, ddCTP, ddTTP)
Seeds are prepared and complementary strand synthesis is performed. Then, they are electrophoresed in different migration paths and fractionated. In this way, the fractionated DNA was transferred from a polyacrylamide gel onto a nylon filter by a vacuum suction device and subjected to blocking treatment. Blocking treatment means to prepare a 1% solution of casein extracted from skim milk powder in order to prevent non-specific adsorption of a fluorescent substance described later to a nylon filter, and
The nylon filter to which the sample A was transferred is dipped to perform blocking.

Subsequently, streptapidine was bound to the biotin at the end of each DNA fragment transferred onto the nylon filter, washed once to remove unreacted streptapidine, and then adsorbed on the remaining streptavidin. Biotinylated alkaline phosphatase was bound to.

In addition, the following compound which is a naphthol derivative phosphoric acid derivative,

Embedded image 3-Hydroxy-N-2′-biphen
yl-2-naphthalene carboxami
dephosphate (hereinafter referred to as HNPP) is 100 mM Tris, 100 mM NaCl
It is dissolved in a buffer (pH 9.5) at a concentration of 50 μg / ml to prepare a substrate solution. Then, the nylon filter was placed on a glass plate, the substrate solution was poured over it, and covered with Saran wrap (company name: Asahi Kasei Corporation) to prevent evaporation of the substrate solution. Thus, the above-mentioned compound, which is a fluorescent substrate, was decomposed with biotinylated alkaline phosphatase after a certain period of time.
Next, the fluorescence signal generated by the enzymatic reaction is DN
After being in a state suitable for decoding the A base sequence, as shown in FIG. 1, the nylon filter 4 is irradiated with ultraviolet rays of 302 nm by the UV lamp 3 to cause fluorescence, and the cut filter 2
An image was taken with a Polaroid camera 1 (manufactured by Polaroid Co.) via the. FIG. 3 shows a polaroid photograph taken.
Thus, the DNA base sequence pattern (fluorescent signal)
The resolution of was sufficient to identify each DNA band.

Since the Saran wrap used in Example 1 transmits ultraviolet excitation light and fluorescence well, it is possible to easily (without problem) monitor the enzyme reaction with the naked eye using a handy type ultraviolet lamp.

(Example 2) DNA was prepared by the dideoxy method in the same manner as in Example 1 to prepare four kinds of DNA samples having different terminal base species which were individually synthesized with complementary strands. And
The DNA sample was fractionated by electrophoresis, then transferred from a polyacrylamide gel onto a nylon filter by a vacuum suction device, and subjected to blocking treatment.

What differs from Example 1 is the step after the blocking treatment. In this example, an alkaline phosphatase-labeled anti-biotin antibody is directly bound to a DNA sample without passing through streptavidin. Then, after washing to remove unreacted anti-biotin antibody, a fluorescent substrate was reacted in the same manner as in Example 1 to detect the DNA base sequence as a fluorescent signal. The signal taken by the Polaroid photograph is shown in FIG. As described above, also in Example 2, as in Example 1, it was possible to obtain a resolution capable of sufficiently identifying each DNA band.

In the conventional method using a chemiluminescent substrate such as AMPPD, the luminescence intensity is so weak that detection with the naked eye is impossible, and in order to monitor the enzyme reaction, exposure to an X-ray film and its development are complicated. You have to carry out various operations. However, in this example, since the DNA base sequence pattern is resolved by the fluorescent substrate, the operation is simple and these complicated operations are unnecessary, the operability of monitoring is improved, and the base sequence can be determined in a shorter time. It will be possible.

In particular, in Example 2, since the two-step process required for the binding of alkaline phosphatase in Example 1 can be made into one step, it can be carried out in a shorter time,
In addition, operability is also improved.

The fluorescent substrate, HNPP, used in this example has a very high sensitivity such that it can detect even a trace amount of DNA. Therefore, at least 5 to 6 exposures are required to cover the entire area of the sequence pattern that conventionally appears over a length of 40 cm (8 for Polaroid film).
However, in this embodiment, it is possible to image the whole from a distant position. For this reason, conventionally, the obtained signal was divided into several pieces and photographed, but in the present embodiment, it is possible to store them in one photograph, and the time required for decoding the base sequence from the signal can be greatly increased. It can be shortened.

In this example, HN was used as the fluorescent substrate.
Although PP was used, the fluorescent substrates shown below are also effective for determining the DNA base sequence and can be used in the same manner as the fluorescent substrates used in this example.

[0043]

[Chemical 1]

[0044]

[Chemical 2]

[0045]

[Chemical 3]

[0046]

[Chemical 4]

[0047]

[Chemical 5]

[0048]

[Chemical 6]

[0049]

[Chemical 7]

[0050]

[Chemical 8]

[0051]

[Chemical 9]

[0052]

[Chemical 10]

[0053]

[Chemical 11]

[0054]

[Chemical 12]

[0055]

[Chemical 13]

[0056]

[Chemical 14]

[0057]

[Chemical 15]

[0058]

[Chemical 16]

[0059]

[Chemical 17]

[0060]

[Chemical 18]

[0061]

[Chemical 19]

[0062]

[Chemical 20]

[0063]

[Chemical 21]

[0064]

[Chemical formula 22]

[0065]

[Chemical formula 23]

[0066]

[Chemical formula 24]

[0067]

INDUSTRIAL APPLICABILITY In the present invention, detection of a signal carried out at the final stage of nucleotide sequence determination, that is, in the monitoring step, fluorescence emitted from a fluorescent substrate is detected,
Got the signal. Therefore, the exposure operation and the development operation of the X-ray film, which have been conventionally required, are not required, the operability of monitoring is improved, and the base sequence of DNA can be determined in an extremely short time.

Further, in the conventional radiolabeling method and chemiluminescence method, the signal was attenuated in a short time and therefore had to be decoded within a few days at the latest, but in the present invention, the fluorescent signal on the nylon filter is Since it is stable for a long time (at least 6 months or more), it can be stored as it is, and there is no problem even if the DNA base sequence cannot be immediately decoded.

Since the fluorescent substance is strongly deposited on the nylon filter, the resolution of the signal does not decrease even if the enzymatic reaction is performed too much, and it can be left for a long time.

Further, all that is required for detection is an inexpensive handy type UV lamp, a camera, and an optical filter, which is very popular.

Further, since it does not have an X-ray film,
A light box (using a fluorescent lamp) is not required for decoding the DNA base sequence, and the fatigue of the eyes of the experimenter is reduced.

[Brief description of drawings]

FIG. 1 schematically shows a photographed state of a DNA base sequence in an example of the present invention.

FIG. 2 shows a fluorescence spectrum in a method for detecting a DNA base sequence using a fluorescent substrate.

FIG. 3 shows DNA bands detected in Example 1 of the present invention.

FIG. 4 shows bands of DNA detected in Example 2 of the present invention.

FIG. 5A and FIG. 5B show steps in Example 1 and Example 2 of the present invention, respectively.

[Explanation of symbols]

 1 Polaroid camera 2 Cut filter 3 UV lamp 4 Nylon filter

─────────────────────────────────────────────────── ───

[Procedure amendment]

[Submission date] September 16, 1993

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be corrected] Claim 1

[Correction method] Change

[Correction content]

[Procedure Amendment 2]

[Document name to be amended] Statement

[Name of item to be corrected] Claim 3

[Correction method] Change

[Correction content]

[Chemical 1]

[Chemical 2]

[Chemical 3]

[Chemical 4]

[Chemical 5]

[Chemical 6]

[Chemical 7]

[Chemical 8]

[Chemical 9]

[Chemical 10]

[Chemical 11]

[Chemical 12]

[Chemical 13]

[Chemical 14]

[Chemical 15]

[Chemical 16]

[Procedure 3]

[Document name to be amended] Statement

[Name of item to be corrected] Claim 4

[Correction method] Change

[Correction content]

[Chemical 17]

[Chemical 18]

[Chemical 19]

[Chemical 20]

[Chemical 21]

[Chemical formula 22]

[Chemical formula 23]

[Chemical formula 24]

[Procedure amendment 4]

[Document name to be amended] Statement

[Name of item to be corrected] 0005

[Correction method] Change

[Correction content]

[0005]

[Constitution of the invention]

In order to solve the above-mentioned problems, the present inventors have (1) individually migrating four kinds of DNA samples having different kinds of terminal bases synthesized for complementary strands in different migration paths. And then fractionating, (2) transferring the fractionated DNA sample onto a filter by suction, (3) each DNA
The steps of binding an enzyme to a sample, (4) reacting the enzyme with a naphthol derivative or anthracene derivative that is a fluorescent substrate, and detecting the emitted fluorescence are sequentially performed, and D
A method for determining the base sequence of NA was found.

[Procedure Amendment 5]

[Document name to be amended] Statement

[Correction target item name] 0007

[Correction method] Change

[Correction content]

[0007]

[Chemical 1] 3-Hydroxy-2-naphthalenecarboxylic acid-2'-phenylaniliderinic acid body 3-Hydroxy-N-2'-biphenyl-2
-Naphthalene carboxamide p
hosphate

[Procedure correction 6]

[Document name to be amended] Statement

[Correction target item name] 0011

[Correction method] Change

[Correction content]

[0011]

[Chemical 5] 3-hydroxy-2-naphthalene-carboxylic acid 2'-methyl-4'-bromo anilinium Dorin acid derivative 3-Hydroxy-N-2'- methyl- 4 '-
Bromophenyl-2-naphthalene
carboxamide phosphate

[Procedure Amendment 7]

[Document name to be amended] Statement

[Correction target item name] 0014

[Correction method] Change

[Correction content]

[0014]

[Chemical 8] 3-Hydroxy-2-naphthalenecarboxylic acid-2'-isopropylanilidenate 3-Hydroxy-N-2'-isopropylp
henyl-2-naphthalene carbox
amide phosphate

[Procedure Amendment 8]

[Document name to be amended] Statement

[Correction target item name] 0018

[Correction method] Change

[Correction content]

[0018]

[Chemical 12] 6-Bromo-3-hydroxy-2-naphthalenecarboxylic acid-2 ′, 4′-dimethylanilide β-D-galactopyranoside 6-Bromo-3-Hydroxy-N-2 ′, 4 ′
-Dimethylphenyl-2-naphtha
Lenecarboxamide β-D-galac
topyranoside

[Procedure Amendment 9]

[Document name to be amended] Statement

[Name of item to be corrected] 0023

[Correction method] Change

[Correction content]

Further, as the anthracene derivative, one represented by the following structural formula can be used.

[Chemical 17] 3-hydroxy-2-anthracenecarboxylic acid-2 ′,
4'-dimethoxyaniliderinic acid body 3-Hydroxy-N-2 ', 4'-dimetho
xyphenyl-2-anthracenecarb
oxamide phosphate

[Procedure Amendment 10]

[Document name to be amended] Statement

[Name of item to be corrected] 0024

[Correction method] Change

[Correction content]

[0024]

[Chemical 18] 3-hydroxy-2-anthracenecarboxylic acid-2'-
Phenylanilidenate 3-Hydroxy-N-2'biphenyl-2-
anthracene carboxamide pho
sphate

[Procedure Amendment 11]

[Document name to be amended] Statement

[Name of item to be corrected] 0025

[Correction method] Change

[Correction content]

[0025]

[Chemical 19] 3-hydroxy-2-anthracenecarboxylic acid-2 ′,
4'-Dimethylaniridolinic acid body 3-Hydroxy-N-2 ', 4'-dimethyl
lphenyl-2-anthracenecarbo
xamide phosphate

[Procedure Amendment 12]

[Document name to be amended] Statement

[Correction target item name] 0026

[Correction method] Change

[Correction content]

[0026]

[Chemical 20] 3-Hydroxy-2-anthracenecarboxylic acid-2-methoxyanilidene acid body 3-Hydroxy-N-2'-methoxyphe
nyl-2-anthracene carboxami
de phosphate

[Procedure Amendment 13]

[Document name to be amended] Statement

[Name of item to be corrected] 0027

[Correction method] Change

[Correction content]

[0027]

[Chemical 21] 3-hydroxy-2-anthracenecarboxylic acid-2 ′,
4′-dimethoxyanilide β-D-galactopyranoside 3-Hydroxy-N-2 ′, 4′-dimetho
xyphenyl-2-anthracenecarb
oxamide β-D-galactopyrano
side

[Procedure Amendment 14]

[Document name to be amended] Statement

[Correction target item name] 0028

[Correction method] Change

[Correction content]

[0028]

[Chemical formula 22] 3-hydroxy-2-anthracenecarboxylic acid-2'-
Phenyl anilide beta-D-galactopyranoside 3-Hydroxy-N-2 ' -b iphenyl-2
-Anthracenecarboxamid e β-
D-galactopyranoside

[Procedure Amendment 15]

[Document name to be amended] Statement

[Name of item to be corrected] 0029

[Correction method] Change

[Correction content]

[0029]

[Chemical formula 23] 3-hydroxy-2-anthracenecarboxylic acid-2 ′,
4'-Dimethylanilide β-D-galactopyranoside 3-Hydroxy-N-2 ', 4'-dimethy
lphenyl-2-anthracenecarbo
xamide β-D-galactopyranosi
de

[Procedure Amendment 16]

[Document name to be amended] Statement

[Name of item to be corrected] 0030

[Correction method] Change

[Correction content]

[0030]

[Chemical formula 24] 3-hydroxy-2-anthracenecarboxylic acid-2 '
-Methoxyanilide β-D-galactopyranoside 3-Hydroxy-N-2'-methoxyphenyl-2-anthracenecarboxami
deβ-D-galactopyranoside

[Procedure Amendment 17]

[Document name to be amended] Statement

[Correction target item name] 0035

[Correction method] Change

[Correction content]

In addition, the following compound which is a naphthol derivative phosphoric acid derivative,

[Chemical 1] 3-Hydroxy-N-2'-biphenyl-2-naphthalenecarboxamide p
hosphate (hereinafter referred to as HNPP) is 100 mM Tris,
Prepare a substrate solution by dissolving it in a buffer consisting of 100 mM NaCl (pH 9.5) at a concentration of 50 μg / ml. Then, the nylon filter was placed on a glass plate, the substrate solution was poured over it, and covered with Saran wrap (company name: Asahi Kasei Corporation) to prevent evaporation of the substrate solution. Thus, the above-mentioned compound, which is a fluorescent substrate, was hydrolyzed with biotinylated alkaline phosphatase after a certain period of time. Next, after the fluorescent signal generated by the enzymatic reaction was in a state suitable for decoding the DNA base sequence,
As shown in (3), the nylon filter 4 was irradiated with ultraviolet rays of 302 nm by the UV lamp 3 to cause fluorescence, and the cut image was taken by the Polaroid camera 1 (manufactured by Polaroid) through the cut filter 2. FIG. 3 shows a polaroid photograph taken. As described above, the resolution of the DNA base sequence pattern (fluorescence signal) was sufficient to identify each DNA band.

[Procedure 18]

[Document name to be amended] Statement

[Correction target item name] 0043

[Correction method] Change

[Correction content]

[0043]

[Chemical 1]

[Procedure Amendment 19]

[Document name to be amended] Statement

[Correction target item name] 0063

[Correction method] Change

[Correction content]

[0063]

[Chemical 21]

[Procedure amendment 20]

[Document name to be amended] Statement

[Correction target item name] 0064

[Correction method] Change

[Correction content]

[0064]

[Chemical formula 22]

[Procedure correction 21]

[Document name to be amended] Statement

[Correction target item name] 0065

[Correction method] Change

[Correction content]

[0065]

[Chemical formula 23]

[Procedure correction 22]

[Document name to be amended] Statement

[Correction target item name] 0066

[Correction method] Change

[Correction content]

[0066]

[Chemical formula 24]

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Kyoko Kondo 2-1, 1-1 Asahi-cho, Kariya city, Aichi Prefecture Aisin Seiki Co., Ltd.

Claims (4)

[Claims] 1. A step of (4) separately migrating four kinds of DNA samples having different terminal base species synthesized with complementary strands in different migration paths, and (2) aspirating the separated DNA samples. And (3) binding an enzyme to each DNA sample, and (4) reacting the fluorescent substrate with a naphthol derivative or anthracene derivative, which is a fluorescent substrate, and detecting the emitted fluorescence. A method for determining a DNA base sequence, which comprises: 2. The DNA sequencing method according to claim 1, wherein the enzyme is alkaline phosphatase, acid phosphatase, or β-galactosidase. 3. The DNA nucleotide sequencing method according to claim 1, wherein the naphthol derivative is represented by the following chemical structural formula. [Chemical 1] [Chemical 2] [Chemical 3] [Chemical 4] [Chemical 5] [Chemical 6] [Chemical 7] [Chemical 8] [Chemical 9] [Chemical 10] [Chemical 11] [Chemical 12] [Chemical 13] [Chemical 14] [Chemical 15] [Chemical 16] 4. The method for determining a DNA nucleotide sequence according to claim 1, wherein the anthracene derivative is represented by the following chemical structural formula. [Chemical 17] [Chemical 18] [Chemical 19] [Chemical 20] [Chemical 21] [Chemical formula 22] [Chemical formula 23] [Chemical formula 24]