JPH1094A - Analysis of nucleic acid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To analyze a nucleic acid through such processes that a doubldstranded DNA specimen is treated with plural restriction enzymes and the difference between both the 3' terminals of the resultant DNA fragments is identified using plural primers, and these fragments are selectively separated by complementary strand synthesis followed by measuring the lengths of the fragments, and the distribution pattern of the lengths is detected. SOLUTION: A double-stranded DNA specimen 40 is cut with plural restriction enzymes into double-stranded DNA fragments 41-1 to 41-3, the difference between the base sequences at both the 3' terminals of these fragments is identified by the respective base sequences of the 1 to 4 bases at the respective 3' terminals of at least two kinds of DNA primers 49', 49", and the DNA fragments each with the base sequences at both the 3' terminals specified are subjected to complementary strand synthesis using the above two kinds of DNA primers in the respective vessels 55-2, 55-3 and selectively separated, and the reaction products are separated by electrophoresis and the respective lengths of the products are measured to detect the pattern of distribution of the above DNA fragment lengths to enable the long double-stranded DNA specimen to be tested, thus accomplishing the objective nucleic acid analysis.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to diagnosis using nucleic acids,
Nucleic acid property evaluation method, analysis method, and DNA used for the same
Regarding the primer set.

[0002]

2. Description of the Related Art The use of DNA for diagnosing diseases and the like is increasing. In this diagnosis, a DNA probe having a base sequence complementary to the target DNA is prepared,
Perform a probe test to see if the NA probe hybridizes with the target DNA,
A specific region of the base sequence of NA is selected, and base sequence information of a DNA fragment generated by PCR amplification is obtained using two DNA probes (primers) and used for diagnosis or the like.
These methods are good for examining one to several kinds of DNA, but are not suitable for examination of DNA containing a very large number of DNA fragments and evaluation of long DNA. However, DNA or genes in the living body work in association with each other,
There is a strong demand to comprehensively grasp and evaluate chromosomes or all contained DNA. For example, in the c-DNA project, which is attracting attention in the genome analysis project, when DNA functions in a living body, the DNA information is first transcribed into m-RNA, proteins are synthesized, and the living body functions. , C-DN which is the complementary strand from m-RNA
Attempts have been made to make A and to comprehensively understand living organisms from information on the type and amount of c-DNA.

[0003] The c-DNA corresponding to the m-RNA functioning in cells is separated, the base sequence of each c-DNA is determined, and the frequency of occurrence of the c-DNA base sequence in one tissue is examined. . For this purpose, first, the c-D
NA is prepared (in a state where various c-DNAs are mixed), and the desired c-DNA is cloned. Escherichia coli containing c-DNA is spread on an agar medium and cultured to obtain a colony. Since any one of the target c-DNA is contained in each colony, the target c-DNA is taken out, the base sequence is determined, and the type of the c-DNA is identified. When the type of c-DNA is identified for each colony one after another, the same type of c-DNA appears. When attention is paid to a specific c-DNA in one tissue, the frequency of appearance in a colony is higher because the larger the amount, the more the gene corresponds to the gene that is strongly expressed in that tissue. Therefore, the base sequence analysis of the c-DNA is performed on a large number of colonies, and the frequency of which c-DNA appears and how many times is determined (Reference: K. Mur)
akawa et. al. , Genomics, 23,
p379-p389 (1994)).

[0004] On the other hand, attempts have been made to perform DNA diagnosis by focusing on the genome (DNA in all chromosomes) or the entire specific chromosome. There is an analysis method called the Gene Scan method (restriction enzyme landmark genome scanning method) (Reference: Y. Hayashizaki et.a).
l. , "DNA POLYMORPHIS
M) ", vol. 3, p10-p15 (1995) (Toyo Shoten)). In this method, DNA is first converted to a first restriction enzyme (for example, an 8-base recognition enzyme such as NotI (average 64 k
Is cleaved once per base)).
A nucleotide labeled with a radioisotope or a fluorescent label is bound to the cut portion, and electrophoresed in a first direction using an agarose gel. After electrophoretic separation, a second restriction enzyme (eg,
The DNA fragment electrophoretically separated in the agarose gel is further cleaved by using a 4-base recognition enzyme (which is cleaved once every 256 bases), and then further cut in a second direction orthogonal to the first direction. Perform electrophoresis to obtain a two-dimensional migration pattern. Using the two-dimensional migration pattern as a fingerprint, the entire DNA is inspected. Attempts have been made to use the DNA in normal cells and the DNA of cells having abnormalities such as cancer for diagnosis by utilizing the fact that the two-dimensional electrophoresis pattern is different. However, at present, there is no good method for examining where large DNA has an abnormality.

[0005]

As described above,
Examining long base length DNA, and grasping and examining the overall picture of a sample containing multiple types of DNA are important for early detection of disease and understanding of the functions that DNA plays in cells. However, at present, there is no good method in the prior art. In the prior art relating to c-DNA analysis, it is necessary to determine the base sequence of a sample contained in a very large number of clones, and it is troublesome and time-consuming, so that it is not practical to apply to various samples. Was. Further, the conventional method using a DNA probe can analyze at most several to ten types of DNA at a time, and is not suitable for testing hundreds to tens of thousands of types of c-DNA and DNA fragments. There is a problem that the method cannot be applied to the inspection of long DNA whose location is unclear.

On the other hand, in the conventional Gene Scan method, a DNA having a long base length can be inspected.
And that the amount of the second restriction enzyme used for cutting the DNA fragment electrophoretically separated in the first direction is enormous, and that each DNA in the two-dimensional migration pattern
Since the positions of the fragments cannot always be obtained quantitatively in the two electrophoresis directions and the length of each DNA fragment subjected to the two-dimensional electrophoresis cannot be accurately determined, there is a problem that it is difficult to create a database as a fingerprint. . An object of the present invention is to provide a new fingerprint method and a DNA test method that overcome these problems of the prior art.

[0007]

In the present invention, first, a double-stranded DNA sample is cut with two or more types of restriction enzymes to obtain a group of double-stranded DNA fragments unique to the sample DNA having a plurality of lengths. Generate. Oligonucleotides having a known base sequence are bound to the 3 ′ ends on both sides of the double-stranded DNA fragment,
A region to which a part of the DNA primer can hybridize is provided. This region and the nucleotide sequence recognized by the restriction enzyme used and the D
Prepare multiple types of NA primer sets. The 1 to 3 bases at the 3 ′ end of the primer of the DNA primer set consist of a combination of substantially all bases (A, T, G, C).
This is a selected base sequence for identifying (classifying) a sequence of 1 to 3 bases following the recognition sequence of the restriction enzyme of the DNA fragment. The number of restriction enzymes used is the same as the number of DNA primer sets to be prepared. A double-stranded DNA fragment group was fractionated into the number of primer combinations of each DNA primer set,
A different combination of primers is added to each fraction, hybridized, and a complementary strand synthesis reaction is performed. The primer of each DNA primer set is labeled with a fluorescent substance or the like.
The product of the complementary strand synthesis reaction obtained in each fraction is separated by gel electrophoresis through separate migration paths, and the length of each DNA fragment is measured to obtain a fingerprint.

[0008] Even when the double-stranded DNA fragment group contains a large number of DNA fragments and the length is difficult to distinguish, in most cases,
Each DNA fragment has a different base sequence following the restriction enzyme cleavage site. Therefore, the DNA fragments are classified using the difference in the base sequence following the restriction enzyme cleavage portion of the DNA fragments. In order to classify DNA fragments, four to sixty-four types of DNA having the base sequence of 1 to 3 at the 3 ′ end having all combinations of base sequences
A complementary strand synthesis reaction is performed using primers. When the terminal 2 to 3 bases of the DNA primer have completely hybridized to the DNA fragment, the complementary strand synthesis reaction proceeds, but otherwise the reaction does not proceed.

When a double-stranded c-DNA is prepared from m-RNA, a complementary strand is bound to the 3 'end of the m-RNA using a primer containing a biotinylated oligo dT.
A double-stranded c-DNA is prepared by a DNA polymerase. Note that a double-stranded c-DNA may be prepared by the same operation with biotin inserted at the end of the c-DNA corresponding to the 5 ′ end of the m-RNA using the cap structure. This double-stranded c-DNA is divided into two fractions, and the first and second restriction enzymes are added to each fraction to cleave the double-stranded c-DNA. Except for the biotin-bound fragment, the first and second oligonucleotides are respectively bound to the cleaved DNA ends.

Next, a second restriction enzyme is added to the fraction to which the first restriction enzyme has been added, and the first restriction enzyme is added to the fraction to which the second restriction enzyme has been added, followed by re-cutting, The first and second oligonucleotides are respectively bonded to the cut portions. When the biotin-bound fragment is removed, both ends are cleaved by the first and second restriction enzymes, and the first and second restriction enzymes are cleaved.
DNA fragments flanked by the oligonucleotides are generated. That is, a fragment is generated in which both ends are cleaved by different restriction enzymes. When complementary strand synthesis was performed using a combination of primers (labeled) of each DNA primer set complementary binding to part or all of the recognition sequence of each restriction enzyme, both ends were cleaved by different restriction enzymes. Of the DNA fragments, the reaction proceeds only with the DNA fragment to which the terminal base of the primer has hybridized.

The length of the synthesized DNA strand is substantially the same as that of the original DNA fragment strand, and thus the length of the DNA strand can be measured by gel electrophoresis or the like. If the DNA fragment synthesized by one complementary strand synthesis reaction is less than the detection sensitivity, a plurality of complementary strand synthesis reactions can be performed using a heat-resistant DNA polymerase to amplify the amount of the synthesized DNA fragment. . In this case, it is possible to control whether or not to synthesize a complementary strand by changing the terminal base sequence of the primer.
The base sequence at the 3 ′ end is called a selection sequence. Only a specific DNA fragment can be selectively amplified using a primer having the selection sequence and complementary to the first and second oligonucleotides. For example, if two bases are used as the selection sequence,
DNA fragments can be obtained by classifying them into 4 ² × 4 ² = 256 types.

The DNA fragments classified into the 256 types are electrophoresed on different migration paths for each type to obtain a fingerprint pattern.

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, a nucleic acid analysis method for providing a fingerprint method which enables a long nucleic acid test according to the present invention will be described in detail with reference to the drawings.

FIG. 1 shows a method for preparing a mixture of DNA fragments from a sample DNA using a plurality of types of restriction enzymes, and performing a complementary strand synthesis reaction using first and second sets of DNA primers. FIG. 4 is a diagram for explaining a procedure for obtaining a DNA fragment having a predetermined base sequence of 2 bases at the 5 ′ end following a base sequence complementary to the entire recognition sequence of the restriction enzyme or a part of the 3 ′ end of the recognition sequence. is there. In the first embodiment, the sample DN
As A, Escherichia coli genomic DNA 1 (〜5 Mb) is used. NotI (8-base recognition restriction enzyme, base sequence G
Recognition sequence of CGGCCGC is 64 kb on average
Appears once, so that Escherichia coli genomic DNA1 is
Cut into about 100 double-stranded DNA fragments 2-
1, 2-2, 2-3,... The resulting double-stranded DN
Both ends of A fragment 2 (namely, NotI cleavage site)
To the DNA fragments 3-1 and 3- by binding a double-stranded biotinylated oligonucleotide (having a known base sequence) 100
2,... (Hereinafter, in each figure, biotin is represented by b for simplicity). The double-stranded biotinylated oligonucleotide (first oligonucleotide) 100 includes a single-stranded oligonucleotide (preferably having a base length of 10 to 30) 10 whose 5 ′ end is biotinylated, and an oligonucleotide 1
It comprises a single-stranded oligonucleotide 11 having a base sequence 101 complementary to 0 and all or a part of a base sequence (CGCCGGCG) 102 complementary to a base sequence recognized by NotI at the 5 ′ end.

Next, a restriction enzyme NlaIII (a 4-base recognition enzyme that recognizes the base sequence CATG)
The DNA fragments 3-1, 3-2, ... are further cut. N
Since the recognition sequence of laIII appears once every 256 bases on average, each DNA fragment 3-1, 3-2, ... is cleaved near both ends. DNA fragments 4-1, 4-2, ..., 4-n, 4'- fragmented by restriction enzyme NlaIII.
1, 4'-2, ..., both ends of 4'-m (that is, Nla
III), a double-stranded second oligonucleotide 110 is bound. The double-stranded second oligonucleotide (having a known base sequence) 110 is a single-stranded oligonucleotide 12 (the base length is preferably 10 to 30).
And a base sequence 111 complementary to the oligonucleotide 12 and a base sequence complementary to the base sequence recognized by NlaIII (G
TAC) 112 is composed of a single-stranded oligonucleotide 13 having all or a part of it at the 5 ′ end. As a result, the DNA fragment 4 ″-having the second nucleotide 110 at both ends was obtained.
DNA fragments 5-1, 5-n,... Having a first oligonucleotide 100 at one end and a second oligonucleotide 110 at the other end.
Is generated.

From these products, using avidin 15 bound to beads 14, a DNA fragment 5-1 having a first oligonucleotide 100 at one end and a second oligonucleotide 110 at the other end, 5-n, ...
To capture. The total number of fragments obtained as described above is
The vicinity of both ends of one fragment cleaved by NotI is cleaved to obtain two fragments, resulting in about 200.

Next, the captured fragment is denatured by heating or denaturing with alkali to obtain a complementary strand (DNA
Fragment) 6 (6-1, 6-2, 6-3, ...) is released. The complementary strand 6 thus obtained has a base sequence 101 complementary to the oligonucleotide 10 and Not at the 3 ′ end.
A nucleotide sequence complementary to the nucleotide sequence recognized by I (CGCCGG
CG) a single-stranded oligonucleotide 11 having 102 at the 5 'end, and an oligonucleotide 12 at the 5' end
This is a fragment having a single-stranded oligonucleotide 13 having a base sequence (GTAC) 112 complementary to the base sequence 111 and a base sequence recognized by NlaIII at the 5 ′ end.

The nucleotide sequence 16-1 consisting of the entire NotI recognition sequence or a part of the 3 ′ end of the NotI recognition sequence
And the primers 16-, 16-,... Of the first DNA primer set having the selected sequence 16-2 consisting of two bases following the base sequence 16-1 (only 16- is shown in FIG. 1). . This first DNA primer set 16 is composed of 16 types of primers having different base sequences of the selected sequence of 2 bases at the 3 ′ end. That is, the base sequence of the selected sequence of 2 bases at the 3 ′ end is A,
4 corresponding to all combinations of 2 bases from T, G, C
× 4 = 16 patterns. Similarly, the base sequence 17-1 consisting of the entire NlaIII recognition sequence or a part of the 3 ′ terminal side of the NlaIII recognition sequence, and the base sequence 17-
The primers 17-, 17- of the second DNA primer set having the selected sequence 17-2 consisting of 2 bases following 1
,... Are prepared (only 17- is shown in FIG. 1).
Similarly to the first DNA primer set 16, the second DNA primer set 17 is also composed of 16 types of primers having different base sequences of the selected base sequence of 2 bases at the 3 ′ end. In addition, each primer of the first DNA primer set 16 is fluorescently labeled at the 5 ′ end (the fluorescent label is indicated by *).

The solution containing the complementary strand 6 is dispensed into 256 vessels (not shown), and each of the 256 vessels contains one kind of primer selected from the first DNA primer set 16; One kind of primer selected from the second DNA primer set 17 is added. First and second
The selection of the types of primers from the DNA primer set is performed for a total of 16 × 16 = 256 combinations of the primers of the first and second DNA primer sets. Two kinds of primers selected by different combinations are added to each container. That is, the first DNA
Since there are 16 types together with the primer set and the second DNA primer set, the combination of primers is 16
× 16 = 256 patterns, and using these 256 combinations of primers, a complementary strand synthesis reaction of the complementary strand 6 is performed in 256 vessels (only one vessel is shown in FIG. 1 for simplicity). . In this complementary strand synthesis reaction, first, an extension reaction proceeds with respect to the DNA fragment 6-i (-strand) using the primer 16- of the first DNA primer set 16 as a primer to generate a double strand. The primer 17− of the second DNA primer set 17 is added to the positive strand that has been denatured by heating to form a single strand.
The elongation reaction proceeds with each of the primers 16-of the first primer set 16 with respect to the-strand, and a double strand is generated. The complementary strand synthesis reaction product thus obtained is subjected to gel electrophoresis and the spectrum is measured.
In gel electrophoresis, using denatured polyacrylamide,
The complementary strand synthesis reaction products in the 256 vessels are electrophoretically separated in a single-stranded state through 256 migration paths corresponding to the vessels. When four different types of fluorescent labels at the 5 ′ end of each primer of the first DNA primer set 16 are used, the primers of the first DNA primer set 16 having four different types of fluorescent labels are used. Can be performed in the same container,
The product of the complementary strand synthesis reaction in the 64 containers can be separated by electrophoresis in a single-stranded state on 64 migration paths corresponding to the containers.

FIG. 2 shows the D value of the above complementary strand synthesis reaction product.
It is a figure which shows a part of gel electrophoresis spectrum of NA fragment. In FIG. 2, reference numeral 21 denotes a spectrum obtained when all the DNA fragments of the above-mentioned complementary strand synthesis reaction product are electrophoretically separated, 22 denotes a spectrum of the above-mentioned complementary strand synthesis reaction product of each container, and 16- 2, 17-2 indicate the base sequence of the selected base portion of the primer used, and 24 indicates the base length of the DNA fragment. R1 and R2 are NotI located on the 5 'end side of the primers 16-, 16-,... Of the first DNA primer set 16, respectively.
Part or all of the base sequence of the recognition sequence of the primers 17-, 17-, of the second DNA primer set 17
... A part or all of the nucleotide sequence of the NlaIII recognition sequence at the 5 'end. When the lengths of all the DNA fragments are measured simultaneously on the same migration path, many peaks overlap as shown in spectrum 21, and the respective DNA fragments cannot be separated and cannot be analyzed. The spectrum 22 has a first and a second
The complementary strand synthesis reaction is performed by changing the combination of the two bases 16-2 and 17-2 of the primer selection sequence (identifying each DNA fragment) of the DNA primer set
Identification is performed based on the base sequences at both ends of the fragment, and DN is determined by a different migration path for each combination of the selected sequences 16-2 and 17-2.
It is a spectrum which separated the A fragment by gel electrophoresis. DN
Since the spectrum was obtained by separating the A fragment group for each combination of the selected sequences 16-2 and 17-2, each peak was separated, and the length (base length 24) of the DNA fragment contained in the spectrum was determined. You can know. In the case of the first embodiment, since the complementary strand synthesis reaction is carried out using 256 combinations of primers of the first and second DNA primer sets, 256 types of electropherogr are used.
am (DNA fragment spectrum) is obtained.
Electropherogram of fragment A is the original DN
It is peculiar to the A sample, and shows a different pattern when the sample DNA is different, so that it can be widely used for DNA diagnosis and the like. That is, a plurality of combinations of selected sequences by the primers of the first and second DNA primer sets (in FIG. 2, one selected sequence is fixed to AA and the other selected sequence is AA, AC, AG,...) , TG, and TT are shown as examples of combinations changed in 16 ways), sample DN
A can be analyzed more accurately. In Example 1, following the generation of DNA fragments 4 ″ -1, 4 ″ -2,..., DNA fragments 5-1, 5-n,.
By using boI (a 4-base recognition enzyme that recognizes the base sequence GATC), DNA fragments 4 ″ -1, 4 ″ -2,.
.., DNA fragments 5-1, 5-n,. In this case, the double-stranded third oligonucleotide may or may not be bound to both ends of the DNA fragment fragmented with the restriction enzyme MboI (that is, the cleavage site with the restriction enzyme MboI). When the DNA fragment is cut by the third restriction enzyme in this manner, for example,
When the third restriction enzyme cleaves 5-1 and 5-n shown in FIG. 1, no double-stranded DNA fragment can be obtained by performing the complementary strand synthesis reaction in FIG. Electrophoresis pattern (electrophoreogr)
Since am) is simple, the identification of the finger print pattern is facilitated, and the analysis of the sample is more accurate.

(Second Embodiment) The second embodiment employs m-RN
This is an example used for the analysis of A.

FIG. 3 shows a sample D obtained by synthesizing double-stranded c-DNA from m-RNA used in the second embodiment of the present invention.
FIG. 4 illustrates a conventional reaction procedure for obtaining NA.
In the second embodiment of the present invention, a predetermined one base following a recognition sequence of a restriction enzyme is cut by a sample DNA with two types of restriction enzymes and a complementary strand synthesis reaction using two types of DNA primer sets. Fig. 3 is a view for explaining a procedure for obtaining a DNA fragment having a base sequence having the following sequence at the 5 'end.

FIG. 3 shows a conventional technique for synthesizing double-stranded c-DNA from m-RNA used in the second embodiment of the present invention (literature: S. Sugano et. Al., "Protein Nucleic Acid Enzyme ( PROTEIN NUCLEIC ACID
AND ENZYME) ", vol. 38, no.
3, p276-p281 (1993) (Kyoritsu Shuppan)). As shown in FIG. 3, m-RNA extracted from the cells had a poly A tail 3 at the 3 ′ end.
And a cap base 31 and a phosphate group 3 at the 5 'end.
2 and full-length m-RNA35-1 having a phosphate group 32 at the 5 'end.
First, the m-RNAs 34-1 and 35-1 were treated with bacterial alkaline phosphatase to remove the phosphate group 32 at the 5 'end of the incomplete length m-RNA 35-1 having no cap structure. The RNA 35-1 is changed to a structure 35-2 in which the hydroxyl group 36 is exposed.

Next, the cells are treated with tobacco acid pyrophosphatase to obtain full-length m-RNA having a cap structure.
Exposing the phosphate group 32 only at the 5 'end of 34-1, m
-RNA34-1 is converted to a structure 3 in which the phosphate group 32 is exposed.
Change to 4-2. Next, using RNA ligase,
An m-RNA34-3 in which a synthetic RNA oligonucleotide 37 has been introduced into the 5 ′ end of the m-RNA34-2 is obtained. R
Since NA ligase binds an RNA oligonucleotide to a phosphate group and does not bind an RNA oligonucleotide to a 5′-terminal hydroxyl group, a synthetic oligonucleotide binds only to full-length m-RNA34-2.

As described above, full-length m-RNA34
After introducing the synthetic oligonucleotide 37 into only -1, the m-RNA is converted to c-DNA 38 using an oligo dT primer and reverse transcriptase. Furthermore, c-DNA3 was synthesized using DNA polymerase.
8 and synthesized various mRs extracted from cells
A mixture of double-stranded c-DNA 39 obtained for each NA is obtained as a double-stranded sample DNA 40.

FIG. 4 shows that a double-stranded sample DNA is cleaved by two kinds of restriction enzymes and a complementary base synthesis reaction is carried out by using two kinds of DNA primer sets to remove a predetermined one base following the recognition sequence of the restriction enzyme. FIG. 4 is a diagram for explaining a procedure for obtaining a DNA fragment having a base sequence at the 5 ′ end. It goes without saying that the method shown in FIG. 4 can also be applied to a single long double-stranded DNA sample. First, a double-stranded sample DNA 40 consisting of a mixture of double-stranded c-DNA was placed in a restriction enzyme NlaIII.
And HhaI. Restriction enzyme NlaIII,
HhaI respectively converts double-stranded DNA into CATG ↓, GCG ↓
Cut as C. 3 'of each fragment generated by cleavage
The terminus is CATG when cut with NlaIII.
(NlaIII-cut end) 46, when cut with HhaI, GCG (HhaI-cut end) 47. 5
0 is one base at the 5 ′ end following the recognition sequence site (NlaIII-cut end, HhaI-cut end) of each restriction enzyme, and N represents any of A, T, G, and C. At the 3 ′ end of each DNA fragment 41-1, 41-2, 41-3,.
Oligonucleotides are introduced by ligation, or poly-chains (poly-A chains, poly-C chains, poly-G chains, poly-T chains) using terminal nucleotidyl transferase.
Chain). In the following, poly A chain 4
The case where 2 is added will be described as an example.

When the poly-A chain 42 is added to the 3 'end of the DNA fragment, as shown in 41'-1 and 41'-2, Nl
The base sequence at the 3 ′ end cleaved with aIII is CATGA
A ... A3 ', and as shown in 41'-3, HhaI
The base sequence at the 3 'end cleaved with is GCGAA ... A3'
Becomes

Two sets of DNA probe (primer) sets 49 'and 49 "are prepared. Each primer of the DNA primer sets 49' and 49" is labeled with a fluorescent substance or the like. As a primer capable of binding a complementary strand to a cleavage site by NlaIII, the base sequence is 5'T ... TTCAT
GX3 ′ (X = AorCorGorT), a first DNA primer set (R
1-X, R1 = * T... TTCATG, * represents a fluorescent label). As a primer capable of binding a complementary strand to a cleavage site by HhaI, the base sequence is 5′T ... TTCG.
A second DNA primer set (R) consisting of four primers of CX3 ′ (X = AorCorGorT)
2-X, R2 = * T... TTCGC, * represents a fluorescent label).

The base sequence from the 5 'end of the primer of the first DNA primer set to CATG is NlaII.
Since it is complementary to all of the DNA fragments cleaved by I, it hybridizes to the end of the DNA fragment cleaved by NlaIII, but the base sequence X hybridizes only to the fragment having a base complementary to base X. Soy. For example, when X = A, the base X portion is the base N portion 5
It hybridizes only to a fragment having a base T in which 0 is complementary. When the portion 50 of the base N is not a complementary base, 5
As in 2, the base X does not hybridize. Similarly, 5 ′ of the primer of the second DNA primer set
Since the base sequence from the end to CGC is complementary to all of the DNA fragments cut with HhaI, it hybridizes to the end of the DNA fragment cut with HhaI, but the base sequence X is complementary to base X. Hybridize only to fragments with different bases. Whether or not the base X has completely hybridized is not clearly understood only by examining the stability of hybridization. This is because most sites of the DNA primer have a common portion that hybridizes to any fragment, and the difference in hybridization stability between only a few terminal bases does not make a sufficiently large difference. However, by using a complementary strand synthesis reaction using a DNA polymerase, it is possible to determine whether or not the terminal bases have completely hybridized.

DNA cut with two kinds of restriction enzymes
A solution containing the fragment in which the poly A chain 42 was added to the 3 ′ end of the fragment was mixed with 16 fractions 55 (55-1, 55-2,
.., 55-16 out of 55-16 are illustrated). 16 fractions 55-1, 55
, ..., 55-16, the first DNA primer set 49 '(R1-X: X = AorCorGorT)
And the second DNA primer set 49 ″
(R2-X ′: X ′ = AorCorGorT) Two kinds of primers composed of 16 kinds of different combinations from the primers were added to 16 fractions corresponding to the 16 kinds of combinations, and complementary strand synthesis was performed. Perform the reaction. In FIG. 4, two types of primers (49-2) (R1-C, R2-T) added to fraction 55-2 and two types of primers (49-R) added to fraction 55-3 are shown.
3) (R1-A, R2-T). The primer is D
Although the primer hybridizes to the NA fragment, only the primer which hybridized at both 3 ′ ends, such as 51 (in a state in which the portion of base N is completely hybridized), the complementary strand extended and extended the complementary strand. Form. A primer hybridized such that only one 3 ′ end is 52 (the state of base N is not hybridized) does not form a complementary strand and does not form an extended complementary strand.

By performing n cycles of the complementary strand synthesis reaction, the complementary strand 53 having the same length as the DNA fragment is amplified 2n times. When only one 3 ′ end of the primer completely hybridizes (52), the complementary strand is amplified n-fold even if the complementary strand synthesis is performed for n cycles. It can be easily distinguished from a completely hybridized case. Since the primer is, for example, fluorescently labeled, the length of the elongated complementary strand can be determined using a fluorescent gel electrophoresis apparatus.

Thereafter, as in the case of the first embodiment, the complementary strand synthesis reaction products obtained from the respective fractions are electrophoresed on separate electrophoresis paths, that is, separate electrophoresis is performed for each combination of primers. The sample DNA can be analyzed by electrophoresis in the migration path and comparing the gel electrophoresis spectra. In this case, since 4 × 4 = 16 combinations of primers, 16 types of DNA fragment spectra are obtained. The spectrum of this DNA fragment is the original m-RNA
Therefore, it can be used not only for analysis but also for diagnosis of m-RNA. As a fluorescent label at the 5 ′ end of each primer of the first DNA primer set, a different 4
When different types are used, the complementary strand synthesis reaction using the primers of the first DNA primer set having four different fluorescent labels can be performed in the same container.
The product of the complementary strand synthesis reaction in each container can be separated by electrophoresis in a single-stranded state on four migration paths corresponding to the containers.

In the second embodiment, a double-stranded sample DNA is cleaved with three types of restriction enzymes, and a complementary strand synthesis reaction is performed using three types of DNA primer sets. A DNA fragment having a base sequence having one base at the 5 'end can also be obtained. In this case, the first and second DNA primer sets (R1-
X, R2-X: X = AorTorGor C)
Third DNA primer set (R3-X: X = Aor
(TorGorC). A solution containing a fragment obtained by adding a poly-A chain 42 to the 3 ′ end of a DNA fragment cleaved by three kinds of restriction enzymes was used to prepare first, second, and third fluorescently labeled DNA primer sets (R1-X, R2-X, R
3-X,: X = AorTorGor), that is, 16 combinations of R1-X and R2-X, 16 combinations of R1-X and R3-X, and R2-X
After dividing into 48 fractions corresponding to a total of 48 combinations of 16 combinations of R3 and R3-X, the same treatment as above was performed, and the complementary strand synthesis reaction product obtained from each fraction was subjected to 48 separate electrophoresis runs. The sample DNA can be analyzed by comparing the gel electrophoresis spectra by electrophoresis on a gel path, that is, by electrophoresis on separate electrophoresis paths corresponding to each combination of primers. In this case, since there are 48 combinations of primers, 48 types of DNA fragment spectra are obtained. The spectrum of this DNA fragment shows not only more detailed analysis of m-RNA but also m-RNA
It can be used for diagnosis and the like.

The first and second DNA primer sets described in FIG. 4 (R1-X, R2-X: X = AorTorG)
orC) instead of R1-XY and R2-ZW, respectively.
(X, Y, Z, and W are any of A, T, G, and C)
First and second primers composed of primers having a base sequence of
May be used. As described in the first embodiment, such first and second DNA primer sets consist of 16 types of combinations of bases X and Y and 16 types of combinations of bases Z and WY. Are 16 × 16 = 256 combinations.
Therefore, after the solution containing the fragment obtained by adding the poly A chain 42 to the 3 ′ end of the DNA fragment cleaved by the two kinds of restriction enzymes is divided into 256 fractions, the same treatment as above is performed. As a result of performing such processing and performing electrophoresis on 256 migration paths, the same result as that shown in FIG. 2 can be obtained. When four different types of fluorescent labels at the 5 ′ end of each primer of the first DNA primer set are used, complementary primers using the primers of the first DNA primer set having four different types of fluorescent labels are used. The strand synthesis reaction can be carried out in the same container, and the products of the complementary strand synthesis reaction in the 64 containers can be separated by electrophoresis in a single-stranded state on 64 migration paths corresponding to the containers.

As described above, a sample DNA is cleaved by a plurality of types of restriction enzymes, and a complementary strand synthesis reaction is carried out using a plurality of types of DNA primer sets, thereby performing all of the recognition sequences of the restriction enzymes or the 3′-terminal of the recognition sequence. A DNA fragment having a predetermined base sequence following the partially complementary base sequence at the 5 ′ end can be obtained by separation, and a plurality of types of DNA fragment spectra obtained from the sample DNA can be obtained. The spectrum of the DNA fragment can be used not only for more detailed analysis of the sample DNA but also for diagnosis and the like.

(Third Embodiment) The third embodiment shows an example in which the present invention is used for analysis of m-RNA extracted from cells using a poly A tail.

FIG. 5 shows a third embodiment of the present invention.
-A double-stranded c-DNA mixture prepared from RNA is cleaved with two types of restriction enzymes, and a complementary strand synthesis reaction is performed to obtain a DNA fragment having a predetermined base sequence following the recognition sequence of the restriction enzyme on the 5 'end side. It is a figure explaining a procedure. Prior Art (Documents: Sambrook et. Al., MOlec
ullar cronong a laboratory
manual 2nd edition, 7.1-
7.36 (1987) (Cold Spring Har)
The cultured cells are treated with a buffer containing 1% SDS to break the cell membrane, and then treated with proteinase K, followed by centrifugation to extract nucleic acids in the cells according to Bor Laboratory Press). Using an oligo dT column, a component having a poly A tail, ie, m-RNA60 (mixture), is obtained from the nucleic acid component. m-RNA
From 60, double-stranded c-DNA 61 (mixture) is prepared using reverse transcriptase, biotinylated oligo dT primer 70, and DNA polymerase. The obtained double-stranded c-DNA61 (mixture) was subjected to fraction A (6
2) Divide into fraction B (63). The double-stranded c-DNA of fraction A (62) was cut with restriction enzyme NlaIII and fraction B
The double-stranded c-DNA of (63) is cut with a restriction enzyme HhaI.

Among the cleavage products, avidin 71 in which a double-stranded c-DNA containing biotin b fragment cleaved by NlaIII and a double-stranded c-DNA containing biotin b fragment HhaI 65 were bound to beads was used. NlaI of double-stranded c-DNA captured without biotin b
The fragment 66 cut by II and the fragment 67 cut by HhaI of the double-stranded c-DNA containing no biotin b are removed.
Double-stranded oligonucleotides 72 (complementary to the NlaIII cleavage site) and 73 (HhaI cleavage site) having a known base sequence complementary to the cleavage site of each restriction enzyme at the end of the double-stranded c-DNA in each fraction That complementarily bind to).

The double-stranded c-DNA of the fraction A (62) was digested with the restriction enzyme HhaI, and the double-stranded c-D of the fraction B (63) was digested.
The NA is cut with the restriction enzyme NlaIII. The beads were removed from each fraction, and the binding between avidin 71 bound to the beads and biotin b caused the double-stranded c-
The DNA is removed from each fraction, leaving a fragment 68 of HhaI not captured by the beads and a fragment 69 of NlaIII not captured by the beads in each fraction. Double-stranded oligonucleotides 72 and 73 having a known nucleotide sequence complementary to the cleavage site of each restriction enzyme were bound,
The main chain c-DNA fragments 81 and 81 'are obtained.

The restriction enzyme recognition site is Nl from the 3 'end.
In the double-stranded c-DNA present in the order of aIII and HhaI, a double-stranded c-DNA fragment 81 cut at two kinds of restriction enzyme sites in fraction B was obtained.
In the case of the double-stranded c-DNA existing in the order of NlaIII, a double-stranded c-DNA fragment 81 ′ which is cleaved at two kinds of restriction enzyme sites in fraction A is obtained. In addition, a double-stranded c-DNA fragment 80 may be generated in which both ends are cleaved with the same restriction enzyme, and such a fragment can be distinguished by a complementary strand synthesis reaction performed below.

Thereafter, the double-stranded c-DNA fragments obtained from Fraction A and Fraction B were mixed, and the first DNA primer set (restriction enzyme) was used in the same manner as in the first or second embodiment. N
having a nucleotide sequence complementary to the laIII recognition sequence),
A complementary strand synthesis reaction was carried out using a second DNA primer set (having a base sequence portion complementary to the recognition sequence of the restriction enzyme HhaI), and corresponding to the primer combination of the first and second DNA primer sets. The product of the complementary strand synthesis reaction is electrophoretically separated on separate electrophoresis paths, and the electrophoretic spectrum is measured, and used for DNA analysis and the like. When the 16 types of DNA primer sets described in the first embodiment are used, the obtained electrophoresis spectrum is 16 × 16
= 256 patterns, and when the four types of DNA primer sets shown in the second embodiment are used, the obtained electrophoretic spectrum is 4 x 4 = 16 patterns. When the first DNA primer set is composed of four types of primers with the selected sequence as one base and the second DNA primer set is composed of 16 types of primers with the selected sequence as two bases, 1, the second D
Primer combination of NA primer set is 4 × 1
Since 6 = 64 patterns, the resulting DNA fragment has 64 electrophoretic spectra.

As described above, in the conventional DNA diagnostic method using DNA primers, only several to ten types of DNA fragments can be examined at a time, and several hundred to several thousand types of c-DNA and Although it is unsuitable for DNA fragment testing and cannot be applied to long DNA testing where the abnormality is unknown, the present invention recognizes the oligonucleotide bound to the 3 ′ end of the DNA fragment and the restriction enzyme. Primer set having a base sequence of 1 to 4 bases (selection sequence) that hybridizes to all or a part of the base sequence to be hybridized and determines whether or not to hybridize with the target DNA fragment at the 3 ′ end Using multiple types, selectively identify DNA fragments, perform complementary strand synthesis, amplify, and support multiple types of DNA primer set primer combinations So it separates by gel electrophoresis in separate migration path, the length of the synthesized complementary strand could be obtained fingerprint from electrophoresis pattern representing the new fingerprinting to overcome the problems of the prior art,
Enables DNA testing. As described above, it is possible to easily classify a sample DNA by using a plurality of DNA primer sets, and to analyze a sample containing many DNA fragments.

The restriction enzyme used in the present invention provides any of 3 'overhang, 3' blunt end and 5 'overhang.
Polynucleotide (any one of poly A chain, poly C chain, poly G chain, poly T chain) as an oligonucleotide to be bound to the end of the DNA fragment by a restriction enzyme giving a 3 ′ blunt end
Is convenient.

In each of the embodiments described above,
When the electrophoretic separation pattern (finger print pattern) by electrophoretic separation is extremely complicated, each DNA primer set used in each of the above-described examples has a different DNA primer set.
Biotin was added to the NA primer, and only the finally synthesized complementary strand was captured. The captured DNA fragment was regarded as a new sample, and the type of the restriction enzyme used in each of the above Examples was changed. Then, the processing in each embodiment is performed again. The resulting electrophoretic separation pattern (finger print pattern) by electrophoretic separation is more easily distinguished, and the sample can be analyzed more accurately. That is, in the present invention, instead of using the length information of a large number of DNA fragments as a fingerprint as it is, the sample DNA is cut using a plurality of restriction enzymes, and the terminal base sequence following the cut portion of the restriction enzyme is used. Since DNA fragments are grouped and the fragment length is measured and fingerprinted for each group, the advantage of obtaining high DNA fragment discrimination ability without overlapping even with the same fragment length, and changing the combination of restriction enzymes Thus, there is an advantage that the discrimination ability can be adjusted.

To summarize the present invention described above, the nucleic acid analysis method of the present invention comprises the following steps: 1) obtaining a DNA fragment by cleaving a nucleic acid sample with two or more types of restriction enzymes; Binding an oligonucleotide containing a deoxynucleotide or an analog thereof to both ends of the DNA fragment; 3) one of the nucleotide sequence of the bound oligonucleotide and the nucleotide sequence recognized by the restriction enzyme following the nucleotide sequence of the bound oligonucleotide; A base sequence complementary to part or all,
A labeled DNA primer set having a selection base sequence consisting of 1 to 3 bases at the 3 ′ end (4 types when the selection base sequence is 1 base, 16 types when the base sequence is 2 bases, 3 types)
(4) a step of conducting a complementary strand synthesis reaction of a region sandwiched between base sequences recognized by different restriction enzymes using at least two or more sets of primers as primers; And a step of detecting a DNA fragment by electrophoretically separating a complementary strand synthesis product, and further has the following characteristics.

A) The nucleic acid sample is RNA, and the RNA is converted into a DNA chain using reverse transcriptase.

B) The base sequence of 1 to 3 bases at the 3 'end of the DNA primer consists of a combination of substantially all base types, and the DNA primer can be any of radioisotopes, biotin, fluorescent substances and the like. Be labeled.

C) Using a plurality of DNA primer sets in which one to three bases at the 3 'end have all possible base sequences at the same time, synthesize a complementary chain in a region sandwiched by base sequences recognized by two kinds of restriction enzymes. Is a labeled DNA fragment generated by the above method.

D) The restriction enzyme should give any of 3 'overhang, 3' blunt end and 5 'overhang.

Each DNA primer of the DNA primer set used in the nucleic acid analysis method of the present invention comprises:
_{5'N 1 N 2 ... N n X} 1 X 2 ... X m Z 1 Z 2 ... Z h Y 1 Y 2 ... Y
_k 3 ′, and N ₁ N ₂ ... N _n (5 ≦ n ≦ 27)
An oligonucleotide substantially complementary nucleotide sequences attached to the ends of NA _{fragments, X 1 X 2 ... X m} (1 ≦ m ≦ 6) is a part complementary to the nucleotide sequence of the restriction enzyme that recognizes a base sequence , Z ₁ Z ₂ ... Z _h (0 ≦ h ≦ 3) are nucleotide analogs capable of hybridizing with plural kinds of nucleotides, and Y ₁ Y ₂ ... Y _k (1 ≦ k ≦ 4) are A, C, G And T (4 to the kth power), characterized in that any of the nucleotides constituting N ₁ N ₂ ... N _n is labeled.

[0051]

As described above, according to the present invention, a sample DNA is cut with two or more types of restriction enzymes, and a predetermined DNA is cut.
Perform a complementary strand synthesis reaction using the A primer set,
The DNA fragments are amplified, a large number of DNA fragments are divided into groups, the number of DNA fragments in each group is reduced, and the composition of the sample DNA can be known from the comparison of the electrophoretic separation spectral pattern of the number of DNA fragments in each group. Sample D
The NA is cleaved with two types of restriction enzymes to clearly identify the nucleotide sequences at both ends of the DNA fragment, determine the fragment length, and increase the number of DNA fragments divided into groups.
Assuming that the number of selected base sequences of the primer for identifying the sequence at the end of the DNA fragment is, for example, 3 bases, 4 ³ = 6
The DNA fragments can be divided into groups in four ways. This 6
DNA consisting of primers having four different base sequences
When two primer sets are used, 64 × 64 = 409
The DNA fragments can be divided into groups in six ways. In the resolution of gel electrophoresis, more than 100 kinds of D
Since the NA fragments can be separated and identified, the total of 4096 x 100 / 400,000 types of DN
The A fragment can be distinguished. In the present invention, it is possible to obtain a fingerprint pattern that enables a long nucleic acid to be tested from a large number of DNA fragments obtained from a long nucleic acid by a restriction enzyme.

[Brief description of the drawings]

FIG. 1 shows a first embodiment of the present invention, in which a DNA fragment mixture is prepared from a sample DNA and DN
The figure explaining the procedure to obtain the A fragment.

FIG. 2 is a diagram showing a part of a gel electrophoresis spectrum of a DNA fragment of a complementary strand synthesis reaction product according to the first embodiment of the present invention.

FIG. 3 shows m-RNA used in the second embodiment of the present invention.
FIG. 4 is a diagram for explaining a conventional reaction procedure for obtaining a sample DNA by synthesizing a double-stranded c-DNA from a DNA.

FIG. 4 is a diagram showing a procedure for obtaining a DNA fragment by a complementary strand synthesis reaction by cleaving a double-stranded c-DNA mixture using two types of restriction enzymes in a second example of the present invention.

FIG. 5 is a third embodiment of the present invention, in which a double-stranded c-DNA mixture prepared from m-RNA is cleaved with two types of restriction enzymes to obtain a DNA fragment by a complementary strand synthesis reaction. FIG.

[Explanation of symbols]

1 ... E. coli genomic DNA, 2-1 2-2, ... ... double-stranded DNA fragment, 3-1, 3-2, ... ... DNA fragment with the first nucleotide bound to both ends, 4-1 4-2,
..., 4'-1, 4'-2, ... DNA fragments cleaved with NlaIII, 4 "-1, 4" -2, ... DNA fragments having second nucleotides at both ends, 5-1, 5-
2,..., A DNA fragment having the first nucleotide bound to one end and the second nucleotide bound to the other end, 6 (6-
1, 6-2, ...) ... complementary strand not containing biotin, 10 ...
A single-stranded biotinylated nucleotide paired with the NotI recognition sequence; 101, a base sequence complementary to the oligonucleotide 10; 102, a base sequence complementary to the base sequence recognized by NotI; A single-stranded oligonucleotide having all or part thereof at the 5 ′ end, 100... A double-stranded biotinylated oligonucleotide,
12: single-stranded oligonucleotide; 111: base sequence complementary to oligonucleotide 12, 112: NlaIII
A single-stranded oligonucleotide having a base sequence complementary to the base sequence recognized at step 13 ... base sequence 111 and all or a part of base sequence 112 at the 5 'end, 110 ... double-stranded second base
Oligonucleotides, 14 ... beads, 15 ... avidin, 16-1 ... all of the NotI recognition sequences or a base sequence consisting of a part of the 3 'end of the NotI recognition sequence, 16-
2 ... Selected sequence consisting of 2 bases, 16 (16-, 16-
, ...): a primer of the first DNA primer set that is fluorescently labeled, 17-1 ... a base sequence consisting of the entire NlaIII recognition sequence or a part of the 3 'end of the NlaIII recognition sequence, 17-2 ... 2 bases Selection sequence, 17
(17-, 17-,...)... The primers of the second DNA primer set, 21... Spectrum when all the DNA fragments are measured simultaneously without segmentation, 22... Spectrum with the segmented DNA fragments, 24. ,
31: cap base, 32: phosphate group, 33: poly A tail, 34-1: full length m-RNA, 35-1: incomplete length m-RNA, 34-2: phosphate of m-RNA 34-1 Structure exposing group 32, 37 ... Synthetic RNA oligonucleotide, 34-3 ... Synthetic RNA oligonucleotide 37
M-RNA, 36 ... hydroxyl, 35-2 ... m-
Structure in which the hydroxyl group 36 of RNA35-1 is exposed, 38 ...
c-DNA, 39: double-stranded c-DNA, 40: double-stranded sample DNA, 41-1, 41-2, ... c-DNA fragment, 42: poly A chain, 41'-1, 41 ' ..., a DNA fragment having a poly A chain 42 added to the 3 'end, 46 ... N
LaIII cut end, 47 ... HhaI cut end, 4
9 ′, 49 ″ DNA primer set 49-1, 49-2, 49-3,..., Two types of primers to be added to the fraction, 50 ... N part, 51 ... N part completely hybridized, 52 ... N-part not hybridized, 53 ... Extended complementary strand,
55 (55-1, 55-2, ...) ... fraction, 60
... m-RNA mixture, 61 ... double-stranded c-DNA mixture,
62 ... fraction A, 63 ... fraction B, 64 ... c containing biotin
-NlaIII digestion fragment of DNA, 65 ... HhaI digestion fragment of c-DNA containing biotin, 66 ... NlaIII digestion fragment of c-DNA containing no biotin, 67 ... HhaI digestion fragment of c-DNA containing no biotin, 68 ...
HhaI cleavage fragment not captured by beads, 69 ... NlaIII cleavage fragment not captured by beads, 70 ...
Biotinylated oligo dT primer, 71 ... avidin bound to beads, 72 ... oligonucleotide corresponding to NlaIII cleavage site, 73 ... oligonucleotide corresponding to HhaI cleavage site, 80 ... c cleaved at both ends by the same restriction enzyme -DNA fragments, 81, 81 '... c-DNA fragments cleaved by two types of restriction enzymes and having oligonucleotides bonded to respective cleavage sites.

Claims (15)

[Claims] (1) a step of obtaining a double-stranded DNA fragment by cleaving a double-stranded DNA sample with a plurality of restriction enzymes; and (2) determining the difference in the base sequences at both 3 'ends of the double-stranded DNA fragment. The double-stranded DNA having at least two types of DNA primers, each having a predetermined base sequence, wherein the base sequences at both 3 ′ ends of the double-stranded DNA fragment are identified by base sequences of 1 to 4 bases at the 3 ′ end. A step of selectively separating fragments using a complementary strand synthesis reaction using the two types of DNA primers; and 3) a step of measuring a length of a product of the complementary strand synthesis reaction, A nucleic acid analysis method, comprising detecting a pattern of distribution of DNA fragment lengths by the plurality of restriction enzymes. 2. A step of obtaining a double-stranded DNA fragment by cleaving a double-stranded DNA sample with a plurality of restriction enzymes; and 2) binding oligonucleotides to both ends of the double-stranded DNA fragment. 3) a base sequence of the oligonucleotide, a base sequence following the base sequence of the oligonucleotide, which is complementary to a part or all of the base sequence recognized by the restriction enzyme, and 3 ′
Using a plurality of sets of labeled DNA primers each having a selected base sequence consisting of 1 to 4 bases at the end, using a plurality of sets of DNA primer sets each comprising a plurality of labeled DNA primers, Performing a complementary strand synthesis reaction in the region of the single-stranded DNA fragment; and 4) electrophoretically separating the product of the complementary strand synthesis reaction, wherein the double-stranded DNA sample is analyzed. Nucleic acid analysis method. 3. The method of claim 2, wherein RN
A method for analyzing nucleic acid, comprising the step of obtaining the double-stranded DNA sample from the A sample using reverse transcriptase. 4. The nucleic acid analysis method according to claim 2, wherein the selected base sequence comprises a combination of substantially all base types. 5. A method comprising: 1) cutting a double-stranded DNA sample with a first restriction enzyme to obtain a double-stranded DNA fragment; 2) attaching a first oligonucleotide to both ends of the double-stranded DNA fragment. Binding; 3) cleaving the product of step 2) with a second restriction enzyme to obtain a double-stranded DNA fragment; 4) attaching a double-stranded DNA fragment obtained in step 3) to the end of the double-stranded DNA fragment. Second
5) a step of capturing the double-stranded DNA fragment having the first and second oligonucleotides at both ends, and 6) a step of capturing the two strands captured in step 5). After converting the single-stranded DNA fragment into single-stranded DNA fragments, fractionating the liquid containing the single-stranded DNA fragment into a plurality of containers; 7) the base sequence of the first oligonucleotide; Following the oligonucleotide base sequence, a base sequence complementary to a part or all of the base sequence recognized by the first restriction enzyme, and a first base consisting of 1 to 4 bases at the 3 ′ end
A first DNA primer set comprising a plurality of labeled first DNA primers having a selected base sequence of:
Following the base sequence of the second oligonucleotide, and the base sequence of the second oligonucleotide, a base sequence complementary to a part or all of the base sequence recognized by the second restriction enzyme, A second DNA primer set comprising a plurality of labeled second DNA primers having a second selected base sequence consisting of 1 to 4 bases,
A DNA primer comprising a combination of the first DNA primer and the second DNA primer is added to each of the containers in correspondence with each of the combinations, so that the first and second restriction enzymes recognize the combination. Performing the complementary strand synthesis reaction of the region of the double-stranded DNA fragment sandwiched by the base sequences to be performed in each of the containers; and 8) electrophoretically separating the product of the complementary strand synthesis reaction in each of the containers. And analyzing the double-stranded DNA sample. 6. The nucleic acid analysis method according to claim 5, wherein in step 8), the products of the complementary strand synthesis reaction in each of the vessels are separated by electrophoresis on different migration paths. 7. The nucleic acid analysis method according to claim 5, further comprising, following the step 5), a step of cleaving the product of the step 5) with a third restriction enzyme to obtain a double-stranded DNA fragment. Nucleic acid analysis method. 8. A method comprising the steps of: 1a) cleaving a double-stranded DNA sample with a first restriction enzyme to retain a double-stranded DNA fragment on a carrier; 2a) attaching a first oligo to the end of said double-stranded DNA fragment. 3a) a step of cleaving the product of step 2a) with a second restriction enzyme to obtain a double-stranded DNA fragment; 4a) a step of cutting the double-stranded DNA fragment obtained in step 3a). Binding a second oligonucleotide to the terminus; 1b) cleaving the double-stranded DNA sample with the second restriction enzyme to retain a double-stranded DNA fragment on a carrier; 2b) step 1b) Bonding the second oligonucleotide to the end of the double-stranded DNA fragment obtained in 3), 3b) cleaving the product of step 2b) with the first restriction enzyme to obtain a double-stranded DNA fragment And 4b) obtained in step 3b) Binding the first oligonucleotide to the end of the double-stranded DNA fragment; and 5) mixing the products of steps 4a) and 4b) to mix the double-stranded DNA fragment held on the carrier. After removal,
Converting the double-stranded DNA fragment into a single-stranded DNA fragment, and fractionating the solution containing the single-stranded DNA fragment into a plurality of containers; 6) the base sequence of the first oligonucleotide; Following the base sequence of the first oligonucleotide, a base sequence complementary to a part or all of the base sequence recognized by the first restriction enzyme, and a first base consisting of 1 to 4 bases at the 3 ′ end.
A first DNA primer set comprising a plurality of labeled first DNA primers having a selected base sequence of:
Following the base sequence of the second oligonucleotide, and the base sequence of the second oligonucleotide, a base sequence complementary to a part or all of the base sequence recognized by the second restriction enzyme, A second DNA primer set comprising a plurality of labeled second DNA primers having a second selected base sequence consisting of 1 to 4 bases,
A DNA primer comprising a combination of the first DNA primer and the second DNA primer is added to each of the containers in correspondence with each of the combinations, so that the first and second restriction enzymes recognize the combination. Performing the complementary strand synthesis reaction of the region of the double-stranded DNA fragment sandwiched by the base sequences to be performed in each of the containers; and 7) electrophoretically separating the product of the complementary strand synthesis reaction in each of the containers. And analyzing the double-stranded DNA sample. 9. The nucleic acid analysis method according to claim 8, wherein in step 7), the products of the complementary strand synthesis reaction in each of the containers are separated by electrophoresis on different migration paths. 10. A step of obtaining a double-stranded DNA fragment by cleaving a double-stranded DNA sample with a plurality of types of restriction enzymes; and 2) adding a plurality of types of oligonucleotides to both ends of the double-stranded DNA fragment. A step of binding; 3) a step of fractionating the solution containing the double-stranded DNA fragment into a plurality of containers; 4) the base sequence of the oligonucleotide, and the restriction enzyme following the base sequence of the oligonucleotide. A base sequence complementary to a part or all of the recognized base sequence;
A DNA primer comprising a combination of the DNA primers from each of a plurality of sets of a DNA primer set comprising a plurality of labeled DNA primers having a selected base sequence consisting of 1 to 4 bases at the end, The complementary strand synthesis reaction of the region of the double-stranded DNA fragment sandwiched between the base sequences recognized by the two types of restriction enzymes was added to each of the containers corresponding to each of the combinations, and the reaction was performed in each of the containers. And 5) electrophoretically separating the product of the complementary strand synthesis reaction in each of the vessels, wherein the double-stranded DNA sample is analyzed. 11. The method of claim 10, wherein the nucleic acid analysis method comprises:
A nucleic acid analysis method, wherein in step 5), the products of the complementary strand synthesis reaction in each of the containers are subjected to electrophoretic separation on different migration paths. 12. The method of claim 10, wherein the nucleic acid analysis method comprises:
A nucleic acid analysis method, wherein the double-stranded DNA sample is cleaved with two types of restriction enzymes in step 1). 13. The nucleic acid analysis method according to claim 10, wherein
A nucleic acid analysis method, wherein the double-stranded DNA sample is cleaved with three types of restriction enzymes in step 1). 14. The method of claim 10, wherein the nucleic acid analysis method comprises:
The oligonucleotide is poly-N (N = AorTorr)
(GorC) chain. 15. A DNA primer set used in the nucleic acid analysis method of claims 2, 5, 8, and 10, wherein the DNA primer set comprises a plurality of DNA primers,
NA _{primer, 5'N 1 N 2 ... N n} X 1 X 2 ... X m Z 1 Z 2
... has the structure of _{Z h Y 1 Y 2 ... Y} k 3 ', N 1 N 2 ... N n (5
≦ n ≦ 27) is a base sequence substantially complementary to the oligonucleotide bound to the end of the double-stranded DNA fragment, and N ₁ N ₂
... are any nucleotide labeling constituting the _{N n, X 1 X 2 ...} X m (1 ≦ m ≦ 6) is a complementary base sequence and part or all of the restriction enzymes that recognize base sequences Yes, Z
₁ Z ₂ ... Z _h (0 ≦ h ≦ 3) are nucleotide analogs capable of hybridizing with a plurality of types of nucleotides,
_{Y 1 Y 2 ... Y k (} 1 ≦ k ≦ 4) is, A, C, DNA primer set characterized by comprising the type of combination of G and T (4 k-th power).