JPH11346766A - Comparative analysis of nucleic acid sequence and reagent kit therefor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for the comparative analysis of nucleic acid sequences through making use of the fragment analysis of fragmented sample nucleic acid. SOLUTION: This method for the comparative analysis of a nucleic acid sequence comprises the following procedure: a wild type-derived sample DNA 1 and a variant type-derived sample DNA 2 are fragmented with restriction enzymes, respectively, to prepare two kinds of 1st fragment group 4 and 5, which are then mixed with each other to effect complete modification followed by complementary strand formation again, thereby affording a 2nd fragment group 8 made up of completely complementary strands 9, 10, and complementary strands 11 formed between variant type-derived fragments and wild type-derived fragments; subsequently, only the single strand 14 portion of the variant part 3 of the fragments 11 is specifically cleaved with an enzyme to afford a 3rd fragment group 15; the fragment analysis of the fragments of the 3rd fragment group and that of the fragments formed of the wild type-derived fragments alone are then performed and the electrophoretic patterns of both kinds of fragment are compared with each other; thereby the presence/absence of the variation in the variant type sample DNA 2 can be confirmed, and the base sequence can be determined by selectively amplifying only the fragment containing the variant part 3.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a nucleic acid sequence comparative analysis method based on the basic techniques of nucleic acid degradation and complementary strand synthesis by an enzyme, and a reagent kit used therefor.

[0002]

2. Description of the Related Art In order to compare the sequences of genes or genomes, it is desirable to perform the comparison after determining the entire sequence of the target sample. However, when comparing only one base such as a point mutant, the method of determining and comparing all sequences is very wasteful and inefficient. It is thought that cancers are caused by point mutations in genes, and in order to spread genetic diagnosis in the future, it is desirable to develop a method for easily detecting point mutants with limited mutation regions. It is.

[0003] Conventionally, point mutants have been detected by SSCP ( S
ingle S trand C onformation
( Polymorphism) method. SSCP is a method in which nucleic acids are completely denatured once and
This is a method in which a two-dimensional structure is formed in a sample nucleic acid after it is brought into a main chain state, and the sample nucleic acid is electrophoretically separated and analyzed on a native gel. The structure of the wild type and the mutant are different from each other, and the mobility by electrophoresis is also different. By utilizing the difference in mobility, a mutant can be detected by comparison with a wild-type electrophoresis pattern. However, in this method, the length of the detectable nucleic acid is about 200 to 300 bases. In the case of a nucleic acid longer than this, the difference in the shape of the two-dimensional structure is small, and the difference in the structure is unlikely to appear in the difference in mobility due to electrophoresis. Further, it is extremely difficult to perform electrophoresis without breaking the two-dimensional structure, and it is difficult to obtain stable data.

[0004] As a method of overcoming the above problems,
A hybrid of a wild type and a mutant (complementary strand forming product between two nucleic acids) is prepared, and only the mutated portion having a different sequence, that is, the portion that cannot form a complementary strand and is in a single-stranded state is specified. Ribonuclease A (Am
Bion; MisMatch Detect II)
And T4 endonuclease VII (Nucl
eic Acid Research, 1995, Vo
l. 23, 5082-5084) and the like have been developed. In the case of this method, 500-
It is possible to analyze a sample nucleic acid up to about 1,000 bases in length. However, even genes that will be analyzed are short, having an average length of 2 to 3 k bases, and a method capable of analyzing longer bases is desired. In any of the above methods, the presence or absence of a mutation can be determined, but in order to obtain the sequence of the mutated portion, it is necessary to determine the full-length nucleotide sequence after all.

[0005]

As described above, the base length of the target nucleic acid is limited to about 1 k base length in any of the conventional methods. In addition, sequencing of the mutated portion cannot be determined without sequencing the full length of the sample nucleic acid. For this reason, it is possible to detect a mutation even in a nucleic acid having a length of 1 k bases or more, and to develop a method of extracting only a portion having a mutation and determining a sequence. An object of the present invention is to provide a nucleic acid sequence comparative analysis method and a reagent kit that solve these problems.

[0006]

According to a first aspect of the present invention, a wild-type sample nucleic acid (double-stranded) and a mutant sample nucleic acid (double-stranded) are separately fragmented in advance to form a double-stranded nucleic acid. Two types of fragments of the first fragment group were obtained, and then two types of the second fragment group of the double-strand in which both ends of each fragment of the first fragment group of the double-strand were aligned were obtained,
After the two kinds of second fragment groups are mixed to form a single strand, hybridization is performed. At this time, a portion having a sequence difference between the wild type and the mutant cannot form a complementary strand, and a single-stranded portion is formed. Enzymatic degradation is performed using the enzyme that specifically recognizes only the single-stranded portion to obtain a double-stranded third fragment group.

A double-stranded fragment (hybrid fragment) containing a mutation in one chain is cleaved at the mutated portion and divided into a plurality of fragments. Subsequently, an oligomer having a known base sequence at both 3 'ends of all fragments of the double-stranded third fragment group is introduced. The third oligomer into which the oligomer thus obtained was introduced
The fragment group of (1) is obtained by a known fragment analysis method (Ele).
crophoresis, vol. 17, 1833-
1840 (1996)) to determine the sequence of the two bases at the 5 ′ end following the restriction enzyme recognition portion of each fragment of the third fragment group into which the oligomer has been introduced, and the fragment length. That is, an electrophoresis pattern (a) is obtained by fragment analysis using a hybrid nucleic acid of a wild-type sample nucleic acid and a mutant sample nucleic acid, a wild-type sample nucleic acid (double-stranded), and a mutant sample nucleic acid (double-stranded) as an analysis sample. Can be

[0008] The fragment group consisting of only the wild type or the fragment group consisting of only the mutant type is similarly analyzed by the fragment analysis method. That is, after fragmenting each sample nucleic acid of the wild-type sample nucleic acid (double-stranded) and the mutant sample nucleic acid (double-stranded),
An oligomer having a known base sequence is introduced into both 3 ′ ends of each fragment, and an electrophoresis pattern (b) is obtained by fragment analysis of a wild-type sample nucleic acid, and an electrophoresis pattern is obtained by fragment analysis of a mutant sample nucleic acid (double-stranded). (C) is obtained. Comparison of the electrophoretic peaks (fragment peaks) appearing between the electrophoretic patterns (a) and (b) or between the electrophoretic patterns (a) and (c).

When no mutation is contained, the electrophoresis patterns (a), (b) and (c) are the same.

[0010] Among the fragments of the third fragment group, the fragment containing the mutation is divided into a plurality of fragments, and in the electrophoresis pattern (a), the pattern of the divided fragments derived from the hybrid fragment is detected. In this way, the presence or absence of a mutation can be easily confirmed from the electrophoresis pattern. In the fragment analysis method, it is possible to easily amplify only a target fragment from a fragment mixture using a selection primer with reference to an electrophoresis pattern. Therefore, referring to the electrophoresis pattern obtained by fragment analysis, only the fragment containing the mutated portion can be amplified and sequenced from the third fragment group into which the oligomer has been introduced.

The first structure of the nucleic acid sequence comparison analysis method of the present invention is as follows. (1) A double-stranded first nucleic acid sample and a double-stranded second nucleic acid sample are each fragmented with a restriction enzyme. ,
Obtaining two double-stranded first fragment groups derived from the first and second nucleic acid samples; and (2) mixing the two first fragment groups to denature all fragments. A step of subsequently performing a complementary strand formation reaction to obtain a double-stranded second fragment group, and (3) a double-stranded third fragment group obtained by decomposing the single-stranded portion generated in step (2) with an enzyme. And a step of (4) obtaining the distribution of fragment lengths by electrophoresing the complementary strand of each fragment of the third fragment group.

In the first configuration, (a) step (1),
In place of (1) and (2), the first and second nucleic acid samples are pre-mixed, the first and second nucleic acid samples are denatured, and a complementary strand formation reaction is performed to limit the complementary strand formation reaction product. (B) fragment length obtained by subjecting each fragment of the fragment group obtained by fragmenting the first or second nucleic acid sample with a restriction enzyme to gel electrophoresis; Comparing the distribution of the fragment length obtained in step (4) with the distribution of the fragment length obtained in the step (4). (D) following step (3), a step of introducing an oligomer having a known base sequence into both 3 ′ ends of each fragment of the third fragment group by ligation,
(E) following the step (3), a step of introducing polyA, polyU, or polyT into both 3 ′ ends of each fragment of the third fragment group using terminal deoxynucleotidyl transferase. , (F) following step (3),
Introducing an oligomer having a known base sequence into both 3 'ends of each fragment of the third fragment group by ligation; and a first base labeled at the 5' end and complementary to at least a part of the base sequence of the oligomer. A sequence, a second base sequence complementary to the restriction enzyme recognition sequence of the restriction enzyme following the 3 ′ end of the first base sequence, and a second base sequence consisting of 1 to 3 bases following the 3 ′ end of the second base sequence. Performing a complementary strand extension reaction using each of the fragments of the third fragment group as a template using a primer having a base sequence of 3 in the step (4). Electrophoresis, (g)
The enzyme of step (3) is any of S1 nuclease, mag bean nuclease, ribonuclease A, and T4 endonuclease VII; (h) the first and second sample nucleic acids are DNA or RNA; Further, it is a reagent kit used for the nucleic acid sequence comparative analysis method of the first configuration,
A restriction enzyme for fragmenting the first and second sample nucleic acids, an enzyme for degrading the single-stranded portion of the double-stranded nucleic acid fragment, and a third fragment group containing at least ligase or terminal deoxynucleotidyl transferase A reagent kit comprising a reagent for introducing an oligomer having a known base sequence into both 3 ′ ends of a fragment, and a 5′-end labeled primer having a base sequence complementary to at least a part of the base sequence of the oligomer There is a feature.

In the second configuration of the present invention, the wild-type sample nucleic acid (double-stranded) and the mutant sample nucleic acid (double-stranded) are separately short-fragmented in advance, and the double-stranded first fragment group 2 After obtaining the seeds and then aligning both ends of each fragment of the two first fragment groups, 2 of the two-chain second fragment group in which the OH groups at both 3 ′ ends of each fragment were modified. Get each seed. In the modification of the 3 'end, for example, a double-stranded chain having a known sequence has a phosphate group at the 5' end of one end, an OH group at the 3 'end and a dideoxynucleotide triad at the 3' end of the other end. Adapters composed of phosphate are added to both ends of each fragment.
Next, the two kinds of second fragment groups whose both 3 ′ ends are modified are mixed to form a single strand, followed by hybridization. At this time, a portion having a sequence difference between the wild type and the mutant cannot form a complementary strand, and a single-stranded portion is formed. Enzymatic degradation is performed using the enzyme that specifically recognizes only the single-stranded portion to obtain a double-stranded third fragment group.

[0014] Of the third fragment group thus obtained, only the 3 'end that has been enzymatically decomposed after the formation of the complementary strand is labeled with a fluorescent substance or the like to obtain a labeled fragment. Fragments other than the fragment that was enzymatically degraded after the formation of the complementary strand were already 3 ′
The end is modified and no label can be added. By detecting only the labeled third fragment thus obtained, the presence or absence of the mutation can be easily confirmed without using a fragment analysis method. Fragments with the detected mutations can be easily sorted.

The second configuration of the nucleic acid sequence comparative analysis method of the present invention is as follows. (1) A double-stranded first nucleic acid sample and a double-stranded second nucleic acid sample are each fragmented with a restriction enzyme. ,
Obtaining two types of double-stranded first fragment groups derived from the first and second nucleic acid samples; and (2) obtaining two fragments of each fragment group of the two first fragment groups. (A) a step of modifying the OH group at the terminal, and (3) mixing the two first fragment groups whose both 3 'terminals are modified with the OH group, denaturing all the fragments, and then performing a complementary strand forming reaction. (4) obtaining a double-stranded second fragment group; (4) obtaining a double-stranded third fragment group by decomposing the single-stranded portion generated in step (3) with an enzyme; And (3) a step of labeling the 3 ′ end of the terminal cleaved by the enzyme in step (4) and a step of (6) detecting the fragment labeled with the 3 ′ end by gel electrophoresis.

In the first configuration, (I) the label in step (5) is a fluorescently labeled nucleotide added using terminal deoxynucleotidyl transferase, and (II) the label in step (5) is (III) detecting a difference in nucleotide sequence between the nucleotide sequences of the first and second nucleic acid samples due to a point mutation, insertion, or deletion. (IV) the enzyme of step (4) is any of S1 nuclease, Magbean nuclease, ribonuclease A, and T4 endonuclease VII; and (V) the first and second sample nucleic acids are DNA Or a reagent kit for use in the nucleic acid sequence comparative analysis method of the first configuration, wherein the restriction enzyme is used to fragment first and second sample nucleic acids. An enzyme that modifies the 3 ′ end of a double-stranded fragment generated by a restriction enzyme, an enzyme that decomposes a single-stranded portion of a double-stranded nucleic acid fragment, and a third fragment group that includes at least ligase or terminal deoxynucleotidyl transferase And a reagent for labeling the 3 ′ end of the fragment with the enzyme (1).

When the present invention is used, the sample nucleic acid is analyzed after being fragmented into short fragments, so that mutations from nucleic acids having a length of 1 k or more can be detected. Further, only the detected mutated portion can be picked up, the nucleotide sequence of the mutated portion can be easily determined, and the problem of the conventional method can be solved.

[0018]

Embodiments of the present invention will be described below in detail with reference to the drawings. FIG. 1 is a diagram illustrating a process of preparing first, second, and third fragment groups from a wild-type sample DNA and a mutant-type sample DNA. The sample DNA (double-stranded) 1
puc19 DNA (about 2.7 kb) into which a fragment of about 200 bp containing exon 7 (110 bp) of wild-type p53 gene was inserted. Sample DNA 2 (double-stranded) was puc19 DNA.
Exon 7 of mutant p53 gene (110
bp) into which a fragment of about 200 bp was inserted. The sequence of the sample DNA2 has a mutated portion 3. Sample DN
The sequences of A1 and A2 are all the same except for the mutated part 3.

(Shortening of Sample DNA) Sample DNA 1,
And 2 are fragmented with a restriction enzyme to prepare a first fragment group.
In this example, the restriction enzyme HhaI was used. However, the restriction enzyme may be any class I, such as SmaI or Hsp92II, and is not limited to HhaI. Restriction enzyme Hha
By the fragmentation using I, a double-stranded first fragment group 4 from the sample DNA 1 and a double-stranded first fragment group 5 from the sample DNA 2 are respectively prepared. The reaction was carried out at 37 ° C. for 1 hour, the sample DNA was completely cleaved with HhaI, and then concentrated and purified by ethanol precipitation for the purpose of inactivating and removing restriction enzymes.

Subsequently, in order to obtain double-stranded first fragment groups 6 and 7 having the same length at both ends of the double-stranded DNA fragments of the first fragment groups 4 and 5, Klenow Fragment was used.
, And then treated with phenol / chloroform for 30 minutes at 37 ° C. for the purpose of deactivating and removing the enzyme.
And an operation of purification and concentration by ethanol precipitation.

(Hybridization and Single-Strand Partial Cleavage) After mixing the two first fragment groups 6 and 7, 94
Each fragment is completely denatured by treatment at 3 ° C. for 3 minutes. Thereafter, the mixture is placed at room temperature and the temperature is slowly lowered. While the temperature is lowered in this manner, the DNA fragment completely denatured by the denaturation treatment forms a complementary strand again (recomplementary strand formation reaction: rehybridizatio).
n). The product of this re-complementary strand formation reaction is the double-stranded second fragment group 8. In the second fragment group, fragment 9 was recomplemented between fragments derived from the mutated sample DNA, and fragment 10 was recomplemented between fragments derived from the mutated sample DNA. By the chain formation reaction, fragment 11
Is the fragment derived from the wild-type sample DNA and the mutant sample DNA
Are formed by a recomplementary strand formation reaction with the fragment derived from

In the fragments other than the fragment 11 in the second fragment group 8, the sequences of the sense strand and the antisense strand are completely complementary to each other, and form a complementary strand exactly over the entire length of the fragment. I have. Fragment 11 forms a completely complementary strand in portions other than the mutated portion 3, but the mutated portion 3 is composed of the sense strand 12 derived from the wild-type sample DNA and the antisense strand 13 derived from the mutated sample DNA. The sequence differs between them, and a complementary chain cannot be formed, and the single-stranded portion 14 is formed. This one
By specifically decomposing and cleaving only the portion 14 in the main chain state, a double-stranded third fragment group 15 can be obtained.
That is, after treating with a single-strand-specific recognition enzyme S1 nuclease at 37 ° C. for 30 minutes, phenol / chloroform extraction and purification / concentration by ethanol precipitation are performed for the purpose of inactivating and removing the enzyme. Was. As a result,
Two double strands 22 are formed from the fragment 11 formed by the recomplementary strand formation reaction between the fragment derived from the wild-type sample DNA and the fragment derived from the mutant sample DNA.

(Introduction of Oligomers of Known Sequence into Both 3 'Ends of DNA Fragment) Next, the third fragment group 15 was analyzed by a fragment analysis method. The procedure of fragment analysis is as follows. An oligomer (hereinafter, referred to as an adapter) 16 having a known sequence at both 3 ′ ends of all the DNA fragments included in the third fragment group 15 is introduced by ligation. The 5 'end of the adapter which is in contact with both 3' ends of the DNA fragment is phosphorylated.
In addition, the phosphate groups at both 5 ′ ends of all DNA fragments included in the third fragment group are removed using CIAP (alkaline phosphatase). Using T4 DNA ligase, the adapter is allowed to react with the DNA fragment concentrated and purified by the ethanol precipitation method at 15 ° C. overnight to bind. As described above, a double-stranded third DNA fragment group 17 into which an adapter having a known base sequence has been introduced is obtained.

FIG. 2 is a diagram showing the principle of the fragment analysis method using 16 kinds of selective primers consisting of a combination of two bases at the 3 'end. Double-stranded third D into which an oligomer (adaptor) having a known base sequence has been introduced
Each fragment of the NA fragment group 31 has an adapter 16, a restriction enzyme recognition sequence 32, and a base sequence 33 used for selection at both 3 ′ ends. The base sequence 33 used for selection has two bases (MM, N, N) that greatly affect the extension reaction when the selection primer forms a complementary strand and extends.
N). In FIG. 2, symbols M and N indicate any of bases A, C, G and T. The length of the base sequence 33 can be selected from a range of 1 to 4 bases, preferably 1 to 3 bases, and more preferably 2 bases. Here, an example of two bases will be described.

The third DNA fragment group 31 into which an adapter having a known base sequence has been introduced is a mixture of a plurality of types of fragments. The fragment length of each fragment contained in this mixture and the sequence of the 2 bases at the 5 ′ end following the restriction enzyme recognition sequence are determined by the fragment analysis method. In the fragment analysis, a fluorescent label selection primer 37 having a fluorescent label 34 at the 5 ′ end is used. There are a total of 16 types of 2 base sequences at the 3 'end of the selection primer from all possible combinations of 2 bases selected from A, C, G and T.
Of the base sequence of the fluorescent label selection primer, a portion 35 complementary to the adapter 16 and a base sequence 36 complementary to the restriction enzyme recognition sequence 32 are the third sequence into which the adapter has been introduced.
Can completely form a complementary strand with the adapter 16 and the restriction enzyme recognition portion 32 of each fragment group.

The fluorescent-labeling selection primer 37 has a two-base selection base sequence 38 (FIG. 2 shows an example of the sequence AA) at the 3 ′ end. The second fragment following the 5 'end of the restriction enzyme recognition sequence 32 of the fragment of the third fragment group into which the adapter was introduced.
The extension reaction of the selection primer 37 proceeds to form the complementary strand 40 only when the base and the base sequence for selection 38 used for selection of the fragment are (1) completely complementary to each other. ) If not complementary, the complementary strand extension reaction does not proceed. Using the difference between (1) and (2), 16
Only a fragment capable of completely forming a complementary strand with each type of fluorescently labeled selection primer is selected from the mixture of fragments,
The sequence of two bases following the restriction enzyme recognition sequence of the fragment can be determined. However, the double-stranded fragments 20 and 21 cut at the mutated portion in the third fragment group 17 shown in FIG. 1 are exceptions unlike ordinary fragment analysis.

FIG. 3 is a diagram for explaining the details of the structure of the fragment containing the mutated portion in the third fragment group into which the adapter has been introduced. When both chains are the double-stranded fragment 18 derived from the wild type, or both chains are the double-stranded fragment 19 derived from the mutant type, both 3 ′ ends of the double-stranded fragments 18 and 19 are the adapter sequence 1
6, and a restriction enzyme recognition sequence 32. However, since the hybrid of wild type and mutant type was
The single-stranded fragments 20 and 21 were added to the double-stranded fragments 18 and 1 at the one-side ends 71 and 72, which were the cleavage ends cut by the restriction enzyme.
9. Adapter sequence 16 and restriction enzyme recognition sequence 32
However, one end 73, 74 of the cleavage site by S1 nuclease has the adapter sequence 16 but does not have the restriction enzyme recognition sequence 32. Therefore, the end 7 of the fragment
On the 3,74 side, the selection primer cannot form a complementary strand.

Actually, fragment analysis was performed by the following method. One kind of fluorescent label selection primer, dATP, dCTP, dGTP, d
TTP and a thermostable DNA polymerase were added to carry out a reaction for 5 cycles. The same reaction was performed for all other 15 types of fluorescently labeled selection primers. Thereafter, the single-stranded fluorescently labeled elongation product obtained by the complementary elongation reaction was analyzed by gel electrophoresis.

FIG. 4 shows the results of fragment analysis. FIG. 4 (a) shows the result obtained by shortening the wild-type sample DNA, introducing adapters into both 3 ′ ends of each fragment and performing fragment analysis (electrophoresis pattern), and FIG. 4 (b). Shows the results (electrophoresis pattern) obtained by shortening wild-type and mutant sample DNAs, introducing adapters into both 3 ′ ends of each fragment, and performing fragment analysis. 4 (a) and 4 (b), the vertical axis 101 shows the sequence of the two bases at the 3 'end of each selected primer, and the horizontal axis 102 shows the base length. Figure (a),
From (b), the length of each fragment contained in the mixture of the third fragment into which the adapter has been introduced, and the sequence information of the 5'-terminal two bases following the restriction enzyme recognition sequence can be obtained.

For example, as shown in FIG. 4 (a), the peak 103 detected when a fluorescent-labeled selection primer having the sequence of AG at the 3 ′ end is used is the two bases following the restriction enzyme recognition sequence in the mixture. Indicates that a TC fragment is present. The peak 104 that appears at the same position as the DNA strand (peak 103) formed by extension of the complementary strand of the primer having the AG terminal sequence is the fragment 103, 1
04 means that each was generated from a double-stranded DNA fragment having a complementary sequence.

When comparing FIG. 4A and FIG. 4B, most of the peaks appear at the same position.
5 and 106 appear only in FIG. 4B. Peaks 105 and 106 are considered to be peaks derived from fragments 20 and 21 shown in FIGS. In this way, the presence or absence of the mutated portion could be easily confirmed by comparing the analysis results of the fragment analysis.

Further, a fragment containing a mutated portion, ie, FIG.
In the fragment 19 shown in FIG. 3, it can be confirmed from the peaks 105 and 106 in FIG. 4B that the two bases following the restriction enzyme recognition sequence of the fragment 19 are both TT. Utilizing the fact that the two bases following the restriction enzyme recognition sequence of fragment 19 are both TT, a fragment containing a mutated portion can be more easily amplified by PCR from a group of restriction enzyme-cleaved fragments derived from a mutant sample. . The PCR product of the fragment containing the mutated portion thus amplified is expected to be peak 107 (in the case of this example, approximately 500 bases from the sum of peaks 105 and 106). By sequencing this fragment of peak 107, the nucleotide sequence of only the mutated portion could be determined.

[0033]

According to the present invention, the wild-type and mutant-type sample nucleic acids are separately short-fragmented separately, two types of first fragments are prepared, and the two types of fragments are mixed to re-complement the complementary strand. (Rehy
and a second set of fragments is prepared. The single-stranded portion of the hybrid containing the mutated portion is decomposed and cleaved with an enzyme to prepare a third fragment group, and the third fragment group is analyzed by a fragment analysis method, and is obtained only from a fragment derived from a wild-type sample nucleic acid. By comparing and analyzing the results of the fragment analysis, the presence or absence of a mutated portion contained in the third fragment group can be confirmed. Also, based on the results of the fragment analysis, only the fragment containing the mutated portion can be selectively amplified by PCR using a selection primer to determine the nucleotide sequence. In the present invention, it is possible to detect a mutation in a nucleic acid having a length of 1 k bases or more, and it is possible to easily determine the base sequence of only the mutated portion.

[Brief description of the drawings]

FIG. 1 shows a wild-type sample DNA,
FIG. 4 is a diagram illustrating a step of preparing first, second, and third fragment groups from a mutated sample DNA.

FIG. 2 is a diagram showing the principle of a fragment analysis method using 16 types of selective primers in an example of the present invention.

FIG. 3 is a view for explaining details of the structure of a fragment containing a mutated portion in a third fragment group in an example of the present invention.

FIG. 4 is a diagram showing the results of fragment analysis in an example of the present invention.

[Explanation of symbols]

1: Sample DNA (pu with inserted wild-type p53 gene)
c19 DNA), 2 ... sample DNA (puc19 DNA with mutant p53 gene inserted), 3 ... mutated part, 4 ...
The first group of double-stranded fragments prepared from sample DNA 1, 5
... a first group of double-stranded fragments prepared from the sample DNA 2,
6: a double-stranded fragment group having the same length at both ends of the first fragment group 4, 7: a double-stranded fragment group having the same length at both ends of the first fragment group 5, 8 ... 2 Second fragment group of chain, 9 ... wild-type sample D
Re-complementary strand formation reaction product between fragments derived from NA, 10... Re-complementary strand formation reaction product between fragments derived from mutant sample DNA, 11... Derived from wild-type sample DNA Re-complementary strand formation reaction product between the fragment and the fragment derived from the mutant sample DNA, 12: sense strand derived from wild-type sample DNA, 13 ... antisense strand derived from mutant sample DNA, 14 ... 1 A part in a single-stranded state, 15... A third fragment group of a double-stranded substance, 16... An adapter, 17... A double-stranded third DN into which an adapter having a known base sequence is introduced.
A fragment group, 18 ... double-stranded fragments derived from wild type in both chains, 1
9 ... a double-stranded fragment derived from the mutant type in both chains, 20, 21 ... a double-stranded fragment 22 ... cleaved at the mutated portion, two double-stranded fragments formed from fragment 11, 31 ... 3 fragments, 32: restriction enzyme recognition sequence, 33: base sequence used for selection, 34: fluorescent label, 35: base sequence complementary to adapter, 36: base sequence complementary to restriction enzyme recognition sequence, 37 ... fluorescent label selection primer, 38 ... 2
Base sequence for base selection, 39: when completely complementary, 40: complementary strand, 41: when not complementary, 71, 7
2 ... cleaved ends cut by restriction enzymes, 73, 74 ...
S1 nuclease cleaved end, 101 ... vertical axis (base sequence of 2 bases at 3 'end of each selected primer),
102 ... horizontal axis (base length), 103, 104, 105, 1
06, 107... Detected peaks.

Claims (17)

[Claims] (1) A double-stranded first nucleic acid sample and a double-stranded second nucleic acid sample are each fragmented with a restriction enzyme to be derived from the first and second nucleic acid samples. (2) mixing the two first-fragment groups to denature all fragments and then performing a complementary strand formation reaction after the denaturation of all fragments; A step of obtaining a second fragment group of the chain; (3) a step of decomposing the single-stranded portion produced in the step (2) with an enzyme to obtain a third group of double-stranded chains; Obtaining a distribution of fragment lengths by electrophoresing the complementary strands of each fragment of the 3 fragment groups. 2. The method according to claim 1, wherein the first and second nucleic acid samples are mixed in advance in place of the steps (1) and (2). A nucleic acid comprising a step of performing a complementary strand formation reaction after denaturing the first and second nucleic acid samples, and fragmenting the complementary strand formation reaction product with the restriction enzyme to obtain the second fragment group. Sequence comparison analysis method. 3. The method according to claim 1, wherein each fragment of a fragment group obtained by fragmenting the first or second nucleic acid sample with the restriction enzyme is obtained by gel electrophoresis. A method for comparing and analyzing nucleic acid sequences, comprising comparing the distribution of fragment lengths with the distribution of fragment lengths obtained in the step (4). 4. The nucleic acid sequence comparison analysis method according to claim 1, wherein the nucleotide sequence difference between the nucleotide sequences of the first and second nucleic acid samples due to point mutation, insertion or deletion. A nucleic acid sequence comparison analysis method, characterized by detecting 5. The method for comparing and analyzing nucleic acid sequences according to claim 1, wherein, after the step (3), an oligomer having a known base sequence is added to both 3 ′ of each fragment of the third fragment group. A method for comparative analysis of nucleic acid sequences, which comprises a step of introducing a compound into a terminal by ligation. 6. The method for comparing and analyzing nucleic acid sequences according to claim 1, wherein, after the step (3), poly A, poly U, Alternatively, a method for comparing and analyzing nucleic acid sequences, comprising a step of introducing poly-T using terminal deoxynucleotidyl transferase. 7. The method for comparing and analyzing nucleic acid sequences according to claim 1, wherein, after the step (3), an oligomer having a known base sequence is added to both 3 ′ of each fragment of the third fragment group. A step of introducing the terminal into the terminal by ligation, a first base sequence labeled at the 5 ′ end and complementary to at least a part of the base sequence of the oligomer, and a restriction enzyme of the restriction enzyme following the 3 ′ end of the first base sequence. Using a primer having a second base sequence complementary to the restriction enzyme recognition sequence and a third base sequence consisting of 1 to 3 bases following the 3 ′ end of the second base sequence, the third fragment Performing a complementary chain extension reaction using each fragment of the group as a template, and electrophoresing the complementary chain extension reaction product in the step (4). 8. The method according to claim 1, wherein the enzyme in the step (3) is S1 nuclease, Magbean nuclease, ribonuclease A, T
4. A method for comparative analysis of nucleic acid sequences, which is any one of 4 endonuclease VII. 9. The method according to claim 1, wherein said first and second sample nucleic acids are DNA or RNA.
A nucleic acid sequence comparative analysis method, characterized in that: 10. A reagent kit for use in the method for comparing and analyzing nucleic acid sequences according to claim 1, wherein a restriction enzyme for fragmenting the first and second sample nucleic acids and a single strand of a double-stranded nucleic acid fragment are provided. An enzyme that decomposes a portion, a reagent for introducing an oligomer having a known base sequence to both 3 ′ ends of each fragment of the third fragment group containing at least ligase or terminal deoxynucleotidyl transferase, A primer having a base sequence complementary to at least a part of the base sequence, the primer being labeled at the 5 ′ end. (1) a double-stranded first nucleic acid sample;
Fragmenting the single-stranded second nucleic acid sample with restriction enzymes to obtain two types of double-stranded first fragment groups derived from the first and second nucleic acid samples, and (2) ) OH at both 3 ′ ends of each fragment of each of the two first fragment groups
A step of modifying a group; and (3) mixing the two kinds of first fragment groups whose both 3 ′ ends are modified with an OH group, denaturing all the fragments, and then performing a complementary strand forming reaction. Second of the chain
(4) a step of (4) decomposing the single-stranded portion produced in step (3) with an enzyme to obtain a double-stranded third fragment group; and (5) the step (4). A method for comparing and analyzing nucleic acid sequences, comprising the steps of: labeling the 3 'end of the end cleaved by the enzyme; and (6) detecting the fragment labeled at the 3' end by gel electrophoresis. 12. The method according to claim 11, wherein the label in the step (5) is a fluorescently labeled nucleotide added using terminal deoxynucleotidyl transferase. Nucleic acid sequence analysis method. 13. The nucleic acid sequence according to claim 11, wherein the label in the step (5) is a fluorescently labeled nucleotide added using a ligation method. Comparative analysis method. 14. The nucleic acid sequence comparative analysis method according to claim 11, wherein the nucleotide sequence difference between the nucleotide sequences of the first and second nucleic acid samples due to point mutation, insertion or deletion. A nucleic acid sequence comparison analysis method, characterized by detecting 15. The method according to claim 11, wherein the enzyme in the step (4) is any one of S1 nuclease, Magbean nuclease, ribonuclease A, and T4 endonuclease VII. A method for comparing and analyzing nucleic acid sequences. 16. The nucleic acid sequence analysis method according to claim 11, wherein the first and second sample nucleic acids are DNA or R.
A nucleic acid sequence comparative analysis method characterized by being NA. 17. A reagent kit used for the method according to claim 11, wherein a restriction enzyme for fragmenting the first and second sample nucleic acids and a 3 ′ end of a double-stranded fragment generated by the restriction enzyme are used. An enzyme to be modified, an enzyme that degrades a single-stranded portion of a double-stranded nucleic acid fragment, and a label containing at least ligase or terminal deoxynucleotidyltransferase and labeling the 3 ′ end of the fragment of the third fragment group with the enzyme A reagent kit comprising: