JPH10174589A - Dna analysis and reagent kit - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for preparing a nested deletion library which does not use cloning and a method for determining a base sequence. SOLUTION: Amplification of each DNA fragment (fragment-1) obtained by cleaving a sample DNA 151 with a restriction enzyme is carried out by using 16 kinds of selection primer sets and DNA 152 (fragment-2) near 5' end of either one of each fragment is obtained by λexonuclease and Klenow fragment. PCR reaction is carried out by using the fragment 2 and the sample DNA 151 to afford a DNA fragment (the third fragment) 158 containing a base sequence complementary to the fragment-2 and longer than the fragment. The starting end of the third fragment is common and the final end is different by a restriction enzyme-cleaving part. The base sequence is determined from the restriction enzyme-cleaving part of each fragment and determination of base sequence in total length of the sample DNA 151 is efficiently carried out.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for preparing a sample to be used in a DNA base sequence determination method based on complementary strand synthesis by an enzyme, and to a base sequence determination reagent kit used therefor.

[0002]

2. Description of the Related Art There is an increasing need for higher efficiency of DNA base sequence determination mainly in genome analysis. Nucleotide sequence determination starts with the DNA contained in the gene being cloned from 10 kbases to 100 kbases in length, and the creation of a DNA library covering all DNAs. Conventionally, base sequence determination of clones thus produced has been roughly divided into three methods (shotgun method, primer walking method, and nested deletion method).

[0003] In the shotgun method, sample DNA is cut at random using ultrasonic waves or the like, and D
An NA fragment is prepared, the base sequence of each fragment is determined, and the entire base sequence is determined by utilizing the overlap between the base sequence-determined fragments. For an explanation of the technology related to the shotgun method,
For example, reference 1 (T. Maniatis et al .:
Molecular Cloning, Cold Sp
ring Harbor Laboratory Pr
ess, 13, 21-33 (1989)).

In the primer walking method, a DNA fragment to be sequenced is analyzed in order from the end. That is, in the primer walking method, the sample DNA
The base sequence is determined from the end of the primer sequence, a primer sequence following the next region is selected from the determined base sequence, and the base sequence is determined again starting from the selected primer sequence. For a description of the technique relating to the primer walking method, see, for example, Reference 2 (T. Mani).
atis et al. : Molecular Clo
ning, Cold Spring Harbor L
laboratory Press, 13, 14-20
(1989)).

In the nested deletion method, the sample DN
The end of the fragment A is decomposed using an enzymatic decomposition reaction to obtain fragments having different lengths little by little, and the starting point of one end of the fragment is aligned. Then, the base sequence is determined from the terminal opposite to the starting point in order of length. . For a description of the technology related to the nested deletion method, see, for example, Reference 3 (T. Maniatis et al.)
l. : Molecular Cloning, Cold
Spring Harbor Laboratory
Press, 13, 34-41 (1989)).

[0006]

In the above-mentioned shotgun method, it is not known which part of the sample DNA is the DNA extracted by the subclone until the entire base sequence is determined. DNA to determine sequence
It is necessary to analyze a DNA fragment corresponding to a DNA length that is 10 to 20 times the length of the strand, which is very time-consuming and troublesome, and is a major obstacle. In addition, the above-described primer walking method can efficiently and efficiently determine a base sequence, but it takes time to determine the base sequence in a time-series manner, and it takes time to prepare primers for each analysis. There is a problem with the point.

In the above nested deletion method, new base sequence information is always obtained by the difference in length between fragments. Also, similar to the primer walking method, the base sequence is determined in order from the end, so that the efficiency is high. Also prepared D
Since the NA fragment is inserted into a plasmid and subcloned and subjected to a nucleotide sequence determination operation, the priming site is obtained from the nucleotide sequence of the plasmid, and it is not necessary to prepare a primer each time the nucleotide sequence is determined. From such a point, it seems that the nested deletion method overcomes the problems in the shotgun method and the primer walking method. However, actually, it is necessary to perform a troublesome operation of finally selecting and aligning samples convenient for base sequence determination in order of length, and this point is the most problematic. Purification of subcloning common to the shotgun method and the nested deletion method is a complicated operation, and is an important problem to be solved.

[0008] Various methods have been tried to solve the problems of the base sequence determination techniques as in the conventional three methods.
A particularly promising method (fragment walking method) for directly determining the base sequence of a DNA fragment of a sample DNA by a restriction enzyme from the state of a mixture will be described below. For a description of the technique related to the fragment walking method, see, for example, Reference 4 (K. Okano et al., Fragment).
walking for large DNAse
quenching by using a libra
ry as small as 16 primers,
Gene, 176: 231-235 (1996)). After the DNA sample is digested with restriction enzymes, an oligomer having a known base sequence is introduced into the end of the DNA fragment by a ligation reaction in order to recognize each DNA fragment from the fragment mixture. Next, a sequencing reaction was carried out using a primer set that can select at least a part of the introduced oligomer and a base sequence complementary to the cut by the restriction enzyme and an unknown base sequence of 1 to 4 bases following the cut. Do. For example, when the unknown base sequence portion has two bases, the primer set includes 16 combinations of possible base sequences of all four bases. When the types of DNA fragments contained in the DNA fragment group are few and there is only one DNA fragment completely complementary to one type of primer, the base sequence of each fragment can be determined using the above primers. When one type of primer forms a complementary strand with two or more DNA fragments, the above-described method is performed after length separation or the like. After determining the nucleotide sequence of each DNA fragment, the fragments are joined to obtain the entire nucleotide sequence. For the joining between fragments, a method of determining the nucleotide sequence of the joining portion by using each fragment and the full-length sample DNA before the cleavage treatment with the restriction enzyme as a template when determining the nucleotide sequence, overcoming the fragment portion, Methods of increasing the types of restriction enzymes to be used and determining the entire nucleotide sequence by utilizing the duplication of the nucleotide sequence between the fragments have been adopted.

As described above, any of the conventional methods has disadvantages. That is, in the shotgun method, it is necessary to read the same base sequence redundantly, and the redundancy is high and the efficiency is low. As methods having low redundancy, there are a primer walking method and a nested deletion method.
However, the primer walking method requires the synthesis of primers or the preparation of a library containing a large number of primers each time prior to base sequencing, and the difficulty of sequencing in series There is. In addition, the nested deletion method has a problem in that it is difficult to prepare a sample convenient for base sequence determination. In addition, the shotgun method, the nested deletion method, and the like require cloning using a culture step that is difficult to automate, which requires time and effort. In addition, the fragment walking method has the advantage over the above three methods that cloning is not required and the redundancy is low, but there are problems such as joining when the base length of a fragment cut with a restriction enzyme and sequenced is long, etc. Remains.

[0010] An object of the present invention is to prepare a nested deletion sample based on the fragments prepared by the fragment walking method and determine the nucleotide sequence. It is an object of the present invention to provide a method for determining a DNA base sequence which can solve the difficulty of sample preparation and the complexity of subcloning operations, which are problems in the splicing and nested deletion methods, and a reagent kit used therefor.

[0011]

[Means for Solving the Problems]

A. In the first configuration of the base sequence determination method of the present invention, (A1): a step of cutting a sample DNA with a restriction enzyme to obtain a DNA fragment; and (A2): a known base sequence at both 3 ′ ends of the DNA fragment. (A3) using a DNA fragment into which the oligomer has been introduced as a template, a first base sequence portion complementary to at least a part of the base sequence of the oligomer, and a base recognized by a restriction enzyme. It is composed of a second base sequence portion complementary to the sequence and a two-base third base sequence portion composed of all possible combinations of A, C, G, and T4 bases, and is labeled (eg, fluorescently labeled). Using each of the primers (hereinafter, an unlabeled primer having this base sequence is referred to as a selection primer), a complementary strand synthesis elongation reaction is performed, and the DNA into which the oligomer has been introduced.
Determining a base sequence of two bases following the restriction enzyme recognition base sequence of the fragment; (A4): using the DNA fragment into which the oligomer has been introduced as a template and selecting based on the base sequence information determined in step (A3) Performing a first amplification reaction to amplify each DNA fragment using two primers; (A5): decomposing the vicinity of one 5 ′ end of the amplified fragment and the vicinity of both 3 ′ ends and decomposing the other 5 ′ end. 'A step of obtaining a single strand near the end, and (A6): an oligomer having a partial base sequence of the sample DNA using the sample DNA before restriction enzyme cleavage as a template and a step (A
5) performing a second amplification reaction using the single strand near the 5 ′ end purified in 5) as a primer, and determining the nucleotide sequence of the product.
In step 2), an oligomer having a known base sequence is introduced by introducing a deoxyribonucleotide oligomer using a ligation reaction or TdT, and one of the two primers in step (A4) is labeled with a phosphate group at the 5 ′ end. In step (A5), the decomposition near the 5 'end is 5' →
Λ exonuclease, which is a 3 ′ exonuclease, is digested near the 3 ′ end by Klenow fragment, which is an enzyme having 3 ′ → 5 ′ exonuclease activity.
4 DNA polymerase, exonuclease III, B
Use any of the AL31 exonucleases.

B. In the second configuration of the base sequence determination method of the present invention, the step (A6) of the first configuration (A) is changed as in the following step (B6). That is, steps (B1) to
(B5) is the same as steps (A1) to (A5), respectively. (B6): Single strand and restriction enzyme near the 5 ′ end purified in step (B5) (ie, step (A5)) Using the sample DNA before cleavage as a template and an oligomer having a partial base sequence of the sample DNA and a kind of selective primer having the same sequence as a part near the 5 ′ end, a second amplification reaction is carried out. And a step of determining a base sequence. In the step (B2) (ie, the step (A2)),
In order to introduce an oligomer having a known base sequence, a ligation reaction or TdT is used to introduce a deoxyribonucleotide oligomer, and the step (B4) (ie, step (A)
One of the two primers in 4)) has a label with a phosphate group at the 5 ′ end, and is labeled with step (B5) (ie, step (A)
In 5)), λ exonuclease which is a 5 ′ → 3 ′ exonuclease is used for degradation near the 5 ′ end, and cleaving which is an enzyme having 3 ′ → 5 ′ exonuclease activity is used for degradation near the 3 ′ end. It is characterized by using any of No Fragment, T4 DNA polymerase, Exonuclease III and BAL31 Exonuclease.

C. In the first configuration of the reagent kit for determining a DNA base sequence of the present invention, a restriction enzyme that degrades a DNA strand, an oligomer having a known base sequence, an oligomer having at least a ligase, or an oligomer having a known base sequence at the end of DNA containing TdT It is characterized by a set of an introduction reagent and a set of 16 selection primers used for complementary strand synthesis, a 5 ′ → 3 ′ exonuclease, and a DNA base sequencing reagent kit comprising an enzyme having 3 ′ → 5 ′ exonuclease activity. .

D. In the third configuration of the method for determining a nucleotide sequence of the present invention, the steps (A5) and (A5) of the first configuration (A) are performed.
The step 6) is changed as in the following steps (D5) and (D6). That is, Steps (D1) to (D4) are the same as Steps (A1) to (A4), respectively. (D5): Decompose the vicinity of both 3 ′ ends of the amplified fragment to obtain one fragment near the 5 ′ end. And (D6): an oligomer having a partial base sequence of the sample DNA and the 5 ′ purified in the step (D5) using, as a template, the sample DNA before restriction enzyme digestion in which only one strand has been purified. Performing a second amplification reaction using the single strand near the end as a primer, and determining the base sequence of the product, and further comprising a step (D2) (that is, a step (A2)). ,
A deoxyribonucleotide oligomer is introduced using a ligation reaction or TdT to introduce an oligomer having a known base sequence, and exonuclease III, which is a 3 ′ → 5 ′ exonuclease, is used for degradation near the 3 ′ end in step (D5). Alternatively, BAL31 exonuclease is used.

E. In the fourth configuration of the method for determining a base sequence of the present invention, the step (D6) of the third configuration (D) is changed as in the following step (E6). That is, steps (E1) to
(E5) is the same as (D1) to (D5), respectively. (E6): Single strand and one strand near the 5 ′ end purified in step (E5) (that is, step (D5)) Using only the purified sample DNA before restriction enzyme cleavage as a template, an oligomer having a partial base sequence of the sample DNA and a kind of selective primer having the same sequence as a part of the single strand near the 5 'end are used. And a step of determining the nucleotide sequence of the product by performing the amplification reaction of Step 2.
2) In step (A2), a deoxyribonucleotide oligomer is introduced using a ligation reaction or TdT to introduce an oligomer having a known base sequence, and in step (E5) (ie, step (D5)), Characterization of the decomposition near the terminus is to use 3 ′ → 5 ′ exonuclease, exonuclease III or BAL31 exonuclease.

F. In the second configuration of the reagent kit for determining a DNA base sequence of the present invention, a restriction enzyme that degrades a DNA strand, an oligomer having a known base sequence, an oligomer having at least a ligase, or an oligomer having a known base sequence at the end of DNA containing TdT Introduction reagent, used for complementary strand synthesis 16
It is characterized by a reagent kit for DNA base sequence determination comprising a species selection primer and a 3 ′ → 5 ′ exonuclease.

G. In the fifth configuration of the nucleotide sequence determination method of the present invention, the steps (A5) and (A5) of the first configuration (A) are performed.
Step 6) is changed as in the following steps (G5) and (G6). That is, steps (G1) to (G4) are the same as steps (A1) to (A4), respectively; (G5): a step of decomposing the vicinity of one 5 ′ end of the amplified fragment; (G6) : A step of decomposing the single-stranded DNA; and (G7): an oligomer and a step (G7) using the sample DNA before restriction enzyme cleavage as a template and having a partial base sequence of the sample DNA.
5) and a step of performing a second amplification reaction using a single strand near the other 5 ′ end purified in (G6) as a primer and determining the nucleotide sequence of the product,
Further, in step (G2) (ie, step (A2)), a deoxyribonucleotide oligomer is introduced using a ligation reaction or TdT to introduce an oligomer having a known base sequence, and the step (G4) (ie, step (A4)) Of the two primers has a label at the 5 ′ end with a phosphate group, and the degradation near the 5 ′ end in step (G5) is 5 ′.
→ It is characterized in that λ exonuclease, which is a 3 ′ exonuclease, is used, and S1 nuclease or Mag Bean nuclease is used for the degradation of the single-stranded DNA in step (G6).

H. In the sixth configuration of the nucleotide sequence determination method of the present invention, the step (G7) of the fifth configuration (G) is changed as in the following step (H7). That is, steps (H1) to
(H6) is the same as steps (G1) to (G6), respectively. (H7): Purified in steps (H5) (ie, step (G5)) and (H6) (ie, step (G6)) Other 5 '
Using a single strand near the end and the sample DNA before restriction enzyme as a template as a template, an oligomer having a partial base sequence of the sample DNA and a type of other oligomer having the same sequence as a part of the single strand near the 5 'end Performing a second amplification reaction using the primers and determining the nucleotide sequence of the product. In addition, the known nucleotide sequence in the step (H2) (that is, the step (A2)) is For the introduction of the oligomer having the compound, a deoxyribonucleotide oligomer is introduced using a ligation reaction or TdT, and then the step (H4) (ie, the step (A)
One of the two primers in 4)) has a label at the 5 ′ end with a phosphate group, and is labeled in step (H5) (ie, step (G)
In step 5)), the 5′-terminal degradation is carried out using a 5 ′ → 3 ′ exonuclease, λ exonuclease, and the single-stranded DN in step (H6) (ie, step (G6)).
It is characterized in that A is degraded using S1 nuclease or mag bean nuclease.

I. In a third configuration of the reagent kit for determining a DNA base sequence of the present invention, a restriction enzyme that degrades a DNA strand, an oligomer having a known base sequence, an oligomer having at least a ligase, or an oligomer having a known base sequence at the end of DNA containing TdT Introduction reagent, used for complementary strand synthesis 16
It is characterized by a DNA base sequence determination reagent kit comprising a species selection primer, 5 ′ → 3 ′ exonuclease, and a single-strand degrading enzyme.

J. In the seventh configuration of the method for determining a base sequence of the present invention, the step (A6) of the first configuration (A), the step (D6) of the configuration (D), and the second configuration in the step (G7) of the configuration (G) are performed. Is changed as in the following step (J1). (J1): Using the sample DNA before restriction enzyme cleavage as a template, the second amplification using a single strand near the 5 ′ end prepared from the sample DNA and a random primer (oligomer having a random base sequence) It is characterized by having a step of performing a reaction and determining the base sequence of the product.

K. Further, in the eighth configuration of the method for determining a base sequence of the present invention, in the step (B6) of the second configuration (B), the step (E6) of the configuration (E), and the step (H7) of the configuration (H). The step of the second amplification reaction is changed as in the following step (K1). That is, (K1): using a single strand near the 5 ′ end prepared from the sample DNA and the sample DNA before restriction enzyme cleavage as a template, a random primer (oligomer having a random base sequence) and one near the 5 ′ end. A second amplification reaction is performed using a kind of selective primer having the same sequence as a part of the main strand,
It is characterized by having a step of determining the base sequence of the product.

In the present invention, a sample DNA is fragmented with a restriction enzyme, and the fragment is amplified by a PCR method. After that, only a single strand near one 5 ′ end is recovered through an enzymatic decomposition reaction, and the fragment is fragmented. Utilizing the fact that it is complementary to one strand of the sample DNA before being subjected. The single-stranded DNA near the 5 'end of the DNA fragment forms a complementary strand with the sample DNA before cleavage with the restriction enzyme, and can be extended in the 3' direction using this DNA as a template. The oligomer having a part of the base sequence of the previous sample DNA is complementary to the single-stranded DNA extension product near the 5 ′ end of the DNA fragment, and this extension product can be used as a template for extension reaction. As in steps (A6), (D6), (G7), and (J1), an oligomer or random having a single strand near the 5 ′ end of the DNA fragment and a partial base sequence of the sample DNA before restriction enzyme cleavage. Oligomers having random base sequences (random primers) can be used as PCR primers, and the site between the two primers can be amplified by PCR. Similarly, D
The single-stranded DNA near the 5 'end of the NA fragment forms a complementary strand with the sample DNA before cleavage with the restriction enzyme, and this DN
A can be used as a template to perform an elongation reaction in the 3 ′ direction.
6), (E6), (H7) and (K1), using the extension reaction product and the sample DNA before restriction enzyme cleavage as a template,
An oligomer having a partial base sequence of a single strand near the 5 'end of a DNA fragment and an oligomer having a partial base sequence of a sample DNA before restriction enzyme cleavage or an oligomer having a random base sequence (random primer) Can be used to amplify the site flanked by both primers by PCR. In addition, the product of the second amplification reaction performed using the template and the primer has one end of the sample DN before the restriction enzyme cleavage.
Same position in A (position with base sequence of oligomer)
The other is determined by the position of the cut of the sample DNA by the restriction enzyme, and a product having a different length by the number of the DNA fragments after the restriction enzyme is generated, so that a library for nested deletion is formed. it can.

[0023]

Embodiments of the present invention will be described below in detail with reference to the drawings.

(First Embodiment) FIG. 1 shows a procedure of a treatment in a first embodiment of the present invention. After a sample DNA is fragmented with a restriction enzyme, a fragment sequence following the restriction enzyme recognition base sequence of each fragment is obtained. FIG. 3 is a diagram illustrating a step of determining a base sequence of a base.
As the sample DNA, a human genomic gene having a length of about 8.9 k bases inserted into the plasmid was used.

<Amplification of Sample DNA> When the amount of sample DNA is small, PCR (Polymerase) is performed in the following procedure.
The sample DNA is amplified by the Chain Reaction method. The following two primers having a base sequence complementary to the plasmid into which the sample DNA was inserted were used.

5'TGTAAAACGACGGGCCAG
T3 '(primer; base sequence No. 1) 5'ACAGGAAACAGCTATGAC 3'(primer; base sequence No. 2) A sample DNA inserted into a plasmid using two types of primers was subjected to heat-resistant polymerase Ex Taq (Takar
a) was used for PCR amplification. Amplified sample D
NA is unreacted primer and dATP, dCTP, d
In order to remove GTP and dTTP, they were separated and fractionated by 1% agarose gel electrophoresis, and a high-purity PCR product consisting of one band of 8.9 k base length was obtained by electrophoresis. Hereinafter, this linear human genomic gene is referred to as a PCR product,
Let it be sample DNA1.

<Fragmentation of Sample DNA> A DNA fragment is prepared by cutting the sample DNA using a restriction enzyme. In this example, the restriction enzyme Hsp92II was used.
Restriction enzymes that cleave 1 are HhaI, MboI, AluI.
Etc. and is not limited to Hsp92II. FIG.
And 2, 3 and 4 indicate fragments generated by restriction enzymes of the sample DNA. The reaction was carried out at 37 ° C. for 1 hour, and Hsp9
After the sample DNA was completely cleaved with 2II, a concentration purification operation by ethanol precipitation was performed for the purpose of inactivating and removing the restriction enzyme. Thereafter, using all of the reaction solution, DN
A known base sequence is introduced according to the method of adding an oligomer to the 3 'end of the A fragment.

<Preparation of Selection Primer> A primer set called a selection primer to be used in the subsequent reaction is prepared. In FIG. 1, 5 indicates a fluorescent-labeled selection primer, and 9 indicates a fluorescent label. This primer has a base sequence 6 complementary to at least a part of the oligomer to be introduced into the fragmented DNA, and a base sequence complementary to the recognition base sequence of the restriction enzyme used to fragment the sample DNA. 7
And a portion of a base sequence 8 consisting of 2 bases of any base sequence of A, C, G, T and four kinds of bases. The number of types of the fluorescent-labeled selection primer is 16 from the number of all possible combinations of any two bases.

<Introduction of Oligomer with Known Base Sequence into Both Ends of DNA Fragment> There are two methods for introducing an oligomer having a known base sequence at both ends of a DNA fragment. The first method is a method in which an oligomer having a known base sequence is linked to a DNA fragment by ligation (for example, Reference 5 (T. Maniatis et al .: Molec).
ullar Cloning, Cold SpringH
arbor Laboratory Press 5,
62-63 (1989))). Among the double-stranded oligomers for ligation (hereinafter referred to as adapters), DN
The 3 ′ end of the adapter that contacts the 5 ′ end of the A fragment is phosphorylated. Similarly, the 5 'end of the adapter on the side in contact with the 3' end of the DNA fragment is also phosphorylated. This is to prevent ligation between adapters. In order to prevent ligation between DNA fragments, the phosphate groups at both 5 'ends of each fragment are removed using CIAP (alkaline phosphatase). The second method is Td
This is a method of sequentially adding dATP (deoxyadenosine triphosphate) or dUTP (uracil triphosphate) to the 3 ′ end of a DNA fragment using T (for example, see Reference 6 (T. Maniatis et al.)).
al. : Molecular Cloning, Col
d Spring Harbor Laborator
y Press, 5, 56-57 (1989))). Since the first method has higher stability in the subsequent reaction than the second method, the first method was used in this example.
The adapters used in this example are 10 and 11 in FIG. One side 11 of the adapter has a three-sided protruding shape so as to bind to the DNA fragment cut with Hsp92II. The adapter and the DNA fragment concentrated and purified by the ethanol precipitation method were allowed to react overnight at 15 ° C. using T4 DNA ligase to bind. After the ligation reaction, only the ten strands linked by a phosphodiester bond are actually linked to the DNA fragment.

<Determination of Base Sequence of Two Bases Following Restriction Enzyme Recognition Base Sequence of Each DNA Fragment> The DNA fragment 131 into which the known base sequence oligomer was introduced into both ends was replaced with the adapter base sequence portion 10 introduced into both ends. , A restriction enzyme recognition portion 12, and base sequence portions 13, 13 'to be selected. The selected base sequence portion (selected base sequence portion) is two bases whose base sequence is identified by the fluorescent label selection primer. In the following description of each drawing, symbols M and N indicate any of bases A, T, G and C, symbol m is a base complementary to base M, and symbol n is a base complementary to base N. Show. The selection base sequence portion consists of 1 to 4 bases, preferably 1 to 3 bases, and more preferably 2 bases.

[0031] DN obtained from the sample by a restriction enzyme cleavage operation
The A fragment is a mixture of a plurality of types of DNA fragments. In order to selectively determine the base sequence of only the target DNA fragment, a fluorescent-labeled selection primer is used. An arbitrary base sequence portion of the fluorescent-labeled selection primer has a total of 1 as described above.
There are six types. The portion 6 complementary to the adapter of the fluorescent label selection primer and the portion 7 complementary to the restriction enzyme recognition base sequence are the adapter base sequence portion 10 of all the DNA fragments.
And a completely complementary strand with the restriction enzyme recognition portion 12. As shown by 14 in FIG. 1, the fluorescent-labeled selection primer has an arbitrary base sequence of 2 bases at the 3 ′ end, and
The complementary strand 16 can be formed only when the A fragment and the two base sequences are completely complementary. If the 2 bases at the 3 'end of the selection primer are not complementary to the DNA fragment as in 15, the extension reaction does not proceed. Using this, DNA
From the mixture of fragments, only those DNA fragments capable of completely forming a complementary strand with each of the 16 types of fluorescently labeled selection primers are selected, and the base sequence of two bases following the restriction enzyme recognition base sequence portion of the DNA fragment is determined.

Actually, a base sequence determination reaction was carried out by the following method. One kind of fluorescent label selection primer and dATP, dCTP, dGTP, dTTP are added to the mixture of DNA fragments.
And a thermostable DNA polymerase were added to carry out a reaction for 5 cycles. Similar reactions were performed for all other fluorescently labeled selection primers. The reaction was then analyzed by electrophoresis. FIG. 2 shows the DN obtained using 16 kinds of selective primers.
The example of the analysis result (gel electrophoresis spectrum) of the product of complementary strand synthesis of A fragment is shown. In FIG. 2, the vertical axis 101 represents the base sequence of the 2 bases at the 3 ′ end of each selected primer, and the horizontal axis 10 represents the base sequence.
2 represents a base length. From FIG. 2, the length of each fragment contained in the DNA fragment mixture and the base sequence information of two bases following the restriction enzyme recognition base sequences at both ends can be obtained. For example, as shown in FIG. 2, a peak 103 detected when a fluorescent-labeled selection primer having an AA base sequence at the 3 ′ end is used.
Indicates that a TT fragment is present in the mixture with a base sequence of two bases following the restriction enzyme recognition base sequence. This A
The peak 104 appearing at the same position as the peak of A means a base sequence complementary to the required base sequence of two bases of the opposite strand of the fragment having the TT base sequence at the end of one strand of a certain DNA fragment. . In this manner, the required base sequence of the two bases on both sides of the DNA fragment contained in the DNA fragment mixture can be determined.

<Amplification of Each DNA Fragment from the DNA Fragment Mixture by PCR Reaction> FIG. 3 shows a nested deletion library using enzymes having 5 ′ → 3 ′ exonuclease activity and 3 ′ → 5 ′ exonuclease activity. Is a view for explaining the steps of sample preparation for use. Based on the base sequence information of the two terminal bases determined as described above, one kind of DNA fragment is selectively amplified from the mixture by PCR using a pair of selective primers for each fragment. 131 in FIG.
Indicates a DNA fragment serving as a template. At this time, one of the two primer pairs 132 and 133 used in the PCR
33 has its 5 ′ end modified with a phosphate group. This is because when the subsequent 5 ′ end of the amplified DNA fragment is decomposed, the phosphate group introduced in FIG.
This is for specifically decomposing the 5 'end shown by. PC
The DNA fragment amplified by the R method is used to remove unreacted primers and dATP, dCTP, dGTP and dTTP (columns: QUIAGEN; QIA quick c).
olumn) for purification and concentration. Hereinafter, this purified product is called a DNA fragment PCR product, and the sample DNA
135.

<Degradation of the DNA fragment PCR product near one 5 ′ end and near both 3 ′ ends> As shown in FIG. 3, the DNA fragment PCR product 135 purified by the above method has one 5 ′ end near the 5 ′ end. In order to obtain a single strand, the other 5 ′ end and the vicinity of both 3 ′ ends were digested with an enzyme having exonuclease activity. This is because the base sequence near the adapter introduced at one 5 ′ end is degraded to obtain only the other single-stranded 5 ′ end used as a primer or template in the second amplification reaction. In this example, after the purified DNA fragment PCR product 135 was reacted at 37 ° C. for 15 minutes using λ exonuclease 136,
An ethanol precipitation operation was performed for the purpose of deactivating the enzyme and purifying and concentrating. Thereafter, the reaction was carried out at 37 ° C. for 30 minutes using Klenow fragment 137, and a column (QUIAGEN; QIA quickcq) was used for the purpose of inactivation removal of enzymes, removal of decomposed oligonucleotides, and purification and concentration of the reaction solution.
The purification operation was carried out using the above-mentioned method. From the above operation, the single-stranded DNA 138 near one of the 5 'ends shown in FIG.
I got Here, in order to obtain a single strand near one 5 ′ end, 5 ′ → 3 ′ exonuclease and 3 ′ → 5 ′
An enzyme having exonuclease activity was used.
3 'Exonuclease 5' instead of the one you need
Biotin may be introduced into the terminal and purified using magnetic beads to which avidin has been added to obtain another single strand.

<Second Amplification Reaction> FIG.
It is a figure explaining the process of amplification. Using the sample DNA 151 before cleavage by the restriction enzyme as a template, the DN
A second amplification reaction was performed using the single-stranded DNA 152 near the 5 ′ end of the A fragment and the oligomer 153 having a partial base sequence of the sample DNA 151 before restriction enzyme cleavage as primers. Oligomers 153 are obtained from sample DN before restriction enzyme cleavage.
It is the base sequence of one 5 'end of A and has the same base sequence as the primer. In the first reaction cycle, DN
The single-stranded DNA 152 near the 5 'end of the A fragment is
Only the portion 155 downstream of the adapter base sequence 154 introduced at the end is a base sequence complementary to the template DNA, only this portion forms a complementary strand with the template, and the extension reaction proceeds to the end of the template DNA, An extension reaction product 156 is obtained. The oligomer 153 is a template DN of the other chain.
A forms a complementary strand with A and the extension reaction proceeds, and the extension reaction product 1
Obtain 57. However, in the second and subsequent reaction cycles, the oligomer 153 also uses the extension product 156 generated in the first cycle reaction as a template to proceed with the extension reaction, and the extension product 1
Obtain 58. In the PCR reaction, the primer was added in a large excess with respect to the amount of the template DNA, and the oligomer 153 having a function as a primer and the single-stranded DNA 152 near the 5 'end were added in a large excess with respect to the template. Therefore, as the cycle proceeds, the oligomer 153 often forms a complementary strand with the extension product 156, rather than forms a complementary strand with the template DNA, and finally the oligomer 153 and the adapter base sequence 154 are formed in a PCR reaction. Only the fragments in between are amplified.

FIG. 5 is a diagram showing a method for determining the nucleotide sequence of a sample DNA using a nested deletion library. DNA fragments 181 and 18 amplified by the above reaction
No. 2 is determined by the restriction enzyme recognition site 183 of the sample DNA 188, and the starting points 184 are all the same, and the opposite ends thereof are determined by the restriction enzyme cleavage site 183. The terminal portion 185 has a nucleotide sequence complementary to the selection primer for each fragment, and the nucleotide sequence was determined using this portion as a priming site. Reference numeral 186 shown in FIG. 5 indicates a determination direction at the time of base sequence determination. As described above, a sample library for nested deletion was prepared, and the nucleotide sequence was determined sequentially, and the full-length nucleotide sequence of the sample DNA 188 before restriction enzyme digestion was determined.

(Second Example) As a sample DNA, a human genomic gene of about 8.9 k base length inserted into a plasmid was used. In this example, cutting of sample DNA with a restriction enzyme,
Introduction of a known base sequence oligomer to both ends of each fragment, each D
Determination of base sequence of 2 bases following restriction enzyme recognition base sequence of NA fragment, amplification of each DNA fragment by PCR reaction, D
The steps up to the degradation near the one 5 terminal and the vicinity of both 3 terminals of the NA fragment PCR product used the same method as in Example 1 shown in FIG. 1, FIG. 2 and FIG.

<Second Amplification Reaction> FIG. 6 is a diagram for explaining the step of the second PCR amplification using the selection primer. As shown in FIG. 6, sample D before cleavage by restriction enzyme
Using the NA201 and the single-stranded DNA 202 near the 5 ′ end of the DNA fragment obtained above as a template, an oligomer 203 having a partial base sequence of the sample DNA before restriction enzyme cleavage.
In FIG. 3, the second primer was selected using the selection primer 133 (204 in FIG. 6) used for PCR amplification of the DNA fragment.
Was performed. The oligomer 203 has the same base sequence as the primer, which is the base sequence at one 5 'end of the sample DNA before restriction enzyme cleavage. In the first reaction cycle, as shown in FIG. 6, the single-stranded DNA 202 near the 5′-end of the DNA fragment has a portion 206 located downstream of the adapter base sequence 205 introduced at the 5′-end thereof as the template DNA. This portion forms a complementary strand with the template, and the extension reaction proceeds to the end of the template DNA to obtain an extension reaction product 207. Oligomer 20
No. 3 forms a complementary strand with the template DNA of the other strand, the extension reaction proceeds, and an extension reaction product 208 is obtained. However, in the second and subsequent reaction cycles, the extension reaction of the oligomer 203 is also performed using the extension product 207 generated in the first cycle reaction as a template, and an extension product 209 is obtained. The product 209 generated in the second cycle reaction is selected primer 2
04 having the base sequence of the priming site 210 and the selection primer 204 was 209 in the third and subsequent cycle reactions.
The elongation reaction proceeds with the DNA strand of
Get 11. In the PCR reaction, the primer was added in a large excess with respect to the amount of the template DNA, and the oligomer 203 having a function as a primer with respect to the template and the selection primer 204 were added in a large excess. For this reason, as the cycle proceeds, the oligomer 203 forms a complementary strand with the extension product 207 more frequently than forms a complementary strand with the template DNA. The fragment is amplified. Each DNA fragment amplified by the above method is the same as the fragment shown in FIG. 5, and a sample library for nested deletion can be prepared as shown in FIG. The full-length nucleotide sequence of the sample DNA188 was determined.

(Third Example) As a sample DNA, a human genomic gene of about 8.9 k base length inserted into a plasmid was used. In this example, cutting of sample DNA with a restriction enzyme,
The steps from the introduction of a known base sequence oligomer to both ends of each fragment and the determination of the base sequence of two bases following the restriction enzyme recognition base sequence of each DNA fragment were carried out in Example 1 shown in FIGS.
This was performed using the same method as described above.

<Amplification of Each DNA Fragment from a DNA Fragment Mixture by PCR Reaction> FIG. 7 is a diagram illustrating a process of preparing a sample for a nested deletion library using 3 ′ → 5 ′ exonuclease. As shown in FIG. 7, each fragment is amplified by the PCR method based on information of two bases following the restriction enzyme recognition base sequence at both ends of each fragment determined by the above method. In FIG. 7, reference numeral 230 denotes a DNA fragment serving as a template. Based on the base sequence information of the terminal 2 bases determined as described above, a kind of DNA fragment is selectively amplified from the mixture using a pair of selective primers for each fragment. From the mixture, a PCR reaction was performed with a pair of selection primers 231 and 232 having both terminal base sequences of the target fragment from the mixture to amplify the fragment. D amplified by PCR
The NA fragment is composed of unreacted primer, dATP, dCT
Column for removing P, dGTP, dTTP (QUAI
Purification and concentration were performed using AGEN (QIA quick column). Hereinafter, this purified product is referred to as a DNA fragment PCR product, and is referred to as sample DNA 233.

<Degradation of the DNA fragment PCR product near both 3 ′ ends> As shown in FIG. 7, in order to obtain a single strand near the 5 ′ end of the purified DNA fragment PCR product 233, 'The near ends 234 and 235 were digested with exonuclease. This is for obtaining the 5 'end used as a primer or template in the second amplification reaction. FIG.
As shown in the figure, the purified DNA fragment PCR product 233
At 37 ° C. using exonuclease III236
After reacting for 5 minutes, a column (QUIAGEN; QIA quick column) is used for the purpose of removing the inactivation of the enzyme, removing the decomposed oligonucleotide, and purifying and concentrating the reaction solution.
mn). From the above operation, FIG.
The single-stranded DNAs 237 and 238 near the 5 'end shown in the following were obtained.

<Second Amplification Reaction> FIG.
It is a figure explaining the process of amplification. As shown in FIG. 8, the sample DNA 250 before cleavage with the restriction enzyme is previously in a single-stranded state, and only one of them is recovered and purified. Sample DNA to P
Amplify by CR reaction. At this time, the primers and primers described in Example 1 were used, and only the primer was modified with biotin at the 5 ′ end. The sample DNA thus amplified was purified using magnetic beads to which avidin was added, and only one strand 251 shown in FIG. 8 was recovered. Using this single-stranded DNA 251 as a template, a single-stranded DNA near both 5 ′ ends of the DNA fragment obtained above
252, 253 and sample DNA 25 before restriction enzyme cleavage
A second amplification reaction was performed using the oligomer 254 having a partial base sequence of 1. Oligomers 254 are single-stranded D
This is the base sequence at the 5 'end of NA251 and has the same base sequence as the primer shown above. In the first reaction cycle, as shown in FIG. 8, only the portion of 256 downstream of the adapter nucleotide sequence 255 introduced at the 5 ′ end of 253 is a nucleotide sequence complementary to the template DNA 251;
Only this part forms a complementary strand with the template, and the extension reaction
Proceed to the end of NA to obtain extension reaction product 257. 2
In the second reaction cycle, the extension reaction of the oligomer 254 proceeds with the elongation product 257 generated in the first cycle reaction as a template, and an elongation product 258 is obtained. Finally PC
In the R reaction, oligomer 254 and adapter base sequence 25
Fragments between 5 are amplified. Each DNA amplified above
The fragment is the same as the fragment shown in FIG. 5, and a sample library for nested deletion can be prepared as shown in FIG. 5, and the nucleotide sequence is determined sequentially to determine the full-length nucleotide sequence of the sample DNA 250 before restriction enzyme digestion. did.

(Fourth Embodiment) As a sample DNA, a human genomic gene of about 8.9 k base length inserted into a plasmid was used. In this example, cutting of sample DNA with a restriction enzyme,
Introduction of a known base sequence oligomer to both ends of each fragment, each D
Determination of base sequence of 2 bases following restriction enzyme recognition base sequence of NA fragment, amplification of each DNA fragment by PCR reaction, D
The steps up to the degradation of the vicinity of both 3 ′ ends of the NA fragment PCR product were carried out using the same method as in Example 3 shown in FIGS. 1, 2 and 7.

<Second Amplification Reaction> FIG. 9 is a diagram for explaining the step of the second PCR amplification using the selection primer. As shown in FIG. 9, sample D before cleavage by restriction enzyme
NA270 is made into a single-stranded state in advance, and only one of the single-stranded is recovered and purified. The sample DNA is amplified by a PCR reaction. At this time, the primer and primer described in Example 1 were used, and only the primer was modified with biotin at the 5 ′ end. The sample DN thus amplified
A was purified using magnetic beads to which avidin was added, and only one strand 271 shown in FIG. 9 was recovered. This single chain D
The oligomer 27 having a partial base sequence of the single-stranded DNA 271 using NA271 and the single-stranded DNAs 272 and 273 near both 5 ′ ends of the DNA fragment obtained above as a template.
A second amplification reaction was performed using the selection primer 231 (275 in FIG. 9) used for PCR amplification of the DNA fragment in FIGS. 4 and 7. The oligomer 274 is a single-stranded DNA2
71 is the base sequence at the 5 'end and has the same base sequence as the primer shown above. In the first reaction cycle, as shown in FIG. 9, only the portion 277 downstream of the adapter nucleotide sequence 276 introduced at the 5 ′ end of 273 is a nucleotide sequence complementary to the template DNA, Only form a complementary strand with the template, the extension reaction proceeds to the end of the template DNA, and an extension reaction product 278 is obtained. In the second reaction cycle, the extension reaction of the oligomer 274 proceeds using the extension product 278 generated in the first cycle reaction as a template, and an extension product 279 is obtained. The product 279 generated in the second cycle reaction has the base sequence of the priming site 280 of the selection primer 275, and the extension reaction proceeds with the selection primer 275 using the DNA strand 279 as a template in the third and subsequent cycle reactions, and the extension product 281 obtain. Finally, a fragment between the oligomer 274 and the selection primer 275 is amplified in a PCR reaction. Each D amplified above
The NA fragment is the same as the fragment shown in FIG. 5, and as shown in FIG. 5, a sample library for nested deletion can be prepared, the nucleotide sequence is determined in order, and the full-length nucleotide sequence of the sample DNA 270 before restriction enzyme digestion is determined. Decided.

(Fifth Embodiment) As a sample DNA, a human genomic gene inserted into a plasmid having a base length of about 8.9 k was used. In this example, cutting of sample DNA with a restriction enzyme,
The steps from the introduction of a known base sequence oligomer to both ends of each fragment and the determination of the base sequence of two bases following the restriction enzyme recognition base sequence of each DNA fragment were carried out in Example 1 shown in FIGS.
The same method as that described above was used.

<Amplification of Each DNA Fragment from the DNA Fragment Mixture by PCR Reaction> FIG. 10 shows a sample preparation for a nested deletion library using 5 ′ → 3 ′ exonuclease and single-strand degrading enzyme. FIG. As shown in FIG. 10, each fragment is amplified by the PCR method based on information of two bases following the restriction enzyme recognition base sequence at both ends of each fragment determined by the above method.
Based on the base sequence information of the terminal 2 bases determined above,
Using a pair of selection primers for each fragment, one kind of DNA fragment is selectively amplified from the mixture. 30 in FIG.
0 indicates a DNA fragment used as a template. At this time, of the two primer pairs 301 and 302 used in the PCR reaction,
02 has its 5 ′ end modified with a phosphate group. This is for the purpose of specifically decomposing the 5 ′ end indicated by reference numeral 303 in FIG. 10 into which a phosphate group has been introduced when the subsequent 5 ′ end of the amplified DNA fragment is decomposed. The DNA fragment amplified by the PCR method contains unreacted primers and dA
Column for removing TP, dCTP, dGTP and dTTP (from QUIAGEN; QIA quick col)
um)). Hereinafter, this purified product is referred to as a DNA fragment PCR product, and the sample DNA 304
And

<Degradation of the DNA fragment PCR product near one 5 ′ end and single-stranded portion> As shown in FIG. 10, the DNA near the one 5 ′ end of the purified DNA fragment PCR product 304 was removed as shown in FIG. To obtain it, the vicinity of the other 5 ′ end was digested with λ exonuclease and then digested with single-stranded DNA portion S1 nuclease. This is for obtaining the other 5 ′ end of the fragment used as a primer or template in the second amplification reaction. As shown in FIG. 10, the purified DNA fragment PCR product 304 was reacted with λ exonuclease 305 at 37 ° C. for 15 minutes, and then subjected to ethanol precipitation for the purpose of inactivating the enzyme and purifying and concentrating it. Thereafter, the reaction was carried out at 37 ° C. for 30 minutes using S1 nuclease 306, and a column (QUIAGEN; QIA quick c) was used for the purpose of inactivation removal of the enzyme, removal of degraded oligonucleotide, and purification and concentration of the reaction solution.
The purification operation was carried out using the above-mentioned method. From the above operation, the double-stranded DNA 30 near one of the 5 'ends shown in FIG.
7 was obtained.

<Second Amplification Reaction> FIG.
It is a figure explaining the process of R amplification. As shown in FIG. 11, using the sample DNA 320 before cleavage by the restriction enzyme as a template, the double-stranded DNA 321 near the 5 ′ end of the DNA fragment obtained above and a partial base sequence of the sample DNA 320 before the restriction enzyme cleavage A second amplification reaction was performed using the oligomer 322 having The oligomer 322 is a base sequence at one 5 ′ end of the sample DNA before restriction enzyme cleavage and has the same base sequence as the primer. In the first reaction cycle, as shown in FIG. 11, 32 downstream of the adapter base sequence 324 introduced at the 5 'end of one strand 323 of the double-stranded DNA 321 near the 5' end of the DNA fragment.
Only part 5 is a base sequence complementary to the template DNA,
Only this part forms a complementary strand with the template, and the extension reaction
Proceed to the end of NA to obtain extension reaction product 326. In addition, the oligomer 322 forms a complementary strand with the template DNA of the other strand, and the extension reaction proceeds to obtain an extension reaction product 327. However, in the second and subsequent reaction cycles, oligomer 3
22 is an extension product 32 generated in the first cycle reaction.
6 also serves as a template, and the extension reaction proceeds to obtain an extension product 328. In the PCR reaction, the primer was added in a large excess with respect to the amount of the template DNA, an oligomer 322 having a function as a primer with respect to the template, and a single-stranded DN near the 5 ′ end.
A323 (double-stranded DNA 3 near the 5 'end of the DNA fragment
21) is a large excess. For this reason, as the cycle proceeds, the oligomer 322 often forms a complementary strand with the extension product 326 rather than forms a complementary strand with the template DNA, and finally the oligomer 322 and the adapter base sequence 324 in the PCR reaction. Only the fragments in between are amplified. Each of the DNA fragments amplified above is the same as the fragment shown in FIG. 5, and a sample library for nested deletion can be prepared as shown in FIG. Was determined.

(Sixth Embodiment) As a sample DNA, a human genomic gene of about 8.9 k bases inserted into a plasmid was used. In this example, cutting of sample DNA with a restriction enzyme,
Introduction of a known base sequence oligomer to both ends of each fragment, each D
Determination of base sequence of 2 bases following restriction enzyme recognition base sequence of NA fragment, amplification of each DNA fragment by PCR reaction, D
The steps up to the decomposition near the one 5 ′ end of the NA fragment PCR product and the single-stranded portion were the same as those in Example 5 shown in FIGS. 1, 2 and 10.

<Second Amplification Reaction> FIG. 12 is a diagram for explaining the step of the second PCR amplification using the selection primer. As shown in FIG. 12, the base sequence of a part of the sample DNA before cleavage with the restriction enzyme was used as a template, using the sample DNA 340 before cleavage with the restriction enzyme and the double-stranded DNA 341 near the 5 ′ end of the DNA fragment obtained above as a template. Oligomer 342 having
A second amplification reaction was performed using the selection primer 301 (343 in FIG. 12) used for PCR amplification of the DNA fragment in FIG. The oligomer 342 is a base sequence at one 5 ′ end of the sample DNA before restriction enzyme cleavage and has the same base sequence as the primer. In the first reaction cycle, as shown in FIG. 12, the 3 'downstream of the adapter base sequence 345 introduced at the 5' end of one strand 344 of the double-stranded DNA 341 near the 5 'end of the DNA fragment.
Only the portion 46 is a base sequence complementary to the template DNA, and only this portion forms a complementary strand with the template, the extension reaction proceeds to the end of the template DNA, and an extension reaction product 347 is obtained. The oligomer 342 is a template DNA of the other strand.
And the elongation reaction proceeds, and the elongation reaction product 34
Get 8. However, in the second and subsequent reaction cycles, the extension product of the oligomer 342 also becomes a template with the extension product 347 generated in the first cycle reaction, and the extension product proceeds.
Get 49. The product 349 generated in the second cycle reaction is the priming site 3 of the selection primer 343.
In the third and subsequent cycle reactions having a base sequence of 50, the extension reaction proceeds with the selection primer 343 using the DNA chains of 349 and 350 as templates, and an extension product 351 is obtained. PC
In the R reaction, the primer was added in a large excess with respect to the amount of the template DNA, and the oligomer 342 having a function as a primer and the selection primer 343 were added in a large excess with respect to the template. Therefore, as the cycle proceeds, the oligomer 342 forms a complementary strand with the extension product 351 more frequently than forms a complementary strand with the template DNA, and finally the oligomer 342 and the selection primer 343 in the PCR reaction.
Are amplified. The DNA fragments amplified as described above are the same as the fragments shown in FIG. 5, and a sample library for nested deletion can be prepared as shown in FIG.
The full-length nucleotide sequence of NA340 was determined.

(Seventh Embodiment) As a sample DNA, a human genomic gene inserted into a plasmid having a base length of about 8.9 k was used. In this example, cutting of sample DNA with a restriction enzyme,
Introduction of oligomers of known sequence to both ends of each fragment, DNA
Sequencing of two bases following the restriction enzyme recognition sequence of the fragment, each D
The steps from the amplification of the NA fragment by PCR reaction to the degradation near the one 5 ′ end and the vicinity of both 3 ′ ends of the DNA fragment PCR product are the same as those in Example 1 shown in FIGS. 1, 2 and 3. Using.

<Second Amplification Reaction> FIG. 13 is a diagram for explaining the step of the second PCR amplification using an oligomer having a random base sequence (random primer). Using the sample DNA 151 before cleavage with the restriction enzyme as a template, the single-stranded DNA near the 5 'end of the DNA fragment obtained above
A second amplification reaction was performed using 152 and an oligomer (random primer) 371 having a random base sequence. The length of the random primer 371 is 6 bases to 1
The length is preferably about 0 bases, and the base sequence is arbitrary.
In the first cycle reaction, the extension product 156 shown in Example 1 and the extension product 372 obtained from the site where the random primer 371 forms a complementary strand with the sample DNA are obtained. In the second and subsequent cycle reactions, the extension reaction of the random primer 371 proceeds with the extension product 156 as a template, and an extension product 373 is obtained. The adapter base sequence portion 154 uses the extension product 373 as a template to obtain an extension reaction product 374. Finally, only the fragment between the adapter sequence portion 154 and the random primer 371 is amplified in the cycle reaction.

[0053]

According to the present invention, the base sequence of two bases following the restriction enzyme recognition site of a fragment obtained by cutting a sample DNA with a restriction enzyme is determined, and PCR is performed based on the base sequence information.
Of the amplified fragment, the 5 ′
Single-stranded DNA near the end and sample DN before restriction enzyme digestion
A can be used to perform a second PCR amplification to produce a nested deletion sample library. Therefore,
The nucleotide sequence can be determined efficiently with much lower redundancy than the shotgun method. Further, since the base sequence of a long sample DNA can be determined with only 16 types of primer sets prepared in advance, it is not necessary to synthesize a primer every base sequence determination unlike the primer walking method.
Furthermore, since a sample is prepared by PCR amplification using the restriction fragment, a library can be prepared uniformly.
Furthermore, in the present invention, since the overlapping information of each fragment can be reliably obtained, the connection between the base sequence-determined fragments can be facilitated. Further, without using subcloning, all reactions are performed only in a test tube, and the base sequence of a long sample DNA can be determined.

(Base Sequence Table) Base sequence number: 1 Base sequence length: 18 Base sequence type: Number of nucleic acid strands: Single strand Topology: Linear Base sequence type: Other nucleic acid, synthetic DNA base Sequence TGTAAAACGACGGCCAGT Base sequence number: 2 Base sequence length: 18 Base sequence type: Nucleic acid Number of strands: Single stranded Topology: Linear Base sequence type: Other nucleic acid, synthetic DNA Base sequence ACAGGAAACAGCTATGAC

[Brief description of the drawings]

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a diagram illustrating a step of, after fragmenting a sample DNA with a restriction enzyme, determining a base sequence of two bases following a restriction enzyme recognition base sequence of each fragment in the example of the present invention.

FIG. 2 is a diagram showing an example of a gel electrophoresis spectrum of a product of complementary strand synthesis of a DNA fragment obtained using 16 types of selection primers in an example of the present invention.

FIG. 3 is a diagram illustrating a process of preparing a sample for a nested deletion library using 5 ′ → 3 ′ exonuclease and an enzyme having 3 ′ → 5 ′ exonuclease activity in an example of the present invention.

FIG. 4 is a diagram illustrating a step of a second PCR amplification in an example of the present invention.

FIG. 5 is a diagram showing a method for determining a base sequence of a sample DNA using a nested deletion library in an example of the present invention.

FIG. 6 is a diagram illustrating a step of a second PCR amplification using a selection primer in an example of the present invention.

FIG. 7 is a diagram illustrating a process of preparing a sample for a nested deletion library using 3 ′ → 5 ′ exonuclease in an example of the present invention.

FIG. 8 is a diagram illustrating a step of a second PCR amplification in an example of the present invention.

FIG. 9 is a diagram illustrating a step of a second PCR amplification using a selection primer in an example of the present invention.

FIG. 10 is a diagram illustrating a process of preparing a sample for a nested deletion library using a 5 ′ → 3 ′ exonuclease and a single-strand degrading enzyme in an example of the present invention.

FIG. 11 is a diagram illustrating a step of a second PCR amplification in an example of the present invention.

FIG. 12 is a diagram illustrating a step of a second PCR amplification using a selection primer in an example of the present invention.

FIG. 13 is a diagram illustrating a step of a second PCR amplification using random primers in an example of the present invention.

[Explanation of symbols]

1, 151, 188, 201, 250, 270, 32
0, 340: sample DNA before restriction enzyme cleavage, 2, 3, 4
... DNA fragments generated by restriction enzyme digestion of sample DNA, 5 ...
Selection primer with fluorescent label, 6: base sequence complementary to at least a part of oligomer, 7: base sequence complementary to restriction enzyme recognition base sequence, 8: base sequence consisting of 2 bases of any base sequence, 9: fluorescence Signs 10, 11, 154, 2
05, 255, 276, 324, 345: Adapter base sequence, 155, 206, 256, 277, 325, 3
46: base sequence portion downstream from the adapter base sequence, 1
2 ... restriction enzyme recognition portion, 13, 13 '... base sequence portion to be selected, 14 ... base pair in which the selection primer and the DNA fragment are completely complementary, 15 ... base pair in which the selection primer and the DNA fragment are not completely complementary , 16, 156, 157, 1
58, 207, 208, 209, 211, 257, 25
8, 278, 279, 281, 326, 327, 32
8, 347, 348, 349, 351, 372, 37
3, 374: complementary strand extension reaction product, 101: vertical axis (base sequence of 2 bases at 3 ′ end of each selected primer), 10
2 ... horizontal axis (base length), 103 ... detected peak, 10
4 ... peaks detected at the same position as the peak of 103, 131, 230, 300 ... DNA fragments serving as templates,
132: Primers used for PCR reaction, 133, 30
2 ... Selection primer used for PCR reaction having a phosphate group label at the 5 'end, 134, 303 ... 5' end to which phosphate group is introduced, 135, 233, 304 ... DN
A fragment PCR product, 136, 305 .lambda. Exonuclease, 137 ... Klenow fragment, 236 ... exonuclease III, 138 ... single strand near one 5 'end, 152, 202, 237, 238, 252, 2
53 single-stranded near the 5 'end, 153, 203, 25
4, 274, 322, 342... Oligomers having a partial base sequence of the sample DNA, 181, 182.
NA fragment, 183: restriction enzyme recognition site, 184, starting point of amplified DNA fragment, 185, terminal portion of amplified DNA fragment, 186, direction of determination of base sequence at the time of base sequence determination, same base as 204, 133 Selection primers having a sequence, 231, 232, 301... Selection primers used for the PCR reaction, 275... Selection primers having the same base sequence as 231. Priming sites of primers, 234, 235... 3 ′ end of DNA fragment, 251, 271.
272, 273 ... single-stranded DNA near the 5 'end, 306
... S1 nuclease, 307, 321, 341 ... double-stranded DNA near the 5 'end, 323, 344 ... one strand of the double-stranded DNA near the 5' end, 371 ... random primer.

Claims (20)

[Claims] 1. A step of (1) fragmenting a sample DNA and amplifying each fragment to obtain a first DNA fragment;
At least the 3 'end of the sample D
Obtaining a second DNA fragment substantially complementary to NA; and (3) using the sample DNA as a template,
A third DNA fragment containing a base sequence complementary to the A fragment and longer than the second DNA fragment is prepared by a complementary strand extension reaction,
Using the sample DNA as a template for analysis. 2. The DNA analysis method according to claim 1, wherein the first DNA fragment is obtained by ligation of an oligomer having a known base sequence after enzymatic cleavage of the sample DNA and PCR amplification. Characteristic DNA analysis method. 3. The DNA analysis method according to claim 1, wherein the first DNA fragment comprises a poly (A) or a poly (U) at the 3 ′ end.
A DNA analysis method characterized by being obtained by PCR amplification after introduction by dylTransferase (TdT). 4. The DNA analysis method according to claim 1, wherein the first DNA fragment is prepared in a form having a phosphate group at one 5 ′ end, and 5 ′ → 3 ′ exonuclease;
And a single-stranded second DNA fragment which is degraded by an enzyme having 3 ′ → 5 ′ exonuclease activity. 5. The DNA analysis method according to claim 4, wherein λ exonuclease is used as the 5 ′ → 3 ′ exonuclease, and the enzymes having 3 ′ → 5 ′ exonuclease activity are exonucleases III and B.
AL31 nuclease, Klenow fragment, T4D
DN characterized by being one of NA polymerases
A analysis method. 6. The DNA analysis method according to claim 1, wherein the first DNA fragment is degraded by 3 ′ → 5 ′ exonuclease to remove the introduced oligomer at the 3 ′ end, thereby removing the second DNA fragment. A DNA analysis method comprising obtaining a DNA fragment. 7. The DNA analysis method according to claim 6, wherein exonuclease III or BAL31 nuclease is used as the 3 ′ → 5 ′ exonuclease. 8. The DNA analysis method according to claim 1, wherein the first DNA fragment is adjusted to a form having a phosphate group at one 5 ′ end, wherein the 5 ′ → 3 ′ exonuclease and the 5 ′ end Single-stranded DN remaining after DNA strand digestion
A DNA analysis method comprising obtaining the second DNA fragment by decomposing part A with a single-strand degrading enzyme. 9. The DNA analysis method according to claim 8, wherein λ exonuclease is used as said 5 ′ → 3 ′ exonuclease, and S1 nuclease or mag bean nuclease is used as said single-strand degrading enzyme. DNA analysis method. 10. The DNA analysis method according to claim 1, wherein the double-stranded sample DNA is used as a template. 11. The DNA analysis method according to claim 1, wherein the single-stranded sample DNA is used as a template. 12. The DNA analysis method according to claim 1, wherein an oligomer having a base sequence complementary to the end of the sample DNA is used as a PCR primer for obtaining the third DNA fragment. DNA analysis method. 13. The DNA analysis method according to claim 1, wherein the same base sequence as the 5 'end of the second DNA fragment or the 5' end of the second DNA fragment is used as a PCR primer for obtaining the third DNA fragment. Characterized by using an oligomer having at least as a PCR primer
A analysis method. 14. The DNA analysis method according to claim 1, wherein the same base as the 5 ′ end of the second DNA fragment or the 5 ′ end of the second DNA fragment is used as a PCR primer for obtaining the third DNA fragment. A DNA analysis method using an oligomer having a sequence and an oligomer having a random base sequence as PCR primers. 15. The DNA analysis method according to claim 1, wherein an oligomer having a selection sequence of 1 to 3 bases at the 3 ′ end is used as a PCR primer for obtaining the first DNA fragment. DNA analysis method. 16. A DNA analysis reagent kit for use in the DNA analysis method according to claim 1, wherein the DNA comprises an enzyme that degrades the sample DNA, and a primer for obtaining the first and third DNA fragments. Analysis reagent kit. 17. A DNA analysis reagent kit for use in the DNA analysis method according to claim 1, wherein the sample DNA is degraded by an enzyme, an oligomer having a known base sequence, a ligase, or a terminal deoxynucleot.
a reagent for introducing an oligomer of a known base sequence to the end of degraded DNA containing idyl transferase (TdT), a primer used for complementary strand synthesis,
A DNA analysis reagent kit comprising 5 ′ → 3 ′ exonuclease and an enzyme having 3 ′ → 5 ′ exonuclease activity. 18. A reagent kit for DNA analysis used in the DNA analysis method according to claim 1, which is an enzyme for degrading the sample DNA, an oligomer having a known base sequence, a ligase, or a degraded DNA containing TdT. A reagent for DNA base sequence determination comprising a reagent for introducing an oligomer having a known base sequence to the end, a primer used for complementary strand synthesis, and 3 ′ → 5 ′ exonuclease. 19. A DNA analysis reagent kit for use in the DNA analysis method according to claim 1, which comprises an enzyme that degrades the sample DNA, an oligomer having a known base sequence, a ligase, or a degraded DNA containing TdT. A reagent for introducing an oligomer having a known base sequence to the terminal, a primer used for complementary strand synthesis, 5 ′ → 3 ′ exonuclease,
A reagent kit for DNA base sequence determination comprising a single-strand degrading enzyme. 20. (1) a step of fragmenting a sample DNA and amplifying each fragment to obtain a first DNA fragment; and (2) at least a 3 ′ terminal side of each of the first DNA fragments is the same as the sample DNA. Obtaining a substantially complementary second DNA fragment; and (3) using the sample DNA as a template and the second DN
A third DNA fragment containing a base sequence complementary to the A fragment and longer than the second DNA fragment was prepared by a complementary strand extension reaction, and a series of deletions in which a plurality of bases were deleted from one end of the sample DNA. Obtaining a dead body and using it as a template for analysis of the sample DNA.