JPH10262699A - Dna analysis - Google Patents

Abstract

PROBLEM TO BE SOLVED: To analyze a DNA by determining the sequences of restriction enzyme-treated fragments of the sample DNA, reconstructing the each fragment by arranging according to the whole permutation, cutting the sample DNA by another restriction enzyme, and comparing the lengths of the cut fragments of the sample DNA with that of the fragments of the reconstructed DNA. SOLUTION: A DNA is analyzed by determining the sequences obtained by forming the sample DNA 1 into fragments 2 by a restriction enzyme and amplifying the respective fragments 2, reconstructing the respective fragments 4 having determined sequence by arranging the fragments according to the whole permutation to produce virtual structural base sequence group 5, cutting the sample DNA by another restriction enzyme and measuring the lengths of the fragments, and comparing the lengths of the cut fragments of the sample DNA with the length of the fragments to be formed by cutting the reconstructed candidate base sequence group to determine the reconstructed base sequence bound in the correct order from the candidate reconstructed base sequence group in the method for analyzing the DNA. The DNA is readily analyzed by the method without time and effort for searching duplex parts for binding respective fragments and useless operation such as plural determinations of the sequence of the same part.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a DNA analysis method.

[0002]

2. Description of the Related Art When determining the entire base sequence of about 50 kb of DNA inserted into a cosmid vector, a long DNA of 500 bases or more can be determined at a time by a DNA sequencer.
When the base sequence is determined, it is necessary to go through a step of making the DNA easy to handle before the sequencing. DNA
There are two conventional methods for making the size easy to handle. The first is to use a restriction enzyme at a specific site
This is a method of cutting NA. The second method is to randomly cut DNA using ultrasonic waves to make the DNA easy to handle.

[0003] D sized to be easy to handle in the first method
The nucleotide sequence of NA is determined after preparing a restriction enzyme map showing the positional relationship of the cut DNA fragments. In other words, the DNA was cut with a plurality of types of restriction enzymes, a restriction enzyme map showing the relative positional relationship between the fragment groups was created, and then the nucleotide sequence of each fragment was determined. Based on the positional relationship, the base sequences obtained from the fragments are connected to determine the entire base sequence.

A second method is a method called a shotgun method. In this method, the DNA is randomly cut to make the DNA easy to handle, and the base sequence of each fragment is determined by utilizing the fact that the cut DNA fragments have an overlapping portion with each other. Based on the nucleotide sequence information, the overlapping portions are overlapped with each other to reconstruct the entire nucleotide sequence.

[0005]

In order to construct a restriction map, DNA is cut with a plurality of types of restriction enzymes, and their fragments are inserted into an appropriate vector for subcloning. Thereafter, the base sequence of a certain fragment is determined from the end to the portion overlapping with another fragment, and a restriction enzyme map showing information on the overlap between fragments is created. When sequencing from one end of a fragment, it is not possible to predict where the overlap with another fragment will be, so it is necessary to sequence from the end until an overlap is found. In most cases, this operation is quite time-consuming because a single sequencing from the end does not reach the overlap. If a restriction enzyme map showing the relative positional relationship of each fragment can be obtained, fragments to be sequenced can be selected efficiently, and waste such as duplicate reading in sequencing can be reduced. However, there is a drawback that creating a restriction map is a very laborious and time-consuming task.

On the other hand, in the shotgun method, DNA fragmented into short fragments by ultrasonic waves is subcloned, and the subclones obtained as a result are randomly picked up to determine the nucleotide sequence. After that, the entire base sequence is reconstructed by superimposing the sequence information from each subclone, but the same type of subclone is picked up in order to pick up the subclone randomly, or almost overlapping. Subclone may be picked up, and to cover the entire sequence and reconstruct the original DNA sequence, 5 to 10 times
You need to read double-length arrays. In this method, the base sequence determination work can be started without the need for information on the positional relationship of the fragments, but the sequenced part may be a part whose sequence is already known, which is wasteful. It is.

[0007] The present invention solves these difficulties, and makes it possible to analyze DNA easily without the trouble of searching for overlapping portions to join each fragment and the waste of sequencing the same portion a plurality of times. is there.

[0008]

In order to achieve the above object, in the present invention, first, DNA to be sequenced is cut with a restriction enzyme. After introducing the DNA cut with the restriction enzyme into the vector, cloning and amplifying each fragment, the size of the DNA fragment introduced into the vector is measured by electrophoresis. Further, a clone containing each fragment is purified by electrophoresis. After purifying each clone again and sufficiently amplifying, the nucleotide sequence of each fragment is determined. In order to determine the nucleotide sequence after re-amplifying the purified clone,
The same part can be sequenced without waste without being sequenced a plurality of times. After determining the sequence of all fragments, the determined nucleotide sequence length was determined by electrophoresis DN
Confirm that it matches the A fragment length.

Next, a group of reconstructed candidate sequences is prepared by rearranging the fragments by all possible permutations. One of the reconstructed candidate sequences is a true reconstructed sequence obtained by actually connecting the fragments in the correct order. Search the positions of the recognition sequences of several types of restriction enzymes for the reconstructed candidate fragment group,
The length of a DNA fragment generated when a certain reconstitution candidate sequence is cleaved with a restriction enzyme is calculated and determined.

Next, the DNA to be actually sequenced is cut with the restriction enzyme whose cleavage site has been searched, and the length of the resulting fragment is measured by gel electrophoresis. By comparing the length of a DNA fragment actually determined by gel electrophoresis with the length of a fragment generated from a group of reconstituted candidate sequences, it is determined that any reconstituted candidate sequence matches the length of a fragment generated from the original DNA sequence. It is determined whether a fragment is to be provided, and it is determined that this reconstructed sequence is a true reconstructed sequence, that is, the original DNA sequence. As a result, the order of joining fragments cut by the restriction enzyme can be determined, and the actual DNA sequence can be obtained from the reconstructed candidate sequence group.

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION

(First Example) Lambda phage DNA was used as a sample. As shown in FIG. 1, lambda phage DNA1 is cut with a first restriction enzyme. Each cut DNA fragment 2 is introduced into a cloning vector for cloning. After amplification of each DNA fragment by cloning, purification is performed by electrophoresis. The purified DNA fragment 3 is again amplified by cloning to obtain an amount necessary for nucleotide sequence determination. Each DNA
The nucleotide sequence of fragment 3 is determined.

Each D resulting from cleavage with the first restriction enzyme
After determining the base sequence of the NA fragment, the sequenced DNA fragments 4 are joined in any order to create a group of candidate reconstructed sequences 5. Since the reconstructed candidate sequence group 5 is a sequence obtained by joining DNA fragments in any order, two candidate sequences are used when the number of DNA fragments is two, six candidate sequences are used when the number of DNA fragments is three, and n N! Consists of candidate sequences.

Next, as shown in FIG. 2, the λ phage DNA 1 is cleaved using a second restriction enzyme having a recognition sequence different from the first restriction enzyme used previously. Gel electrophoresis is performed on the λ phage DNA fragment 8 generated by the second restriction enzyme. From the migration pattern 12 and the mobility of the molecular weight marker 11, the length of the DNA fragment generated by the second restriction enzyme is measured. Further, for restriction enzymes having different recognition sequences, λ phage DNA is similarly cut, and the length thereof is measured by gel electrophoresis.

As shown in FIG. 1 and FIG. 3, the location of the second restriction enzyme or the recognition sequence of the restriction enzyme used in addition to the second restriction enzyme is searched on a computer using a search program. As a result of the search, the cleavage position 6 by each restriction enzyme is found. For each of the sequences of the reconstructed candidate sequence group, information 20 on the length of each DNA fragment generated by each restriction enzyme is obtained from the cleavage position of the second restriction enzyme or another restriction enzyme used by calculation.

Finally, information 20 on the length of the cut fragment of each sequence of the reconstructed candidate sequence group obtained by calculation and the actual DN
A is cut with restriction enzyme B, and the length of each DNA fragment obtained from electrophoresis pattern 12 of gel electrophoresis is compared. Of the reconstructed candidate sequences, only the sequence 21 obtained by joining the fragments in the correct order gives a digested fragment length that matches the fragment length resulting from actually cutting the DNA with the second restriction enzyme,
It is possible to determine the reconstructed sequence 22 obtained by rearranging in the correct order from the reconstructed candidate sequence group.

Second Example In the second example, a human gene of 8.9 k base length inserted into a plasmid was used as a sample.

(Fragmentation of Sample DNA and Introduction of Known Sequence into Fragment End) As shown in FIG.
The DNA fragment 31 is obtained by cleaving NA30. D obtained
An oligomer having a known sequence at both ends of the NA fragment 31 is introduced. Oligomers can be introduced by binding a double-stranded oligomer synthesized in advance using DNA ligase to the end of a DNA fragment, or by introducing dATP or dUTP using terminal deoxynucleotidyl transferase.
There is a method of adding a specific base such as P to the end of the DNA fragment. Either of them may be used, but the former is used in this example.

In order to form a phosphodiester bond between the DNA end and the oligomer, the 5 'end of the oligomer 40 in contact with the 3' end of the DNA fragment is phosphorylated. DNA
The phosphate group at the 5 ′ end of the DNA fragment is removed using alkaline phosphatase to prevent the binding between the fragments, and the 3 ′ end of the oligomer 41 located at the 5 ′ end of the DNA fragment is phosphorylated to prevent the binding between the oligomers. Keep it. DN
The oligomer 40 to be bound to the fragment A and the oligomer 41 having a sequence complementary thereto are hybridized to form a double-stranded adapter 42, and the DNA is ligated using DNA ligase.
Ligation with fragment 31. No phosphodiester bond is formed between the oligomer 41 whose 3 'end is phosphorylated and the 5' end of the DNA fragment 31, and the oligomer 40 whose 3 'end and 5' end of the DNA fragment 31 are phosphorylated. A phosphodiester bond is formed between them. As a result, DNA fragment 3
DN with an oligomer introduced at the 3 'end of both + and-chains
A fragment 32 is formed.

(Measurement of Fragment Length of DNA Fragment) The fragment length of the adjusted DNA fragment is measured by denaturing polyacrylamide gel electrophoresis or agarose electrophoresis. In denaturing polyacrylamide gel electrophoresis, a DNA fragment length of about 1000 bases can be measured with a resolution of about 1 base length, and in agarose electrophoresis, a DNA fragment length of about 3000 bases can be measured with a resolution of about 10 bases. If the length of the DNA fragment is shorter than 1000 bases, use the former,
If the length is longer than 1000 bases, the latter is used to measure the fragment length. If the DNA fragment length exceeds 3,000 bases, the measurement accuracy will decrease, but if the sample DNA is fragmented using a plurality of restriction enzymes, the DNA fragments exceeding 3,000 bases will be fragmented to a fragment length with high measurement accuracy. Can be.

The fragment length was measured by agarose electrophoresis, as shown in FIG. 2, by separating the fragmented DNA 8 by electrophoresis and labeling it with a reagent called an intercalator that binds to double-stranded DNA such as ethidium bromide. The fragment length is measured from the position of the detected molecular weight marker 11.

In the measurement of fragment length by denaturing polyacrylamide gel electrophoresis, the DNA fragment is electrophoresed in a single-stranded state, and therefore cannot be labeled with an intercalator. Therefore, the fragment length is measured using the primer 48 that has been fluorescently labeled in advance. A fluorescently labeled primer 48 having the following characteristics is prepared.

As the primer 48 used for measuring the fragment length, a primer which can be used for a base sequence determination reaction is used. That is, a portion of the sequence 50 which can be an anchor primer so that the sequence near the restriction enzyme cleavage site can be read, a portion of the sequence 51 complementary to at least a portion of the oligomer introduced into the DNA fragment, The part of sequence 52 complementary to the recognition site of the restriction enzyme used, and A, C, G,
It is composed of a part of sequence 53 consisting of two bases of an arbitrary sequence of T4 bases. Since the portion of the arbitrary sequence is composed of 16 types, which are all possible combinations of 2 bases, a total of 16 types of primers are prepared. If the base at the 3 'end of the primer is not hybridized, the elongation reaction of the DNA strand does not occur. For this reason, in the case of a primer having an arbitrary sequence at the 3 'end, it is possible to select a DNA fragment 60 in which an extension reaction occurs selectively from a plurality of types of DNA fragments.
Due to such properties, a primer having such an arbitrary sequence at the 3 'end is called a selection primer.

A complementary strand synthesis reaction is performed using one of the prepared selection primers. In this reaction, when the extension reaction proceeds to the end of the template DNA, no further extension is possible because there is no template DNA. Therefore, denaturation of the DNA strand using an excessive amount of the fluorescent labeling primer and the thermostable DNA polymerase → D
When the elongation reaction is performed a plurality of times in a heat cycle of binding of NA to the primer → elongation reaction of the primer, a fluorescently labeled single-stranded DNA having the same fragment length as the DNA fragment is obtained as a reaction product. Single-stranded DNA for all DNA fragments
Are difficult to separate when there are many types of DNA fragments. However, when the reaction is carried out using the selection primer, a fluorescently labeled single-stranded DNA fragment having the same fragment length only for the DNA fragment of the template DNA that has hybridized with an arbitrary sequence at the 3 'end of the primer and the template DNA is obtained. Is synthesized, so that fragments can be easily separated.

When extension reactions are similarly performed on the 16 types of primers prepared, single-stranded DNs for all template DNAs are obtained.
The A fragment can be synthesized. The reaction products for the 16 types of primers are subjected to denaturing polyacrylamide gel electrophoresis in separate lanes to measure the fragment length.

The fragment length of the DNA fragment is measured according to the above procedure. Even when the sample DNA is fragmented with different restriction enzymes by repeating the same procedure, the fragment length of the DNA fragment is measured, and the DNA fragment length generated when the sample DNA is cut with various restriction enzymes is measured. .

(Determination of DNA Base Sequence) In DNA base sequence determination, the sequences of DNA fragments fragmented with restriction enzymes are sequentially determined. DNA having a known sequence introduced into the end by the above method
The sequence of the fragment may be determined using a fluorescently labeled selection primer (DNA Research Vol. 1, 231-237, 19).
94). An anchor sequence may be used as a primer to determine the sequence of the end of the DNA fragment. In this case, a DNA fragment to be sequenced is amplified by PCR amplification using information on the two bases at the end of the DNA fragment obtained from the measurement of the fragment length.

Since the lengths of the + and-chains of the DNA restriction enzyme fragments are the same, those having the same fragment length among the fragments that have undergone the extension reaction during the measurement of the fragment length are the + and-chains of the DNA fragment, respectively. Therefore, if PCR amplification is performed by combining the selection primers that have undergone the extension reaction, only a specific DNA fragment can be amplified from a mixture of various DNA fragments. When performing a sequencing reaction by labeling a primer at the time of DNA base sequencing, sequencing of a site adjacent to the primer may be difficult due to the effect of an unreacted primer, but the anchor sequence portion of the selected primer used for the amplification reaction may be difficult to determine. If a base sequence determination reaction is performed as a primer, the site adjacent to the anchor for sequencing will be the portion of the selection primer used for amplification, and the end of the DNA fragment will not be close to the end of the anchor for sequencing. Errors at the time of determination can be reduced.

Even if the terminator labeling method is used in the sequencing reaction, the reading error at the end of the DNA fragment can be similarly reduced.

As described above, the base sequence of each DNA fragment obtained by treating the sample DNA with restriction enzymes is determined. Thereafter, in the same manner as in the first embodiment, a group of reconstructed candidate sequences is prepared by joining the obtained DNA fragments in all the permutations, and a restriction enzyme recognition site present in the reconstructed candidate sequence is searched. The sample DNA is cleaved, the recognition sequence of the restriction enzyme used for measuring the length of the resulting DNA fragment is searched, and the cleavage pattern of the reconstructed candidate sequence is obtained. Since one of the reconstructed candidate sequence groups is a correct reconstructed sequence, the correct reconstructed sequence is obtained by comparing the cleavage pattern of each reconstructed candidate sequence with the actual sample DNA cleavage pattern, and the base sequence of the sample DNA is determined. To determine.

[0030]

According to the present invention, the joining of fragments at the time of base sequence determination can be carried out from the base sequence information and the information on the length of the fragment cut with another restriction enzyme. As a result, DNA analysis can be performed without creating a restriction enzyme map which has conventionally required much labor and time. In addition, since it is not necessary to perform duplicate reading of the same portion as in the shotgun method, it is possible to determine the base sequence efficiently without reading the number of bases which is many times the number of base sequences to be determined.

[Brief description of the drawings]

FIG. 1 is an explanatory diagram of creation of a reconfiguration candidate sequence group in a first embodiment of the present invention.

FIG. 2 is an explanatory view of the measurement of the fragment length of a DNA fragment in the first embodiment of the present invention.

FIG. 3 is an explanatory diagram of reconfiguration sequencing in the first embodiment of the present invention.

FIG. 4 is an explanatory diagram of a DNA base sequence determination method according to a second embodiment of the present invention.

[Explanation of symbols]

1 ... lambda phage DNA, 2 ... lambda phage DNA fragment, 3 ... lambda phage DNA fragment, 4 ... sequence of DNA fragment, 5 ... reconstructed candidate sequence group, 6 ... cleavage site, 8 ... lambda phage DNA fragment.

Claims (5)

[Claims] 1. A process in which a sample DNA is fragmented using a restriction enzyme to amplify each fragment, followed by sequencing, and a virtual reconstructed base sequence group in which the sequenced fragments are arranged in all permutations and connected. The process of preparing, the process of cutting the sample DNA with another restriction enzyme and measuring the fragment length, and the process of cutting the fragment length and the sample DNA generated when the reconstructed candidate nucleotide sequence group is cut with the restriction enzyme A DNA analysis method comprising a step of comparing the obtained fragment lengths and determining a reconstructed base sequence joined in a correct order from a group of reconstructed candidate base sequences. 2. A fragment is generated from a sample DNA by using a restriction enzyme, and nucleotide sequence information obtained by determining the nucleotide sequence of each fragment is compared with DNA fragment length information obtained by a plurality of different restriction enzymes. A DNA analysis method characterized by determining the positional relationship between restriction fragments. 3. The method according to claim 1, wherein a reconstructed candidate sequence group is created by linking the sequenced fragments to all possible permutations, and the positional relationship between the fragments is determined using the sequence information. DNA analysis method to determine 4. The DNA analysis process according to claim 1, wherein the sequenced fragments are connected to all possible permutations to form a group of reconstructed candidate sequences. 5. A DNA analysis method comprising determining the positional relationship between fragments without performing overlapping reading of a base sequence when joining fragmented DNAs.