JP2002085097A - Method for determination of dna base sequence - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for the determination of a DNA base sequence enabling the determination of the base sequence of a long DNA which has hitherto been difficult to determine. SOLUTION: The base sequence of a double-stranded DNA 4 is determined by (1) a step to bond an oligomer 6 having the recognition sequence of a class IIs restriction enzyme to a terminal of a double-stranded DNA 4, (2) a step to cleave the double-stranded DNA 8 having the oligomer bonded by the step (1) with a class IIs restriction enzyme, (3) a step to bond the oligomer to the fragment of the double-stranded DNA cleaved by the step (2) and (4) a step 20 to repeat the step (2) and the step (3) multiple times to obtain a series of double-stranded DNA fragments 9 composed of several number of bases and having a definite base length difference between the fragments. The present process enables easy determination of the base sequence of a long DNA fragment.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for preparing a sample for DNA analysis for DNA base sequence determination, genetic diagnosis and the like.

[0002]

2. Description of the Related Art Conventionally, DNA base sequence determination has been carried out by preparing a group of DNA fragments having different lengths by one base by the dideoxy method and separating the DNA fragments by gel electrophoresis. I have. DN that does this automatically
A sequencer is commercially available.

[0003] Separation of DNA length by gel electrophoresis requires separation of one base difference, and separation of up to about 600 bases can usually be performed. To determine the base sequence of longer DNA, shorter fragments are sequenced and joined together.

[0004] A method using such gel electrophoresis is as follows.
It is used for a wide range of applications, including the determination of nucleotide sequences of genomes and the like. With the progress of genome analysis, short DNA or DN
There has been a growing demand for rapid sequencing of the A sequence in large quantities. Therefore, in addition to gel electrophoresis, various new DNA sequencing methods have been proposed.

One of them is that when DNA synthesizes a complementary strand,
This method involves combining dNTPs one by one with a DNA polymerase, but converting pyrophosphate generated as a by-product to ATP and reacting with luciferin to obtain chemiluminescence, which is monitored to determine the base sequence. A complementary strand is repeatedly synthesized, and the type of base incorporated is monitored to determine the DNA base sequence. Recently, Uhle
have improved this method to a practical level and named it Pyro Sequencing (Reference 1: Scien).
ce 281, 363-365, 1998).

[0006] On the other hand, Brenner et al. Cut a double-stranded DNA to be sequenced with a class IIs restriction enzyme, and obtained an adapter (encoded ada) having a known sequence.
ptor) is bound to the enzyme cleavage site, and a fluorescently labeled probe (decoded probe) is attached to the adapter (encoded adapter) bound to the enzyme cleavage site.
or), and a double-stranded DN generated by cleavage of class IIs restriction enzyme at each repetition.
The nucleotide sequence of the 5 'protruding end of A is determined (Reference 2: Nat)
ure Biotechnology, 18,630-
634 (2000)).

[0007]

The techniques described in the prior art and in References 1 and 2 are of a length that allows base sequence determination for gene expression profile analysis, gene diagnosis, or screening for diagnosis. Although it is as short as about 20 bases, it is characterized by the ability to analyze a large amount of DNA at a time, and has been attracting attention.

[0008] Each of the current DNA analysis methods including DNA sequencing has its own problems. DN prepared by Sanger method (dideoxy method)
In the method of separating A by gel electrophoresis and determining the base sequence,
The upper limit of the base sequence determination is up to the DNA size at which a difference in length of one base can be separated by gel electrophoresis, which is about 600 bases in a usual apparatus, and about 1000 bases in an apparatus using a long gel.

There is a need to separate longer DNAs up to a single base difference or to detect DNAs in a state where each DNA band does not overlap, but this is not possible at present.

On the other hand, pyrosequencing and class IIs
In a sequencing method using a restriction enzyme, a cyclic reaction in which a reaction cycle is repeated (cyclic reaction) is performed.
Since the base sequence is determined by n), the base length for which the base sequence can be determined is determined by the reaction efficiency. At present, the base sequence can be analyzed with good reproducibility from 20 to 30 bases.

In pyrosequencing, the base sequence is determined by using a cyclic reaction in which pyrophosphate generated each time a complementary strand is synthesized is converted to ATP and luciferin is illuminated. The reaction yield of each cycle reaction is 1
It is not 00%, but as the reaction proceeds, there are cases where the chain is extended and cases where it is not extended, and it becomes impossible to determine the nucleotide sequence. For this reason, there has been a problem that the length at which a base sequence can be determined at one time is short.

If the base sequence can be determined for a longer base, it is expected to be used for more detailed analysis or DNA diagnosis. The problem with pyrosequencing is to be able to determine even longer nucleotide sequences.

[0013] An object of the present invention is to provide a DNA sequence capable of determining the nucleotide sequence of long DNA, which has been difficult to determine the nucleotide sequence.
An object of the present invention is to provide a method for determining an NA base sequence.

[0014]

According to the DNA sample preparation method of the present invention, a fixed base length of 2 bases or more, that is, a fixed base length ranging from 2 bases to 16 bases or 20 bases in length. Is used as a unit, a series of DNA fragments having different lengths by one unit, that is, a series of DNA fragments having different lengths by a fixed base length are prepared from a DNA sample using a class IIs restriction enzyme. The analysis solves the problems in the prior art.

For example, in gel electrophoresis, large DNA
Is determined by gel electrophoretic separation with a difference in length of one base, the base sequence of long DNA cannot be determined.

A DNA sample is prepared by a series of DNs having different lengths by 1 unit, with 3 or 5 bases as 1 unit.
If it is separated into fragment A, DN that forms a series of DNA fragments
The A-band spacing (spacing) increases,
Even the NA fragment can identify the length for each unit.

When ddNTP fluorescently labeled to the end of this series of DNA fragments is bound with a DNA polymerase and analyzed, for example, if one unit is composed of 5 bases, the series of DNA fragments are fragments having different lengths by one unit. As a result, the DNA fragments can be separated and detected by identifying the difference in the length of 5 bases instead of the difference in the length of 1 base. Since the difference in DNA fragment length is 5 bases, it is possible to identify DNA fragments that are much longer than in the case of 1 base difference.

Since the DNA base sequence can be determined every five bases, the cleavage site of the class IIs restriction enzyme is shifted by one base. A group of five DNA fragments was prepared, and a series of DNA fragments in which the cutting positions of the class IIs restriction enzymes were shifted by one base between the groups of five DNA fragments were prepared and included in each DNA fragment group. The entire base sequence can also be known by knowing the terminal base sequence of the DNA fragment to be obtained and synthesizing the sequence information.

In pyrosequencing using the synthesis reaction of an extended complementary strand, a large number of copies of the DNA to be sequenced are made, and this is used as a template to determine the base sequence by a cyclic reaction in which the complementary strand synthesis reaction is repeated using primers. However, if the reaction yield is not 100%, the reacted and unreacted products will coexist, and the reaction will be delayed and various bases will be incorporated due to the unreacted products. It will be low.

Therefore, at the time when the cycle reaction has progressed for 20 or 30 cycles, four types of complementary strand synthesis substrates dNTPs are added to advance the complementary strand, thereby preparing a long double-stranded DNA starting from the primer. . Next, Class II
After cutting with a s restriction enzyme, an oligomer having a priming region at the cut portion is bonded, the size of the DNA double strand is adjusted, and then the single strand is formed. This gives
Alignment of DNA chains having irregular lengths enables highly reliable nucleotide sequence determination.

According to the present invention, a class IIs restriction enzyme that recognizes and cuts a certain length of base length from the position of the starting point of complementary strand synthesis when the complementary strand is extended by performing complementary strand synthesis.
The difficulty is overcome by cutting the NA and arranging the lengths of the respective fragments to enable the sequencing of long DNA. This situation is the same in the method of analyzing the base sequence by adding the class IIs restriction enzyme cleavage and fluorescently labeled ddNTP, and it is possible to obtain highly reliable base sequence information.

[0022]

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a method for preparing a DNA sample,
And a DNA sequencing method and a DNA analysis method using the same. A large number of copies of the DNA sample to be analyzed are prepared, and from the copies of the DNA sample, a series of DNA fragments having different lengths by one unit are prepared, with a predetermined length of several bases as one unit. Is analyzed.

DNA fragments having different lengths by one unit can be easily analyzed by gel electrophoresis even if the length of the DNA fragments is longer.
The length of the A fragment becomes longer.

On the other hand, by applying the present invention to a new DNA analysis method using a cycle reaction, a DNA base sequence several times longer than that of the prior art can be determined, which hinders practical use. A new DNA analysis method can be widely used as a DNA base sequence determination method without any problem.

Example 1 Example 1 discloses a sample preparation method according to the present invention for base sequence determination using gel electrophoresis separation or DNA analysis.

FIG. 1 is a diagram for explaining a DNA preparation method according to the present invention, and is a diagram for explaining the principle of a method for preparing a series of DNA fragments having different lengths by a fixed base length. Here, BpmI and BsgI were used as class IIs restriction enzymes.
Of course, other class IIs restriction enzymes may be used. Primer 1 that hybridizes to DNA sample 40, and a class at the 5 'end of the portion that hybridizes to DNA sample 40
The DNA sample 40 is PCR-amplified using the primer 2 having the recognition part 6 of the IIs restriction enzyme BpmI to obtain a PCR amplification product 3. Retain 1% of the amplified DNA sample and 99% of the amplified DNA fragment
Cleavage with pmI yields DNA fragment 4.

Next, one end of the DNA fragment 4 cut with the class IIs restriction enzyme BpmI was added to the class IIs restriction enzyme BsgI
5 with Class IIs Restriction Enzyme BpmI
Oligomers comprising a mixture of oligomers 6 having a recognition site of 1: 100 in a ratio of 1: 100 are bound by ligation.

As a result, out of the generated DNA fragments, about 99 DNA fragments 8 that can be cleaved with the class IIs restriction enzyme BpmI were obtained.
%, And the remaining DNA fragment 7 is a class IIs restriction enzyme B
Since it has an sgI recognition site, it is not cleaved by the class IIs restriction enzyme BpmI.

Of course, the present invention also works effectively when only the oligomer 6 having the recognition portion of the class IIs restriction enzyme BpmI is bound to the DNA fragment 4 by the above ligation (in this case, the following mixture 27). Is oligomer 6
-Only DNA fragment 8 to which-has been bound).

Next, the mixture 27 of the DNA fragments 7 and 8 to which the oligomer was bound was added to the class IIs restriction enzyme BpmI.
Step 20 is repeated (N-1) times, in which the cleavage of the DNA fragment generated by the cleavage and the reaction of enzymatically binding the oligomer to the cut portion of the DNA fragment by ligase are performed. Finally, DN
A fragment 9-1 is added to the reaction solution. DNA fragment 9-1 is
This is a 1% portion of the DNA sample (PCR amplification product 3) that has not been cut by the class IIs restriction enzyme BpmI of the PCR amplification product 3 and has been left above.

As a result, assuming that a given base length is one unit, a series of DNA fragments 9 having different lengths by one unit is obtained.
(9-1, 9-2, ..., 9-N).

D at the site where the oligomer ligates
The terminal nucleotide sequence of the NA fragment (terminal nucleotide sequence of the ligation site) may have various nucleotide sequences as the nucleotide sequence of the cleavage site by the class IIs restriction enzyme BpmI. Oligomers are mixed so as to have a base sequence.

That is, as oligomers 5 and 6,
A mixture of oligomers having a base sequence capable of complementarily binding to all of the base sequences that may appear in the terminal base sequence of the ligation site is used.

FIG. 2 is a diagram showing an example of the structure (base sequence) of the oligomers 11 and 11 'which are connected by ligation to the cut portion of a DNA fragment generated by cleavage with a class IIs restriction enzyme.

The oligomer 11 is an example of the oligomer 6, and has a common base sequence 12, a class IIs having a known sequence common to each oligomer regardless of the type of XY of each oligomer.
Recognition sequence of restriction enzyme BpmI 13, Class IIs restriction enzyme BpmI
The base sequence of the linker portion 10 having a known sequence for controlling the cleavage site caused by the base sequence (an example of two bases is shown in FIG. 2), and an arbitrary two base sequence site 14.

The oligomer 11 'is an example of the oligomer 5, and has a common base sequence 12' having a known sequence common to each oligomer regardless of the type of XY of each oligomer.
Recognition sequence 13 'of class IIs restriction enzyme BsgI, class IIs restriction enzyme
It is composed of a base sequence of a linker portion 10 'having a known sequence for controlling a cleavage site by BsgI (FIG. 2 shows an example of two bases), and an arbitrary two base sequence site 14'.

X and Y are any of A, T, G, and C. XY has 16 combinations of base sequences.
Are each a mixture of oligomers having 16 different types of arbitrary two base sequence sites.

FIG. 3 shows that a series of DNA fragments having different lengths by one unit is defined as one unit having a fixed base length.
BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a diagram for explaining a method of preparing by class IIs restriction enzyme cleavage.

In the example shown in FIG. 3, the class IIs restriction enzyme Bpm
By using an oligomer 6 containing a base sequence of 9 bases and an arbitrary base sequence 14 of 2 bases as the linker 10 for controlling the cleavage site of the recognition sequence 13 of I and the class IIs restriction enzyme BpmI, the base length is fixed. Has a length of 5 bases.

That is, by repeating the step of cutting with the class IIs restriction enzyme BpmI and the reaction of binding the oligomer to the cut portion of the DNA fragment generated by the cutting, a DNA fragment further shortened by 5 bases can be obtained.

In FIG. 3, M9 and M'9 indicate nine sequences of M and nine sequences of M ', respectively, and M and M' are arbitrary sequences. Sequence M'9 'is complementary. Xn
Yn, X'nY'n, Xn + 1Yn + 1, X'n + 1Y'n + 1, Xn + 2Yn +
2, X'n + 2 Y'n + 2 indicates an arbitrary two base sequence site selected from A, T, G, C, and XnYn, X'nY'n, Xn + 1
Yn + 1 and X'n + 1Y'n + 1, Xn + 2Yn + 2 and X'n + 2Y'n + 2 are
Each is complementary. N, N 'are A, T, G, C
Any of the bases.

Class II in the nth repetition step 20
s Cleavage site of DNA fragment generated by cleavage with restriction enzyme BpmI (site where oligomer binds by ligation)
When the ligation reaction of the oligomer 6 with the DNA 15 is completed, a DNA fragment 18 is obtained.

When the DNA fragment 18 was cleaved again with the class IIs restriction enzyme BpmI, a site shorter by 5 bases (class
A DNA fragment 17 cleaved with the IIs restriction enzyme BpmI) 16 can be obtained. However, the remaining 1% of D which does not have the recognition sequence of this class IIs restriction enzyme BpmI.
NA fragment 7 remains uncut.

Again, oligomer 6 was ligated to D
The DNA fragment 19 was ligated to the cleavage site (the site where the oligomer binds by ligation) 15 'of the NA fragment 17,
Get.

Subsequently, in the (n + 1) -th repetition step 20, the DNA fragment 1 was again treated with the class IIs restriction enzyme BpmI.
When Cleavage 9 is cut, a site shorter by 5 bases (Class IIs
Part cut with restriction enzyme BpmI) D cut with 16 '
NA fragments can be obtained.

As described above, by repeating step 20, a DNA fragment 9 having a series of lengths reduced by 5 bases can be prepared.

DN cleaved with class IIs restriction enzyme BpmI
Based on the cleavage site of fragment A, class IIs restriction enzyme B
By repeating Step 20 of cutting the DNA fragment with pmI and ligation of the oligomer 6 to the cut portion, a series of DNA fragments having different lengths by 5 bases is prepared.

When the oligomer 6 shown in FIG. 3 is used in the sample preparation method shown in FIG. 1, DNA fragments 9 having different lengths by one unit can be obtained with five bases as one unit.

By controlling the base sequence of the oligomer 6 that binds to the DNA fragment 4 by changing the base length of the linker portion 10, the cleavage site by the class IIs restriction enzyme is shifted by one base for each DNA fragment group. And DN
Groups of A fragments can also be prepared.

FIG. 4 shows the results obtained by cutting the class IIs restriction enzyme.
FIG. 3 is a diagram illustrating a method for preparing a group of a series of DNA fragments having different lengths for each base. The five oligomers 6-0,6-1,6-2,6-having the base sequence of the linker portion 10 shown in FIG.
Prepare 3, 6-4. The configuration of the oligomer 6-0 is the same as the configuration of the oligomer 6.

A portion of 5% of the DNA sample (PCR amplification product 3) is left, and 95% of the PCR amplification product 3
The solution of the container 30 containing the solution 27 'containing the DNA fragment 4 obtained by cleaving the DNA fragment 4 with the class IIs restriction enzyme BpmI was added to the containers 35-1, 35-2, 35-3, 35-4, 35-5. Dispense into For each container 35-m (m = 1 to 5),
When the base sequence of the linker portion 10 is (m + 8) bases (m = 1 to
5), the oligomer 6-m (m = 0 to 4) having the recognition sequence of the class IIs restriction enzyme BpmI is bound to the cleavage site of the DNA fragment 4 by the class IIs restriction enzyme BpmI by a ligation reaction.

That is, the oligomer 6-0 is placed in the container 35-1.
, The oligomer 6-1 in the container 35-2, and the container 35-3.
In the container 35-4.
3 and the oligomer 6-4 are added to the container 35-5 to perform a ligation reaction.

As a result, between the containers 35-m (m = 1 to 5), a mixture 27-m (m = 1) containing a DNA fragment in which the cleavage site by the class IIs restriction enzyme BpmI was shifted by one base at a time.
1-5) are obtained.

In each of the containers 35-m (m = 1 to 5), the mixture 27-m (m = 1 to 5) was cleaved with the class IIs restriction enzyme BpmI as described above. Step 20 of repeating the reaction of enzymatically connecting the oligomer 6-0 (oligomer 6) having a base sequence of 9 bases of the linker portion 10 with the ligase to the cut portion of the DNA fragment generated by the cleavage. Finally, the container 35-m
To each of the containers (m = 1 to 5), a 5% portion of the DNA sample (PCR amplification product 3) that has been left in advance is divided into five equal portions and added.

As a result, DNA fragment group 21-m
(M = 1-5) is obtained. As shown in FIG. 4, in each group (container), a series of fragment groups 9 (21-1 to 21-5) having different lengths by 5 bases are generated, and each base (container) has one base. DNA cut at shifted positions (different positions between groups (containers))
Fragment 21 is obtained.

As described above, by repeating step 20 in each container, DNA fragments 21 differing in length by one base
Can be obtained.

FIG. 5 shows the result obtained by the method shown in FIG.
FIG. 2 is a diagram illustrating the principle of adding a fluorescently labeled ddNTP to the end of a series of DNA fragments having different lengths by one base to determine a base sequence.

The end of the DNA fragment 21 obtained by repeating the step 20 shown in FIG.
I recognition sequence 13 is included. The DNA fragment 21 is cleaved with the class IIs restriction enzyme BpmI, and any one of the ddNTPs is cut using four types of fluorescent-labeled dideoxynucleotides (ddNTP *, * are different fluorescent labels for each type of N) and DNA polymerase. * 23 extends the complementary strand to the protruding end of the cleavage site 22.

As a result, the group of DNA fragments 24-m (m = 1 to 5) incorporating ddNTP * complementary to the single-stranded base sequence at the 5 ′ protruding end was placed in the container 35-m (m = 1 to 5).
Generated in each container of 5). As a result, ddNTPs * each having a different fluorescent label * corresponding to the base type are added, and a fluorescently labeled DNA fragment 24 is obtained.

Therefore, by observing the fluorescence emitted from the fluorescent label *, the type of base incorporated at the 3 ′ end of the cleavage site 22 of the DNA fragment 24 can be determined. By separating the length of the DNA fragment 24 by electrophoresis and detecting the fluorescence generated from the fluorescent label of the DNA fragment subjected to electrophoresis, the base sequence can be known every five bases.

The DNA fragment group 24-m (m = 1 to
5) is injected into the sample injection end 25-m (m = 1 to 5) of the electrophoresis path corresponding to each m (m = 1 to 5). By independently analyzing the length and terminal base type of the DNA fragment group 24-m (m = 1 to 5), the base sequence of each base of DNA can be known.

The DNA fragment group 24-m (m = 1 to
Needless to say, 5) may be separated by electrophoresis using a capillary electrophoresis path corresponding to each m (m = 1 to 5). Adjacent DNA fragments 24 separated and detected in each migration path
The difference in length is as large as 5 bases, and the size of a DNA fragment that can be separated by gel electrophoresis is much larger than when one base is separated, which facilitates separation.

The method for preparing a sample using the class IIs restriction enzyme described above is not limited to the above-described example of determining the base sequence of a DNA sample. For example, it can be applied to genetic diagnosis. Based on the sample preparation method of the present invention, a series of DNA fragments having different lengths by a predetermined base length are prepared, gel electrophoresis is performed, and the terminal base species of the DNA fragments is identified by fluorescence detection. This gives D with a certain length
In addition to knowing the base type of each NA fragment, it is possible to examine whether or not there is a base substitution from the electrophoresis pattern as follows. This is based on the fact that a change occurs in the migration pattern of a DNA fragment longer than the length of the DNA fragment where the base substitution exists.

That is, when comparing two DNA samples,
According to the method described above, a series of DNA fragment groups having different lengths by a fixed base length are prepared from each DNA sample. By comparing the DNA fragment groups prepared from the two DNA samples, the gel electrophoresis band patterns of the original long DNA sample are compared. If a base sample is substituted in a DNA sample, the distance between adjacent DNA bands in the electrophoresis direction is slightly shifted due to a difference in base type.

Although it is often difficult to understand by comparing the electrophoresis band pattern for separating one base of the original long DNA sample or the electrophoresis speed of the DNA fragment, the electrophoresis band pattern of the DNA fragment according to the present invention is as follows. By comparing the band pattern of the DNA fragment having the position where the base substitution is present at the end with the band patterns of the DNA fragments before and after it, the difference in the base sequence between the two DNA samples can be accurately found.

Example 2 Example 2 is an example in which the sample preparation method using the class IIs restriction enzyme according to the present invention was applied to pyrosequencing. First, pyrosequencing will be described below.

FIG. 6 is a diagram showing the principle of the conventional DNA sequencing method by pyrosequencing used in Example 2. A complementary strand of the DNA to be sequenced is obtained as a single-stranded DNA sample 400. A primer 401 that hybridizes to a site where a base sequence is to be determined is prepared.

Single-stranded DNA sample 400, primer 40
1, a substrate (deoxynucleotide dNTP402; dATP, dGTP, dCTP,
dTTP), enzyme (DNA polymerase), etc.
03 is formed and complementary strand synthesis occurs.

At this time, if only one kind of dNTP is added, the complementary strand elongation occurs only when it is a base type to be incorporated in complementary strand synthesis. In the complementary strand extension reaction, pyrophosphate (PPi) 404 is released. In a series of reactions 405 occurring in pyrosequencing, pyrophosphate (PP
i) and APS (adenosine 5'phosphophosphate) in the presence of the enzyme ATP sulfurylase to produce ATP, and the resulting ATP and luciferin to react in the presence of oxygen and luciferase to produce chemiluminescence. 407 and a reaction to generate pyrophosphoric acid (PPi). This newly generated pyrophosphate (PPi)
Reacts with APS to produce new ATP. Surplus dN
TP402 is converted to dNDP by ATPase,
dNDP is converted to dNMP by ADPase.
On the other hand, the generated ATP is converted to ADP by ATPase, and ADP is converted to AMP by ADPase. The ATP generation reaction after the complementary chain elongation reaction is in a steady state if there is sufficient APS, luciferin, and O ₂ , and the luminescence reaction continues because the luminescence reaction continues.

For details of reactions related to pyrosequencing, see Reference 1 (Science 281, 363-36).
5, 1998).

The presence or absence of the complementary strand synthesis of dNTP 402 added in a series of complementary strand synthesis reactions 406 is determined by chemiluminescence 40.
7 by monitoring the occurrence of dNTP402, the type of dNTP402 used for complementary strand synthesis can be sequentially known.
The nucleotide sequence of NA can be determined. For example, in the example shown in FIG. 6, when dATP, dCTP, dGTP, and dTTP are added in this order, when dATP, dCTP, and dTTP are added, the complementary strand elongation occurs to generate chemiluminescence 407, and the extended complementary strand ACT is formed. Is done. As a result, the base sequence ACT can be determined.

Since the pyrosequencing uses a cycle reaction, the length of a base sequence that can be determined in a series of reactions is 20 to 30. In pyrosequencing, if the same base is continuously present in the base sequence of a single-stranded DNA sample, two deoxynucleotides will be incorporated in succession. The amount of generated pyrophosphate differs between when one deoxynucleotide is incorporated and when two or three deoxynucleotides are incorporated, and there is a difference in the resulting luminescence intensity.

Using this difference in light emission intensity, it is possible to determine how many deoxynucleotides have been incorporated, but the accuracy is not very good. In particular, as the complementary chain elongation reaction proceeds, the luminescence intensity decreases and the number of spurious signals due to the reaction not being 100% increases, so that it becomes more difficult to understand how many bases have the same base sequence.
The sample preparation method of the present invention is effective for overcoming these difficulties.

The method for utilizing the sample preparation method using the class IIs restriction enzyme according to the present invention for pyrosequencing is as follows.
There is a street.

The first use method is the same as in the first embodiment.
This is a method in which a series of DNA fragments having different lengths by a predetermined base length are prepared, and pyrosequencing is performed using these as a template. The first usage will be obvious with reference to the other embodiments, so the second usage will be described.

FIG. 7 is a diagram for explaining a second method of use in the second embodiment, in which DNA by pyrosequencing is used.
FIG. 3 is a diagram illustrating an example of a method of alternately performing base sequence determination and class IIs restriction enzyme cleavage.

As shown in FIG. 7, first, the 5 ′ end of the DNA sample whose base sequence is to be determined is fixed on the surface of a solid carrier such as beads 300. After adding an oligomer (adaptor) 307 having a cleavage recognition site for the class IIs restriction enzyme BpmI to the 3 'end of the DNA sample, the single-stranded DNA was added.
The sample 301 is used. At this point, the single-stranded DNA 301 to which the oligomer 307 has been added is immobilized on the beads 300.

The extended complementary strand 303 is synthesized using the region containing the base sequence of the oligomer 307 as a priming site. That is, a pyrosequencing process is performed using the primer 302 that hybridizes to the oligomer 307 introduced into the single-stranded DNA 301, the base sequence is determined, and the extended complementary strand 303 is synthesized.

When the nucleotide sequence having a length of about 20 to 30 bases has been determined, the reaction is stopped after all the dNTPs are added to further elongate the complementary strand. This last process of complementary strand elongation may optionally be omitted.

After removing the reaction solution, the class IIs restriction enzyme B
pmI and its buffer solution are added, and the DNA is cut with a class IIs restriction enzyme BpmI at a predetermined position (a cleavage site of a class IIs restriction enzyme) 304 from the end of the double-stranded DNA. An oligomer 305 having a cleavage recognition sequence of the class IIs restriction enzyme BpmI is added to the cleavage portion by a ligation reaction.

After addition of the oligomer 305 to the cleavage site 304 of the class IIs restriction enzyme by a ligation reaction,
Wash with single-stranded DNA. Using this single-stranded DNA 306 shortened by a certain length as a template, primer 3
Pyrosequencing is performed again using 02 '.
By repeating step 308 of the above process, DNA sequencing by pyrosequencing and class IIs
Restriction enzyme cleavage is performed alternately, and the base sequence is reliably determined at a fixed length.

FIG. 8 is a diagram showing an example of the structure of an oligomer which is ligated to a cut portion of a DNA fragment generated by cleavage with the class IIs restriction enzyme BpmI in Example 2.

As described above, when the class IIs restriction enzyme BpmI was used, as shown in FIG.
Template DNA that is as short as bases is created That is, after the adapter 305 containing the recognition part (sequence) 311 of the class IIs restriction enzyme BpmI is ligated to the DNA fragment 317,
The base sequence is determined from the portion of the base sequence 315 which is subsequently removed by the cleavage of the IIs restriction enzyme BpmI to the portion of the base sequence 316 to be newly determined, and the complementary strand is synthesized.

Since cleavage with the class IIs restriction enzyme BpmI cuts position 313, the template DNA which is 12 bases shorter is used.
Can be. When this operation (ligation of an adapter and cleavage with the class IIs restriction enzyme BpmI) is performed twice, a template DNA shorter by 24 bases is generated. The length of the template DNA to be prepared can be determined by adjusting the length of the base sequence of the linker portion 314 that controls the cleavage site of the class IIs restriction enzyme, and can be determined depending on the purpose of the analysis.

Using the shortened DNA as a template DNA, the base sequence is determined again by pyrosequencing. The determined base sequence is from the thirteenth base of the already determined base sequence, and can be advanced while performing a cross-check with the previously determined base sequence, thereby increasing the accuracy.

Since the base sequence analysis is repeated with a shift of exactly 12 bases, the base sequence analysis is performed repeatedly in 12 base steps even if several identical bases are connected and the base sequence analysis is hindered. Using the condition described above, correction can be performed, and an accurate nucleotide sequence can be determined. By repeating this operation, a long base sequence can be determined, and the inconvenience caused by the continuation of the same base can be solved.

The length of the DNA chain extended by the pyrosequencing reaction is such that the reaction efficiency used here is 100%.
However, the length of the DNA becomes constant again due to cleavage by the class IIs restriction enzyme.

Of course, since the efficiency of this class IIs restriction enzyme cleavage reaction is not 100%, irregularities will occur when the reaction is repeated. However, an enzyme in which the recognition site of the class IIs restriction enzyme and the cleavage site are as far apart as possible is used. This makes it possible to increase the number of steps (cut base length) per reaction, so that a base sequence in a long DNA range can be analyzed with an acceptable accuracy.

For example, pyrosequencing requires 100 elongation reactions to determine the length of 100 bases. When pyrosequencing is combined with class IIs restriction enzyme cleavage, About 9
The number of times of the pyrosequencing extension reaction after each cleavage with the class IIs restriction enzyme is thirteen times, which makes it possible to avoid difficulties in determining the nucleotide sequence due to incomplete reaction.

In the above description, pyrosequencing and cleavage of class IIs restriction enzymes were performed alternately.
As described above, by using a class IIs restriction enzyme, a series of DNA fragments having different lengths by a predetermined base length are prepared in advance, and pyrosequencing is performed using these DNA fragments as templates, thereby performing the same as described above. You can do it.

In FIG. 8, XX, Y'Y ', A,
Arbitrary two nucleotide sequence site 312 selected from T, G, C
XX and Y′Y ′ are complementary to each other. Also,
H, H ', M, M', N, N ', Z, Z' are A, T, G,
Indicates any base of C.

Example 3 Example 3 is a method using a class IIs restriction enzyme and a fluorescent-labeled dideoxynucleotide (ddNTP *) for DNA base sequence determination.

FIG. 9 is a view for explaining the principle of the method for determining a DNA base sequence by a cycle reaction using a class IIs restriction enzyme in Example 3. A number of copies 501 of a double-stranded DNA sample are made and immobilized on a solid surface (beads) 502. An oligomer 503 having a recognition sequence for a class IIs restriction enzyme is bound to the end of the DNA 501. 5 'when digested with class IIs restriction enzyme
An enzyme that generates a fragment 504 having a protruding end at the end is used. For example, class IIs restriction enzyme BsgI and the like. Fluorescently labeled deoxynucleotide 505 is bound to the 3 'end of this cut using DNA polymerase. After washing and removing the reagent, irradiate the laser and observe the generated fluorescence.

The four dideoxynucleotides (ddN)
TP *) is mixed and allowed to react. The four kinds of dideoxynucleotides each have a different fluorescent substance * added according to the base type, and which fluorescent substance, that is, which base type has been incorporated. Can be found by identifying and detecting the wavelength of the fluorescence.

After measuring the fluorescence, the oligomer 50 having a linker having a known sequence for controlling the recognition site of the class IIs restriction enzyme and the cleavage site of the class IIs restriction enzyme was used.
6 is ligated to the end using a ligase. The recognition site of the class IIs restriction enzyme was smaller than that of the previous cleavage site by beads 502.
It is designed such that it can be cleaved at the base site one base ahead of the terminal side fixed to the base, and this operation cuts bases from the 3 'end one base at a time.

The second cleavage of the class IIs restriction enzyme, the addition of a fluorescently-labeled dideoxynucleotide, and the subsequent detection of fluorescence allow the base species one base ahead of the terminal fixed to the beads 502 to be detected from the first round. Can be determined. Hereinafter, a repetitive process 507 of binding the oligomer 506, cleavage with the class IIs restriction enzyme, incorporation of dideoxynucleotide by mixing four kinds of deoxynucleotides, and detection of fluorescence is performed.

Each time Step 507 is repeated, the recognition site of the class IIs restriction enzyme is changed from the previous cleavage site by using the oligomer 506 having a linker portion in which the length of the oligomer 506 is shorter by one base. It should be possible to cut at the base site one base ahead on the terminal side fixed to the beads 502.

Although the nucleotide sequence of DNA can be sequentially determined by repeating step 507, the efficiency (yield) of the reaction is 10%.
Rather than 0%, 20 repetitive reactions are near the limit.

In the reaction of step 507 repeated 20 times or more, the contribution from the unreacted DNA fragment increases, and it is difficult to determine the exact nucleotide sequence. Such obstacles can also be solved with the method according to the invention.

In the present invention, instead of removing bases one by one, a predetermined number of bases are removed at a time and a base sequence is determined (first method). Alternatively, a series of fragments having different lengths by a fixed base length are prepared, and the base sequence is determined for each fragment (second method). The second method is the same as in the first and second embodiments.

FIG. 10 shows a third embodiment in which a predetermined number of bases are removed at a time to determine the base sequence.
It is a figure which shows the example of the method of. FIG. 10 is a diagram illustrating an example in which the sample preparation method of the present invention is used for DNA base sequence determination by a cycle reaction using a class IIs restriction enzyme.

In the method of cutting the DNA strand by one base and determining the base sequence, it is impossible to analyze a long strand. Therefore, in the first method, as shown in FIG. Nucleotide sequencing is now possible.

First, a large number of copies of the DNA sample to be sequenced were made, and the five groups 61-1 and 61-
2, 61-3, 61-4 and 61-5.

An oligomer consisting of the base sequence of the common base sequence 12, the recognition sequence 62 of the class IIs restriction enzyme, and the base sequence of the linker portion 10 for controlling the cleavage site of the class IIs restriction enzyme was added to the ends of the DNA samples of each group. Have been. Site 63 cleaved by class IIs restriction enzyme
Are shifted one base at a time, so that groups 61-1, 6
The base sequence of the linker portion 10 of the oligomer added to 1-2, 61-3, 61-4, and 61-5 differs by one base.

The DNA samples belonging to the five groups can be fixed to different positions on the solid surface for each group, or the DNA samples belonging to the five groups can be fixed to different solid surfaces and distinguished from each other. To do. In the example shown in FIG. 10, the DNA samples 61-1, 61-2, 61-3, 61-
4,61-5 are respectively different solids (beads) 60-1, 60-2, 60-3, 60-4,
It is fixed to the surface of 60-5.

First, as in the example shown in FIG.
The DNA sample was cut at the site 63 with the IIs restriction enzyme, and at the protruding end of the cut portion, a mixture of four types of ddNTP * (fluorescent-labeled dideoxynucleotide: * indicates a different fluorescent label for each type of N) and DNA Either ddNTP * is subjected to complementary strand extension using a polymerase.

Since the cleavage sites 63 of the five groups are prepared so as to be shifted by one base at a time, the laser is irradiated so that the dd with the complementary strand extended to the DNA terminal of each group.
By detecting the fluorescence emitted from the NTP * fluorescent label, the base sequence of 5 bases can be read.

Next, the above-mentioned oligomer is added to the terminal, and a predetermined length is cut from the terminal of the DNA fragment using a class IIs restriction enzyme.

At this time, a class IIs restriction enzyme was selected so that DNAs of all groups were cleaved at a position 5 bases away from the first cleavage site 63, and the base sequence of the linker portion 10 of the oligomer was selected. Set the length in advance.

Again, a mixture of four types of ddNTP * and D
Either ddNTP * is subjected to complementary strand extension at the protruding end of the cut using NA polymerase. DdNTs with complementary strands extended to the DNA ends of each group by laser irradiation
By detecting the fluorescence emitted from the P * fluorescent label,
Again, the next five bases can be sequenced. This means that the base sequence of 10 bases could be analyzed by two reaction operations.

In the same manner, the step 64 of repeating the addition of the oligomer to the end of the DNA fragment and the cutting of the DNA fragment by the class IIs restriction enzyme is repeated 20 times, whereby the example shown in FIG. Where only the base sequence analysis of bases could be performed, base sequence analysis up to 100 bases can be performed. The number of bases to be cut by the class IIs restriction enzyme can be changed depending on the purpose.

FIGS. 11 and 12 show Example 3, which is a second method for preparing a series of DNA fragments having different lengths by a constant base length and determining the base sequence of each fragment. FIG. FIG. 11 is a diagram illustrating an example in which the sample preparation method of the present invention is used for DNA sequencing by pyrosequencing.

This is a method in which a series of single-stranded DNA fragments differing by a predetermined length of 10 to 20 bases are prepared in advance and used. DNA sample 101-i (i = 1, 2, ...,
m) is immobilized on one end of the 5 ′ end of the bead 106. DNA sample 102-i fixed on the beads (i = 1, 2, 2)
To m), an oligomer having a priming site 105 on one chain is added by a ligation reaction.

DNA sample 102-i immobilized on beads
(I = 1, 2,..., M) are converted to the reaction wells 104-ij (i = 1, 2,.
1, 2,-, n). That is, the i-th DNA sample 102-i is dispensed into n reaction wells 104-ij (j = 1, 2,..., N) on the i-th row of the titer plate 103.

Reaction wells 104-ij (i = 1, 2, 2)
~, M: j = 2, ~, n))
In the same manner as in Example 1, the step of repeating the cleavage with the class IIs restriction enzyme and the above-mentioned ligation of the oligomer to the cleavage site is repeated (j-1) times, for example, the length differs by 16 bases, for example. A series of DNA fragments having a length is prepared. Further, the double-stranded D in each reaction well 104
The single-stranded DNA released from the beads 106 is removed by washing to make the NA single-stranded, and
The 5 'end is fixed to the beads 106, leaving a single-stranded DNA having the priming site 105 at the 3' end. class
BpmI is used as the IIs restriction enzyme.

As a result, each DNA-i (i = 1, 2, 2)
~, M) A series of single-stranded DNA fragments 107-i (i = 1, 2, ~, m) differing in length by 16 bases for each sample
Are obtained independently and simultaneously. DNA sample 102-i (i = 1, 2, 2) not cleaved with the class IIs restriction enzyme
~, M) (DNA fragment 107-i-1) is in reaction well 1
One class at 04-i-1 (i = 1, 2, ..., m)
DNA with a short base length of 16 bases that is cut by IIs restriction enzyme
Fragment 107-i-2 is the reaction well 104-i-2 (i =
1, 2, ..., m), ... (n-1) times of class IIs restriction enzyme digestion, and a short 16 (n-1) base length D
NA fragment 107-in is in reaction well 104-in
(I = 1, 2,-, m).

As shown in FIG. 12, in each reaction well of the titer plate 103 in which a series of single-stranded DNA fragments 107-i differing in length by 16 bases were prepared, a complementary strand synthesis primer 108 was used. dNTP (N = A,
C, G, and T) are sequentially added, and the DNA base sequence is determined by pyrosequencing. Titer plate 1
The chemiluminescence is detected from the transparent bottom of each reaction well 03 by the CCD camera 109 under the control of the controller 110, and the signal detected by the data processor 111 is processed to determine the base sequence.

As described above, DNA fragments having a length different from each other by 16 bases are sorted and arranged, and a plurality of DNs are arranged.
The base sequence analysis of a series of DNA fragments having different lengths by a fixed base length obtained for each DNA sample of the A sample is simultaneously performed. Thus, for each DNA fragment, 16
The base sequence is analyzed for each base, and the obtained base sequences are integrated to determine the entire base sequence for each DNA sample.

(Embodiment 4) FIG. 13 shows Embodiment 1 of the present invention.
Example 4 is a modified series of DNs, in which a series of DNs differing in length by a fixed base length according to the recognition sequence of the class IIs restriction enzyme.
FIG. 3 is a diagram illustrating a sample preparation method in which each DNA fragment of the A fragment can be distinguished from each other.

In Example 1 of the present invention, if each DNA fragment of a series of DNA fragments having different lengths by a fixed base length can be distinguished from each other, it is not necessary to fix them on a solid surface in a spatially separated state. Absent. For example, the recognition sequence of the class IIs restriction enzyme contained in the oligomer base sequence added to the end of the DNA fragment is set as a different base sequence for each DNA fragment having a different length. Thereby, the DNA fragment is selected and the class IIs restriction enzyme digestion can be performed.

The class IIs restriction enzymes usable herein include AlvI, Bb, which forms a 5 ′ protruding end by cleavage.
sI, BbvI, BsmFI, BspMI, EarI, FokI, HgaI, PleI, Sf
aNI, 3 'protruding end formed by cutting, BciVI, Bm
There are rI, BpmI, BseRI, BsgI, HphI, MboII, MnlI and the like.

As shown in FIG. 13, a primer 1 that hybridizes to the DNA sample 40 and an oligomer having a base sequence of the recognition site of the class IIs restriction enzyme BpmI at the 5 ′ end of the portion that hybridizes to the DNA sample 40 ( (Primary oligomer) 6′-1 and primer 2
The NA sample 40 was subjected to PCR amplification and the PCR amplification product 9′-1
Get. As the oligomer 6'-1, for example, the oligomer 6 shown in FIG. 3 is used.

The DNA fragment 9'-1 was ligated with the class IIs restriction enzyme B
Cleavage with pmI (first class IIs restriction enzyme) yields DNA fragment 4. Next, one end of the DNA fragment 4 cut with the class IIs restriction enzyme BpmI was added to the class IIs restriction enzyme BsgI
An oligomer (second oligomer) 6′-2 having a recognition part of (second class IIs restriction enzyme) is bound by ligation.

Next, the DNA fragment 9'-2 to which the oligomer 6'-2 (the second oligomer) has been bound is referred to as the n-th (n = 2, 3,..., (N-1)) Class IIs
The cleavage with the restriction enzyme and the cleavage of the DNA fragment (9′-n) (n = 3, 4,.
Step 2 of repeating the reaction of enzymatically coupling oligomers (6′-n) of (n = 3, 4,..., N) with a ligase
0 ′ is performed (N−1) times. For example, the n-th (n = 2,
3, ..., (N-1)) as class IIs restriction enzymes
I, BciVI, BmrI, BpmI, BseRI, HphI, MboII, MnlI,
Use…, etc.

As a result, the DNA fragments 9'-n differing in length by one unit with a certain base length as one unit.
(= 1, 2,..., N) to obtain a series of DNA fragments 9 ′.

The length of the base sequence of the linker portion of the n-th (n = 1, 2,..., N) oligomer (6′-n) is determined by the length of each DNA fragment 9′-n ( = 1, 2,
.., N) are controlled so that the lengths differ by one unit.

As in Example 1, various nucleotide sequences may appear as the nucleotide sequence of the terminal fragment of the DNA fragment at the site to be ligated with the oligomer (terminal nucleotide sequence of the ligation site) at the cleavage site by the class IIs restriction enzyme. Therefore, oligomers are mixed so as to have all the base sequences corresponding to the possible base sequences.

That is, as the n-th (n = 2, 3,..., N) oligomer (6′-n), it can complementarily bind to all the nucleotide sequences that may appear in the terminal nucleotide sequence at the ligation site. A mixture of oligomers having different base sequences is used.

As in the example shown in FIG. 2, the n-th (n = 2,
The oligomers (3 ′,..., N) (6′-n) are a common base sequence having a known sequence, a class IIs restriction enzyme recognition sequence,
It comprises a base sequence of a linker having a known sequence for controlling a cleavage site by a class IIs restriction enzyme, and an arbitrary base sequence site having a predetermined number of bases (eg, an arbitrary two base sequence site).

For example, when an arbitrary 2-base sequence site is used, XY has 16 combinations of base sequences and the n-th (n = 2, 3,..., N) oligomer (6′-
n) is a mixture of oligomers having 16 different arbitrary 2 nucleotide sequence sites, respectively.

Each DNA fragment 9'-n (= 1, 2,...,
N), the end of the DNA strand of the first DNA fragment 9′-1 of the series of DNA fragments is cleaved with the first class IIs restriction enzyme, A mixture of dideoxynucleotides (ddNTP *) labeled with different fluorescent labels * is added to the base, and the type of base that has undergone complementary chain extension adjacent to the cleavage site is determined by irradiating the reaction solution with excitation light at a wavelength of fluorescence emitted by the reaction solution. Identify and know.

The ligation of the first oligomer 6'-1 and the cleavage with the first class IIs restriction enzyme are repeated, and the base sequence of the first DNA fragment 9'-1 is determined by about 20 bases in the same manner.

Next, in the same manner as in the determination of the base sequence of the first DNA fragment 9'-1, the second DN
The end of the DNA strand of the A fragment 9'-2 was replaced with the second class IIs
The nucleotide sequence is determined by digestion with a restriction enzyme.

Hereinafter, this operation is performed on each DNA fragment 9'-n (= 3, 4,..., N) of the series of DNA fragments.
Class II of nth different (n = 3,4, ..., N)
The DNA base sequence can be determined over a range of 100 bases or more by repeatedly using the s restriction enzyme and the n-th (n = 3, 4,..., N) oligomer (6′-n).

There are not so many types of class IIs restriction enzymes that can be used here. Primers were selectively hybridized to the oligomer portion added to the end of the DNA fragment in a manner dependent on the base sequence of the oligomer, and selected. DN
The complementary strand is synthesized from the A fragment into a double strand,
The above-mentioned base sequence determination reaction can also be performed by digestion with the IIs restriction enzyme.

[0136]

According to the DNA sequencing of the prior art by gel electrophoresis, a difference of one base in the length separation by gel is used.
Since the length of the NA needs to be identified, the length of the base sequence can be determined, but according to the present invention, every 5 bases,
Or make a series of fragments with different base length by several bases,
By comparing the lengths, the nucleotide sequence of a longer DNA fragment can be easily determined as compared with the prior art.

In addition, SNPs (single nucl)
eotide polymorphisms) analysis,
New sequencing methods that will be important in large-scale DNA analysis, such as pyrosequencing and cycle reactions using class IIs restriction enzymes, can increase the length of sequencing and improve analysis efficiency. It is very useful. The short length of base sequence determination of these new methods is a drawback for a wide range of applications, but the present invention can easily overcome this drawback and is a powerful tool for high-speed, high-throughput DNA analysis. New methods become available.

[Brief description of the drawings]

FIG. 1 is a diagram for explaining the principle of a method for preparing a series of DNA fragments having different lengths by a constant base length in Example 1 of the present invention.

FIG. 2 is a diagram showing an example of the structure of an oligomer that binds by ligation to a cut portion of a DNA fragment generated by cleavage with a class IIs restriction enzyme in Example 1 of the present invention.

FIG. 3 shows that a fixed base length was 1 in Example 1 of the present invention.
As a unit, a series of D units with different lengths
The figure explaining the method of preparing a NA fragment by class IIs restriction enzyme digestion.

FIG. 4 is a diagram illustrating a method for preparing a series of DNA fragments having different lengths by one base in Example 1 of the present invention.

FIG. 5: In Example 1 of the present invention, fluorescently labeled d was added to the end of a series of DNA fragments having different lengths by one base.
The figure explaining the principle of adding dNTP and determining a base sequence.

FIG. 6 is a diagram showing the principle of a DNA sequencing method using pyrosequencing of the prior art used in Example 2 of the present invention.

FIG. 7 is a diagram illustrating an example of a method for alternately performing DNA base sequence determination by pyrosequencing and class IIs restriction enzyme cleavage according to Example 2 of the present invention.

FIG. 8 is a diagram showing an example of the structure of an oligomer that binds by ligation to a cut portion of a DNA fragment generated by cleavage with a class IIs restriction enzyme in Example 2 of the present invention.

FIG. 9 is a view for explaining the principle of a DNA base sequence determination method by a cycle reaction using a class IIs restriction enzyme in Example 3 of the present invention.

FIG. 10 is a diagram showing an example of a first method of Example 3 of the present invention, in which a predetermined number of bases are removed at a time and a base sequence is determined.

FIG. 11 is a diagram showing an example of a second method of preparing a series of DNA fragments having different lengths by a fixed base length and determining a base sequence for each of the DNA fragments according to the third embodiment of the present invention.

FIG. 12 is a diagram illustrating an example of a second method according to the third embodiment of the present invention, in which a series of DNA fragments having different lengths by a fixed base length are prepared and a base sequence is determined for each of them.

FIG. 13 is a fourth embodiment obtained by modifying the first embodiment of the present invention, in which a series of DNA fragments having different lengths by a certain base length can be distinguished from each other by a recognition sequence of a class IIs restriction enzyme. FIG.

[Explanation of symbols]

1 ... primer, 2 ... primer, 3 ... PCR amplified DNA fragment sample, 4 ... DNA fragment cut with class IIs restriction enzyme, 5,11 '... oligomer having recognition sequence of class IIs restriction enzyme BsgI, 6, 11,6-0,6-
1,6-2,6-3,6-4 ... Class IIs restriction enzyme BpmI
Oligomers having the recognition sequence of 6′-1, 6′-2,
..., 6'-N: oligomer having recognition sequence of class IIs restriction enzyme, 7: DNA fragment not cleaved by class IIs restriction enzyme BpmI, 8: DNA fragment cleaved by class IIs restriction enzyme BpmI, 9, 9-1 , 9-2, ..., 9-N, 9 ',
9′-1, 9′-2,..., 9′-N: a series of DNA fragments having different lengths by a fixed base length, 10, 10 ′ ... class
Base sequence of the linker that controls the cleavage site of the IIs restriction enzyme, 12, 12 '... common base sequence, 13, 1
3 ': Class IIs restriction enzyme recognition sequence, 14, 14': Arbitrary nucleotide sequence, 15, 15 ': Portion to which oligomer binds by ligation, 16, 16': Class IIs restriction enzyme B
Part cut with pmI, 17: DNA fragment shortened by cutting with class IIs restriction enzyme BpmI, 18, 19 ... Class
A DNA fragment in which an oligomer is bound to a cleavage site of a DNA fragment produced by cleavage of the IIs restriction enzyme BpmI, 20, 20 ′
... step of repeating cleavage with class IIs restriction enzyme BpmI and ligation of oligomer to the cleavage site, 21 ... 1
DNA fragments of different length by base, 21-1, 21-
2,21-3,21-4,21-5 ... By step 20,
Containers 35-1, 35-2, 35-3, 35-4, 35-
Group of DNA fragments generated in 5, 22 ... Class II
s cleavage site by restriction enzyme BpmI, 23 ... ddNTP *,
24 ... DNA fragment incorporating ddNTP *, 24-
1,4-2,24-3,24-4,24-5 ... container 3
Groups of fluorescently labeled DNA fragments generated in 5-1, 35-2, 35-3, 35-4, 35-5, 25 ...
Electrophoresis path, 25-1, 25-2, 25-3, 25-
4, -25-5 ... sample injection end of electrophoresis path, 27,27
-1,27-2,27-3,27-4,27-5 ... mixture of DNA fragments to which oligomers are bound, 27 '... DN
A solution containing the fragment A, 30, a container containing a solution containing the mixture 27, 35-1, 35-2, 35-3, 35-
4, 35-5 ... Container into which mixture 27 is dispensed, 40 ... D
NA samples, 60-1, 60-2, 60-3, 60-4,
60-5: beads, 61-1, 61-2, 61-3, 6
1-4, 61-5: group of DNA samples, 62: recognition sequence of class IIs restriction enzyme, 63: cleavage site by class IIs restriction enzyme, 64: addition of oligomer to the end of DNA fragment, and class IIs restriction enzyme Repeating 101-i (i = 1, 2,-, m)
... DNA sample, 102-i (i = 1, 2, ..., m) ... DNA sample fixed on beads, 103 ... titer plate, 104-ij (i = 1, 2, ..., m: j = 1,
2, ..., n) ... reaction well, 105 ... priming site, 106 ... beads, 107-i (i = 1, 2, ...,
m): A series of DNAs of different lengths by a fixed base length, 1
08: primer for complementary strand synthesis, 109: CCD camera, 110: controller, 111: data processor, 300: beads, 301: single-stranded DNA sample, 30
2 ... primer, 302 '... sequence of primer 302,
A primer having a class IIs restriction enzyme recognition sequence, a linker sequence, and an arbitrary two-base sequence; 303, an extended complementary strand; 304, a cleavage site of a class IIs restriction enzyme;
05: Examples of oligomers that bind to cleavage sites of class IIs restriction enzymes, 306: Single-stranded D shortened by a certain length
NA, 307: oligomer introduced into single-stranded DNA sample, 307 ': oligomer introduced into single-stranded DNA sample, oligomer having sequence of class IIs restriction enzyme recognition sequence and linker portion, 308 ... DNA by pyrosequencing A step of repeating base sequence determination and cleavage of class IIs restriction enzyme, 311 ... recognition sequence of class IIs restriction enzyme BpmI, 312 ... arbitrary two base sequence site, 313 ... cutting position of class IIs restriction enzyme BpmI, 314 ... class IIs restriction Sequence of the linker part controlling the cleavage site of the enzyme BpmI, 315: base sequence to be removed by cleavage of the class IIs restriction enzyme BpmI, 316: base sequence to be newly determined, 317: DNA fragment to which the oligomer binds , 400: DNA sample, 401: primer, 402
... nucleotide of complementary strand synthesis substrate, 403 ... extended D
NA chain, 404 ... pyrophosphate released by complementary strand synthesis, 405 ... series of reactions occurring in pyrosequencing, 406 ... series of complementary strand synthesis reactions, 407 ... chemiluminescence, 501 ... double-stranded DNA sample, 502 ... Beads, 50
3. An oligomer having a recognition sequence for a class IIs restriction enzyme,
504: a fragment having a protruding end at the 5 'end; 505, a fluorescently labeled deoxynucleotide; 506, an oligomer having a recognition site for a class IIs restriction enzyme; 507, a binding of an oligomer; cleavage by a class IIs restriction enzyme; Iterative process of incorporation of deoxynucleotide by mixing of deoxynucleotides and detection of fluorescence.

[Procedure amendment]

[Submission date] May 23, 2001 (2001.5.2)
3)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0037

[Correction method] Change

[Correction contents]

X and Y are any of A, T, G, and C. XY has 16 combinations of base sequences.
Are each a mixture of oligomers having 16 different types of arbitrary two base sequence sites. In FIG. 2, the DNA sequence GTGCAGAAXY is the sequence number in the sequence listing.
No. 1 and the DNA sequence CTGGAGAAXY corresponds to SEQ ID NO:
Equivalent to 2.

[Procedure amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0041

[Correction method] Change

[Correction contents]

In FIG. 3, M9 and M'9 indicate nine sequences of M and nine sequences of M ', respectively, and M and M' are arbitrary sequences. Sequence M'9 'is complementary. Xn
Yn, X'nY'n, Xn + 1Yn + 1, X'n + 1Y'n + 1, Xn + 2Yn +
2, X'n + 2 Y'n + 2 indicates an arbitrary two base sequence site selected from A, T, G, C, and XnYn, X'nY'n, Xn + 1
Yn + 1 and X'n + 1Y'n + 1, Xn + 2Yn + 2 and X'n + 2Y'n + 2 are
Each is complementary. N, N 'are A, T, G, C
Any of the bases. The sequence shown in FIG.
The sequence shown in the lower part of FIG. 3 corresponds to the sequence number 3 in the sequence listing.
It corresponds to No. 4.

[Procedure amendment 3]

[Document name to be amended] Statement

[Correction target item name] 0091

[Correction method] Change

[Correction contents]

In FIG. 8, XX, Y'Y ', A,
Arbitrary two nucleotide sequence site 312 selected from T, G, C
XX and Y′Y ′ are complementary to each other. Also,
H, H ', M, M', N, N ', Z, Z' are A, T, G,
Indicates any base of C. The sequence shown in FIG.
This corresponds to SEQ ID NO: 5.

[Procedure amendment 4]

[Document name to be amended] Statement

[Correction target item name]

[Correction method] Change

[Correction contents]

In addition, SNPs (single nucl)
eotide polymorphisms) analysis,
New sequencing methods that will be important in large-scale DNA analysis, such as pyrosequencing and cycle reactions using class IIs restriction enzymes, can increase the length of sequencing and improve analysis efficiency. It is very useful. The short length of base sequence determination of these new methods is a drawback for a wide range of applications, but the present invention can easily overcome this drawback and is a powerful tool for high-speed, high-throughput DNA analysis. New methods become available.

[Sequence List] SEQUENCE LISTING <110> Hitachi, Ltd. <120> A process for determining a DNA base sequence <130> H00010121A <140> JP2000 / 281748 <141> 2000-09-12 <160> 5 <170> PatentIn Ver. 2.1 <210> 1 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <220> <221> misc_feature <222> (9) .. (10 ) <223> n is A, C, G or T <400> 1 gtgcagaann 10 <210> 2 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA < 220> <221> misc_feature <222> (9) .. (10) <223> n is A, C, G or T <400> 2 ctggagaann 10 <210> 3 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <220> <221> misc_feature <222> (8) .. (28) <223> n is A, C, G or T <400> 3 tctggagnnn nnnnnnnnnn nnnnnnnn 28 <210> 4 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <220> <221> misc_feature <222> (8) .. (26 ) <223> n is A, C, G or T <400> 4 tctggagnnn nnnnnnnnnn nnnnnn 26 <210> 5 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <220> <221> misc_feature <222> (1). 7) <223> n is A, C, G or T <220> <221> misc_feature <222> (14) .. (32) <223> n is A, C, G or T <400> 5 nnnnnnnctg gagnnnnnnn nnnnnnnnnn nn 32

[Sequence List Free Text] SEQ ID NOS: 1 to 5: Synthetic DNA SEQ ID NO: 1: n represents A, C, G or T (location: 9 and
10) SEQ ID NO: 2: n represents A, C, G or T (location: 9 and
10) SEQ ID NO: 3: n represents A, C, G or T (location: 8 to 2)
8) SEQ ID NO: 4: n represents A, C, G or T (location: 8 to 2)
6) SEQ ID NO: 5: n represents A, C, G or T (location: 1 to
7) SEQ ID NO: 5: n represents A, C, G or T (Location: 14 to 3)
2)

 ──────────────────────────────────────────────────続 き Continued on the front page F term (reference) 2G045 AA35 DA12 DA13 FB01 FB02 FB05 FB12 FB13 GC15 4B024 AA11 AA20 CA01 CA09 HA19 4B063 QA13 QQ42 QR08 QR42 QR84 QS03 QS04 QS25 QS32 QX02

Claims (20)

[Claims] (1) A class is added to one end of double-stranded DNA.
(2) cleaving the double-stranded DNA to which the oligomer has been bound in step (1) with the class IIs restriction enzyme, 2 generated by cutting in step (2)
Binding the oligomer to a single-stranded DNA fragment;
(4) The steps (2) to (3) are repeated a plurality of times to prepare a series of double-stranded DNA fragments having different lengths by one unit, with a fixed base length of 2 bases or more as one unit. And (5) a step of determining the base sequence of the double-stranded DNA in the step (1). (2) A class is added to one end of the double-stranded DNA.
(2) cleaving the double-stranded DNA to which the oligomer has been bound in step (1) with the class IIs restriction enzyme, 2 generated by cutting in step (2)
Binding the oligomer to a single-stranded DNA fragment;
(4) The steps (2) to (3) are repeated a plurality of times to prepare a series of double-stranded DNA fragments having different lengths by one unit, with a fixed base length of 2 bases or more as one unit. (5) cutting the series of double-stranded DNA fragments obtained in the step (4) with the class IIs restriction enzyme, and (6) cutting the series of the double-stranded DNA fragments cut in the step (5). A step of performing a complementary chain extension reaction for extending a fluorescently labeled deoxynucleotide at the end of the double-stranded DNA fragment; and (7) performing an electrophoretic separation of the fragment obtained in the step (6) to perform the step (1). Determining the nucleotide sequence of the double-stranded DNA according to (1).
Nucleotide sequencing. 3. The method according to claim 2, wherein the double-stranded DNA in the step (1) is fixed on a solid surface. 4. A step of (1) dividing the double-stranded DNA into a plurality of groups; and (2) having a class IIs restriction enzyme recognition sequence at one end of the double-stranded DNA of each of the groups.
Binding the oligomer prepared by adjusting the length so that the cleavage site by the class IIs restriction enzyme differs by one base between the groups; and (3) performing the step (2) for each of the groups. 2 in which the oligomer is bound
Cutting the single-stranded DNA with the class IIs restriction enzyme, (4) binding the oligomer to the double-stranded DNA fragment generated by the cutting in step (3) for each group, and (5) ) For each of the groups, the step (3)
Repeating the step (4) a plurality of times to prepare a series of double-stranded DNA fragments having different lengths by 1 unit, with a fixed base length of 2 bases or more as 1 unit,
(6) a step of cleaving the series of double-stranded DNA fragments prepared in the step (5) with the class IIs restriction enzyme for each of the groups; and (7) a step of cleaving the ( Performing a complementary strand extension reaction to extend a fluorescently labeled deoxynucleotide to the end of the series of double-stranded DNA fragments cleaved in 6); and (8) performing the step (7) for each of the groups. Electrophoretically separating the reaction product of step (8). Based on the results of electrophoretic separation obtained for each of the groups in step (8), the double-stranded DNA of step (1) A method for determining a DNA base sequence, comprising: 5. The method according to claim 4, wherein the double-stranded DNA in the step (1) is fixed on a solid surface. (1) A class is added to one end of double-stranded DNA.
(2) cleaving the double-stranded DNA to which the oligomer has been bound in step (1) with the class IIs restriction enzyme, 2 generated by cutting in step (2)
Binding the oligomer to a single-stranded DNA fragment;
(4) The steps (2) to (3) are repeated a plurality of times to prepare a series of double-stranded DNA fragments having different lengths by one unit, with a fixed base length of 2 bases or more as one unit. And (5) determining a base sequence for each of the series of double-stranded DNA fragments prepared in the step (4). In the step (5), the series of the double-stranded DN
A method for determining a DNA base sequence, comprising determining the base sequence of the double-stranded DNA in the step (1) based on the base sequence obtained for each A fragment. 7. The method for determining a DNA base sequence according to claim 6, wherein in the step (5), (5a) the series of double-stranded DNA fragments prepared in the step (4) are used. Performing a complementary chain elongation reaction using a DNA polymerase as a template, and (5b) converting pyrophosphate generated by the complementary chain elongation reaction in the step (5a) to ATP;
Detecting the generated luminescence by performing a luminescence reaction using an enzyme, and determining the base sequence, wherein the DNA base sequence is determined. 8. The method for determining a DNA base sequence according to claim 7, wherein the double-stranded DNA in the step (1) is fixed on a solid surface. 9. The method for determining a DNA base sequence according to claim 6, wherein in the step (5), (5a) the series of double-stranded DNA fragments prepared in the step (4) are used. Cleaving with the class IIs restriction enzyme, and (5b) performing a complementary strand extension reaction to extend a fluorescently labeled deoxynucleotide to the end of the double-stranded DNA fragment generated in the step (5a); (5c) a step of irradiating the double-stranded DNA fragment generated in the step (5b) with a laser beam to detect fluorescence emitted from the fluorescent label, and (5d) a double-stranded DNA generated in the step (5b). Linking the oligomer to the DNA fragment by ligation; (5e)
A step of cleaving the double-stranded DNA fragment generated in the step (5d) with the class IIs restriction enzyme; and (5f) a step of labeling the end of the double-stranded DNA fragment generated in the step (5e) with the fluorescent label. Performing a complementary chain extension reaction to extend the deoxynucleotides, and (5g) the step (5f).
Irradiating the laser light to the double-stranded DNA fragment generated in step (a) to detect fluorescence emitted from the fluorescent label;
h) a step of repeating the above (5d) to the above (5g) a plurality of times, wherein the base sequence is determined. 10. The method for determining a DNA base sequence according to claim 9, wherein the double-stranded DNA in the step (1) is fixed on a solid surface. (1) A DNA sample having a first priming site containing a sequence of a first oligomer having a recognition site for a class IIs restriction enzyme at the 3 ′ end and having a 5 ′ end fixed to a solid surface. A complementary strand is synthesized using a first primer complementary to the first priming site as a template, pyrophosphoric acid generated during the complementary strand synthesis is converted into ATP, and a luminescence reaction is performed using an enzyme. Performing a base sequence determination reaction to determine the base sequence of a part of the DNA sample by detecting the generated luminescence, and (2) converting the DNA chain generated by the base sequence reaction into the class II
(4) a step of cleaving with the s restriction enzyme, (3) binding a second oligomer having a recognition sequence of the class IIs cleavage enzyme to a cleavage site of the DNA strand cleaved in the step (2), Making the DNA strand, to which the second oligomer generated in the step (3) is bound, a single strand,
(5) A second priming site is formed at the 3 ′ end, generated at the step (4), the 5 ′ end is fixed to the solid surface, and the length of the DNA strand generated at the step (2) is determined. Using a single-stranded DNA having a length shorter than a predetermined length as a template, a second strand complementary to the second priming site is used.
The complementary strand synthesis is performed using the primers described above, the pyrophosphate generated during the complementary strand synthesis is converted into ATP, the luminescence reaction is performed using the enzyme, and the generated luminescence is detected. A step of performing a base sequence determination reaction for determining a part of the base sequence; and (6) a step of repeating the steps (2) to (5) a plurality of times to determine a base sequence of the DNA sample. DN characterized by the following:
A base sequencing method. 12. A method comprising: (1) preparing a series of double-stranded DNA fragments having different lengths by 1 unit from a DNA sample with a fixed base length of 2 bases or more as one unit;
(2) separating the length of the series of DNA fragments,
Determining the base sequence of the A sample. 13. A method comprising: (1) preparing a series of double-stranded DNA fragments each having a different length by one unit from a DNA sample, taking a fixed base length of at least two base lengths as one unit;
(2) a step of separating the length of the series of DNA fragments. 14. A method comprising the steps of: (1) binding an oligomer having a recognition sequence of the class IIs restriction enzyme to one end of a double-stranded DNA obtained by cutting a DNA sample with a class IIs restriction enzyme; A) cutting the double-stranded DNA bound to the oligomer in the step (1) with the class IIs restriction enzyme; and (3) applying the oligomer to the double-stranded DNA fragment generated by the cleavage in the step (2). A bonding step; and (4) repeating the steps (2) to (3) a plurality of times, with a fixed base length of at least two base lengths as one unit, and a series of double strands having different lengths by one unit. DNA
And a step of preparing a fragment.
Sample preparation method. 15. A step of (1) binding an oligomer having a recognition sequence of a class IIs restriction enzyme to double-stranded DNA;
(2) 2 in which the oligomer is bonded in the step (1)
A step of cleaving the single-stranded DNA with the class IIs restriction enzyme, a step of (3) binding the oligomer to the double-stranded DNA fragment generated by the cleavage of the step (2), and (4)
The double-stranded D to which the oligomer is bonded in the step (3)
Cleaving NA with the class IIs restriction enzyme;
(5) Double-stranded DN generated by the cleavage in step (4)
A step of binding the oligomer to the fragment A and (6) repeating the steps (4) to (5) a plurality of times, with a fixed base length of 2 bases or more as one unit, and Preparing a series of different double-stranded DNA fragments. 16. A step of (1) binding an oligomer having a recognition sequence of a class IIs restriction enzyme to double-stranded DNA;
(2) 2 in which the oligomer is bonded in the step (1)
A step of cleaving the single-stranded DNA with the class IIs restriction enzyme, a step of (3) binding the oligomer to the double-stranded DNA fragment generated by the cleavage of the step (2), and (4)
The double-stranded D to which the oligomer is bonded in the step (3)
Cleaving NA with the class IIs restriction enzyme;
(5) Double-stranded DN generated by the cleavage in step (4)
Bonding the oligomer to the A fragment, and preparing a series of double-stranded DNA fragments having different lengths by one unit, with a fixed base length of at least two base lengths as one unit. A method for preparing a DNA sample, comprising: (1) The double-stranded DNA contains the first class IIs
Binding a first oligomer having a recognition sequence for a restriction enzyme, and (2) cleaving the double-stranded DNA bound to the first oligomer in the step (1) with the first class IIs restriction enzyme. And (3) adding an n-th (n = 2) class IIs restriction enzyme different from the first class IIs restriction enzyme to one end of the double-stranded DNA fragment cleaved in the step (2). Binding the n-th (n = 2) oligomer having an enzyme recognition sequence; and (4) cleaving the double-stranded DNA bound to the n-th oligomer with the n-th class IIs restriction enzyme. And (5) the double-stranded DNA fragment generated by the cleavage in the step (4) has a recognition sequence for the n-th class IIs restriction enzyme used in the step (4) to be executed next. Bonding the n-th oligomer; (6) performing the step (4); It said step (5), oligomers of the first n, the class IIs restriction enzyme of the first n,
The above steps (4) to (5) are made different every time the steps (4) to (5) are repeated a plurality of times, and a fixed base length of 2 bases or more is defined as one unit. A series of double-stranded DNAs differing in length by one unit
And a step of preparing a fragment.
Sample preparation method. (18) The double-stranded DNA contains the first class IIs
Binding a first oligomer having a recognition sequence for a restriction enzyme, and (2) cleaving the double-stranded DNA bound to the first oligomer in the step (1) with the first class IIs restriction enzyme. And (3) adding a recognition sequence for a second class IIs restriction enzyme different from the first class IIs restriction enzyme to one end of the double-stranded DNA fragment generated by the cleavage in the step (2). (2) a step of bonding a second oligomer having the double oligomer D to which the second oligomer is bonded;
Cutting the NA with the second class IIs restriction enzyme, wherein a fixed base length of at least two bases is defined as one unit, and a series of double-stranded DNAs having different lengths by one unit.
And a step of preparing a fragment.
Sample preparation method. 19. A method comprising: (1) binding an oligomer having a class IIs restriction enzyme recognition sequence to double-stranded DNA;
(2) 2 in which the oligomer is bonded in the step (1)
A step of cleaving the single-stranded DNA with the class IIs restriction enzyme; and (3) a double-stranded DN obtained by binding the oligomer to the double-stranded DNA fragment generated by the cleavage in the step (2).
A fragmentation of the fragment A with the class IIs restriction enzyme is repeated a plurality of times, and a fixed base length of 2 bases or more is defined as one unit, and a series of double-stranded Ds having different lengths by one unit
A step of preparing an NA fragment.
NA sample preparation method. 20. (1) The double-stranded DNA contains the first class IIs
Binding a first oligomer having a recognition sequence for a restriction enzyme, and (2) cleaving the double-stranded DNA bound to the first oligomer in the step (1) with the first class IIs restriction enzyme. And (3) adding an n-th (n = 2) class IIs restriction enzyme different from the first class IIs restriction enzyme to one end of the double-stranded DNA fragment cleaved in the step (2). Cutting the double-stranded DNA obtained by binding the n-th (n = 2) oligomer having an enzyme recognition sequence with the n-th class IIs restriction enzyme is performed by the n-th oligomer and the n-th oligomer. While changing the class IIs restriction enzyme of
A step of preparing a series of double-stranded DNA fragments having different lengths by one unit by repeating a plurality of times several times and setting a fixed base length of 2 bases or more as one unit.