WO2018003220A1 - Method for producing dna library and method for analyzing genomic dna using dna library - Google Patents

Abstract

According to the present invention, a DNA library exhibiting excellent reproducibility is easily produced. A nucleic acid amplification reaction is carried out in a reaction solution containing a genomic DNA and a high concentration of a random primer, and a fragment of the DNA obtained by the nucleic acid amplification reaction is obtained by using the genomic DNA as a template.

Description

Method for preparing DNA library and method for analyzing genomic DNA using DNA library

The present invention relates to a method for preparing a DNA library that can be used for DNA marker analysis, for example, and a genomic DNA analysis method using the DNA library.

Generally, in genome analysis, genetic information included in the genome, for example, base sequence information, is comprehensively analyzed. However, there is a problem that the analysis for determining the base sequence of the entire genome takes man-hours and costs. In addition, in organisms with a large genome size, there is a limit to genome analysis based on nucleotide sequence analysis due to the complexity of the genome.

Patent Document 1 discloses a method of incorporating a sample-specific identifier into a restriction enzyme-treated fragment ligated with an adapter in the amplified fragment length polymorphism (AFLP) marker technique and determining only a part of the sequence of the restriction enzyme-treated fragment. Disclosure. In the method disclosed in Patent Document 1, the complexity of genomic DNA is reduced by restriction enzyme treatment on genomic DNA, and the restriction enzyme-treated fragment is sufficiently obtained by determining the base sequence for a part of the restriction enzyme-treated fragment. I have identified. However, the method disclosed in Patent Document 1 requires man-hours such as a restriction enzyme treatment for genomic DNA and a ligation reaction using an adapter, and it is difficult to reduce costs.

On the other hand, Patent Document 2 discloses that the DNA extracted from rice samples is amplified by PCR in the presence of appropriate primers by the so-called RAPD (Randomly Amplified Polymorphic DNA) method. It has been disclosed that a high discriminating DNA marker has been found. The method disclosed in Patent Document 2 discloses the use of a plurality of STS (SequenceequTagged Sites) -ized primers limited by a specific sequence. In the method disclosed in Patent Document 2, a DNA marker for identification amplified using an STS primer is detected by electrophoresis. However, the RAPD method as disclosed in Patent Document 2 has remarkably low reproducibility of PCR amplification and cannot generally be employed as a DNA marker technique.

Patent Document 3 discloses a method for preparing a genomic library, in which PCR is performed using one kind of primer designed based on a sequence that appears relatively frequently in the target genome. It is disclosed that the entire region is amplified almost evenly, so that a genomic library can be generated. Although Patent Document 3 describes that a genomic library can be prepared by performing PCR using a random primer containing a random sequence, it does not describe any actual protocol or experimental results. Therefore, the method described in Patent Document 3 requires genome base sequence information in order to specify the genome appearance frequency, and man-hours and costs for that purpose are expected. Furthermore, the method described in Patent Document 3 has a problem that the complexity of genomic DNA cannot be reduced because amplification is attempted over the entire genome.

Japanese Patent No. 5389638 JP 2003-79375 A Japanese Patent No. 3972106

By the way, for genome information analysis such as gene linkage analysis using DNA markers, it is desired to prepare a DNA library by a method that is simpler and excellent in reproducibility. As described above, various methods are known as methods for preparing a DNA library, but there are currently no methods that are sufficient in terms of simplicity and / or reproducibility. Therefore, in view of such circumstances, an object of the present invention is to provide a DNA library preparation method that is simpler and more reproducible and a genomic DNA analysis method that uses the DNA library.

As a result of intensive investigations to achieve the above-described object, the present inventors have determined that the concentration of the random primer in the reaction solution is within a predetermined range in PCR using a random primer, so that excellent reproducibility is achieved. It has been found that it can be achieved, and the present invention has been completed.

The present invention includes the following.
(1) A method for preparing a DNA library, wherein a nucleic acid amplification reaction is performed in a reaction solution containing genomic DNA and a high concentration of random primers, and a DNA fragment obtained by the nucleic acid amplification reaction is obtained using genomic DNA as a template.
(2) The method for preparing a DNA library according to (1), wherein the reaction solution contains 4 to 200 μM of the random primer.
(3) The method for preparing a DNA library according to (1), wherein the reaction solution contains 4 to 100 μM of the random primer.
(4) The method for producing a DNA library according to (1), wherein the random primer is a nucleotide having a length of 9 to 30 bases.
(5) The method for producing a DNA library according to (1), wherein the DNA fragment has a length of 100 to 500 bases.
(6) A genomic DNA analysis method using a DNA library prepared by the DNA library preparation method according to any one of (1) to (5) as a DNA marker.
(7) A step of determining the base sequence of a DNA library prepared by the method for preparing a DNA library according to any one of (1) to (5), and confirming the presence or absence of the DNA marker based on the base sequence (6) The genomic DNA analysis method according to (6).
(8) The genomic DNA analysis method according to (7), wherein in the step of confirming the presence or absence of the DNA marker, the presence or absence of the DNA marker is confirmed from the number of reads in the base sequence of the DNA library.
(9) The base sequence of the DNA library is compared with the base sequence of the DNA library prepared using known sequence information or genomic DNA derived from other organisms or other tissues. The genomic DNA analysis method according to (7), wherein the presence or absence of a DNA marker is confirmed.
(10) A step of preparing a pair of primers that specifically amplify the DNA marker based on the nucleotide sequence of the DNA marker, and using the genomic DNA extracted from the target organism as a template and using the pair of primers The genomic DNA analysis method according to (6), comprising a step of performing a nucleic acid amplification reaction and a step of confirming the presence or absence of the DNA marker in the genomic DNA from the result of the nucleic acid amplification reaction.
(11) performing a nucleic acid amplification reaction in a first reaction solution containing genomic DNA and a high concentration of random primers, and obtaining a first DNA fragment obtained by the nucleic acid amplification reaction using genomic DNA as a template;
Nucleic acid amplification in the second reaction solution containing, as a primer, the first DNA fragment obtained and a nucleotide sequence containing at least 3% of the nucleotide sequence at the 5 'end of the random primer at the 3' end Carrying out a reaction to obtain a second DNA fragment in which the nucleotide is linked to the first DNA fragment.
(12) The method for preparing a DNA library according to (11), wherein the first reaction solution contains 4 to 200 μM of the random primer.
(13) The method for preparing a DNA library according to (11), wherein the first reaction solution contains 4 to 100 μM of the random primer.
(14) The method for producing a DNA library according to (11), wherein the random primer is a nucleotide having a length of 9 to 30 bases.
(15) The method for producing a DNA library according to (11), wherein the first DNA fragment has a length of 100 to 500 bases.
(16) The primer for amplifying the second DNA fragment includes a region used for the base sequence determination reaction, or is used for a nucleic acid amplification reaction using the second DNA fragment as a template or a repeated nucleic acid amplification reaction. The method for producing a DNA library according to (11), wherein the primer includes a region used for the base sequence determination reaction.
(17) The second DNA fragment obtained by the method for producing a DNA library according to any one of (11) to (15), or used for the base sequence determination reaction by the method for producing a DNA library according to (16) A method for analyzing a DNA library, comprising a step of determining a base sequence of a DNA fragment obtained using a primer containing a complementary region to a primer for a sequencer.
(18) A genomic DNA analysis method using a DNA library prepared by the DNA library preparation method according to any one of (11) to (17) as a DNA marker.
(19) A step of determining the base sequence of a DNA library prepared by the method for preparing a DNA library according to any one of (11) to (17) and confirming the presence or absence of the DNA marker based on the base sequence (18) The genomic DNA analysis method according to (18).
(20) The genomic DNA analysis method according to (19), wherein in the step of confirming the presence or absence of the DNA marker, the presence or absence of the DNA marker is confirmed from the number of reads in the base sequence of the DNA library.
(21) The base sequence of the DNA library is compared with the base sequence of the DNA library prepared using known sequence information or genomic DNA derived from another organism or tissue, and based on the difference in base sequence (19) The genomic DNA analysis method according to (19), wherein the presence or absence of a DNA marker is confirmed.
(22) A step of preparing a pair of primers that specifically amplify the DNA marker based on the base sequence of the DNA marker, and using the pair of primers with the genomic DNA extracted from the target organism as a template The genomic DNA analysis method according to (18), comprising a step of performing a nucleic acid amplification reaction, and a step of confirming the presence or absence of the DNA marker in the genomic DNA from the result of the nucleic acid amplification reaction.
(23) A DNA library prepared by the method for preparing a DNA library according to any one of (1) to (5) and (11) to (16).

This specification is described in the specification and / or drawings of Japanese Patent Application No. 2016-129048, which is the basis of the priority of the present application, and described in the specification and / or drawings of Japanese Patent Application No. 2016-178528 And contents described in the specification and / or drawings of Japanese Patent Application No. 2017-071020.

Since the DNA library preparation method according to the present invention is based on a nucleic acid amplification method using random primers, a DNA library can be prepared very simply. In addition, the method for preparing a DNA library according to the present invention is excellent in reproducibility of nucleic acid fragments to be amplified even by a nucleic acid amplification method using random primers. Therefore, according to the method for producing a DNA library according to the present invention, the prepared DNA library can be used as a DNA marker, and can be used for genomic DNA analysis such as gene linkage analysis.

Therefore, since the genomic DNA analysis method using the DNA library according to the present invention uses a DNA library prepared with ease and excellent reproducibility, genomic DNA analysis can be performed at low cost and with high accuracy. it can.

It is a flowchart which shows the preparation method of the DNA library which concerns on this invention, and the genomic DNA analysis method using a DNA library. FIG. 3 is a characteristic diagram showing the relationship between the amplified fragment length obtained from an electrophoresis image of a DNA library amplified by PCR under normal conditions using sugarcane NiF8 DNA as a template and the fluorescence unit (FU). FIG. 5 is a characteristic diagram showing the relationship between the amplified fragment length and the fluorescence unit (FU) obtained from an electrophoresis image of a DNA library amplified using sugarcane NiF8 DNA as a template at an annealing temperature of 45 ° C. FIG. 4 is a characteristic diagram showing the relationship between the amplified fragment length obtained from an electrophoresis image of a DNA library amplified using sugarcane NiF8 DNA as a template at an annealing temperature of 40 ° C. and the fluorescence unit (FU). FIG. 4 is a characteristic diagram showing the relationship between the amplified fragment length and the fluorescence unit (FU) obtained from an electrophoresis image of a DNA library amplified using sugarcane NiF8 DNA as a template at an annealing temperature of 37 ° C. FIG. 6 is a characteristic diagram showing the relationship between the amplified fragment length obtained from an electrophoresis image of a DNA library amplified with sugarcane NiF8 DNA as a template and an enzyme amount of 2.5 μunit and the fluorescence unit (FU). FIG. 5 is a characteristic diagram showing the relationship between the amplified fragment length obtained from an electrophoretic image of a DNA library amplified with sugarcane NiF8 DNA as a template and an enzyme amount of 12.5 μunit and the fluorescence unit (FU). FIG. 3 is a characteristic diagram showing the relationship between the amplified fragment length obtained from an electrophoresis image of a DNA library amplified using sugarcane NiF8 DNA as a template at a MgCl ₂ concentration of 2 times and the fluorescence unit (FU). FIG. 4 is a characteristic diagram showing the relationship between the amplified fragment length obtained from an electrophoretic image of a DNA library amplified with sugarcane NiF8 DNA as a template at a MgCl ₂ concentration of 3 times and the fluorescence unit (FU). FIG. 5 is a characteristic diagram showing the relationship between the length of amplified fragments obtained from an electrophoresis image of a DNA library amplified using sugarcane NiF8 DNA as a template at a MgCl ₂ concentration of 4 times and the fluorescence unit (FU). FIG. 5 is a characteristic diagram showing the relationship between the amplified fragment length obtained from an electrophoresis image of a DNA library amplified with an 8-base long random primer using sugarcane NiF8 DNA as a template and a fluorescence unit (FU). FIG. 5 is a characteristic diagram showing the relationship between the amplified fragment length and the fluorescence unit (FU) obtained from an electrophoresis image of a DNA library amplified using a sugarcane NiF8 DNA as a template and a random primer of 9 bases in length. FIG. 6 is a characteristic diagram showing the relationship between the amplified fragment length and the fluorescence unit (FU) obtained from an electrophoresis image of a DNA library amplified with an 11 base long random primer using sugarcane NiF8 DNA as a template. FIG. 5 is a characteristic diagram showing the relationship between the amplified fragment length and the fluorescence unit (FU) obtained from an electrophoresis image of a DNA library amplified with a sugarcane NiF8 DNA as a template and a 12-base long random primer. FIG. 5 is a characteristic diagram showing the relationship between the amplified fragment length and the fluorescence unit (FU) obtained from an electrophoresis image of a DNA library amplified with a 14-base long random primer using sugarcane NiF8 DNA as a template. FIG. 3 is a characteristic diagram showing the relationship between the amplified fragment length and the fluorescence unit (FU) obtained from an electrophoresis image of a DNA library amplified using a sugarcane NiF8 DNA as a template and a 16-base random primer. FIG. 5 is a characteristic diagram showing the relationship between the amplified fragment length and the fluorescence unit (FU) obtained from an electrophoresis image of a DNA library amplified with an 18 base long random primer using sugarcane NiF8 DNA as a template. FIG. 6 is a characteristic diagram showing the relationship between the amplified fragment length and the fluorescence unit (FU) obtained from an electrophoresis image of a DNA library amplified with 20 nucleotides of random primer using sugarcane NiF8 DNA as a template. FIG. 4 is a characteristic diagram showing the relationship between the length of amplified fragments obtained from an electrophoresis image of a DNA library amplified with sugarcane NiF8 DNA as a template and a random primer concentration of 2 μM and the fluorescence unit (FU). FIG. 4 is a characteristic diagram showing the relationship between the length of amplified fragments obtained from an electrophoresis image of a DNA library amplified with sugarcane NiF8 DNA as a template and a random primer concentration of 4 μM and the fluorescence unit (FU). It is a characteristic diagram showing the relationship between the amplified fragment length obtained from an electrophoresis image (first time) of a DNA library amplified with sugarcane NiF8 DNA as a template and a random primer concentration of 6 μM and the fluorescence unit (FU). FIG. 3 is a characteristic diagram showing the relationship between the length of an amplified fragment obtained from an electrophoresis image (second time) of a DNA library amplified with sugarcane NiF8 DNA as a template and a random primer concentration of 6 μM and the fluorescence unit (FU). It is a characteristic diagram showing the relationship between the amplified fragment length obtained from an electrophoresis image (first time) of a DNA library amplified with sugarcane NiF8 DNA as a template and a random primer concentration of 8 μM and the fluorescence unit (FU). FIG. 3 is a characteristic diagram showing the relationship between the length of an amplified fragment obtained from an electrophoresis image (second time) of a DNA library amplified at a random primer concentration of 8 μM using sugarcane NiF8 DNA as a template and the fluorescence unit (FU). It is a characteristic diagram showing the relationship between the amplified fragment length obtained from an electrophoresis image (first time) of a DNA library amplified with sugarcane NiF8 DNA as a template at a random primer concentration of 10 μM and the fluorescence unit (FU). It is a characteristic diagram showing the relationship between the amplified fragment length obtained from an electrophoresis image (second time) of a DNA library amplified at a random primer concentration of 10 μM using sugarcane NiF8 DNA as a template and the fluorescence unit (FU). It is a characteristic diagram showing the relationship between the amplified fragment length obtained from an electrophoresis image (first time) of a DNA library amplified with sugarcane NiF8 DNA as a template at a random primer concentration of 20 μM and the fluorescence unit (FU). FIG. 5 is a characteristic diagram showing the relationship between the length of an amplified fragment obtained from an electrophoresis image (second time) of a DNA library amplified with sugarcane NiF8 DNA as a template and a random primer concentration of 20 μM and the fluorescence unit (FU). FIG. 3 is a characteristic diagram showing the relationship between the length of an amplified fragment obtained from an electrophoresis image (first time) of a DNA library amplified with sugarcane NiF8 DNA as a template and a random primer concentration of 40 μM and the fluorescence unit (FU). It is a characteristic diagram showing the relationship between the amplified fragment length obtained from the electrophoresis image (second time) of a DNA library amplified with sugarcane NiF8 DNA as a template at a random primer concentration of 40 μM and the fluorescence unit (FU). It is a characteristic diagram showing the relationship between the amplified fragment length obtained from an electrophoresis image (first time) of a DNA library amplified with sugarcane NiF8 DNA as a template at a random primer concentration of 60 μM and the fluorescence unit (FU). It is a characteristic diagram showing the relationship between the amplified fragment length obtained from an electrophoresis image (second time) of a DNA library amplified with sugarcane NiF8 DNA as a template at a random primer concentration of 60 μM and the fluorescence unit (FU). It is a characteristic diagram showing the relationship between the amplified fragment length obtained from an electrophoresis image (first time) of a DNA library amplified with sugarcane NiF8 DNA as a template at a random primer concentration of 100 μM and the fluorescence unit (FU). It is a characteristic diagram showing the relationship between the amplified fragment length obtained from an electrophoresis image (second time) of a DNA library amplified with sugarcane NiF8 DNA as a template at a random primer concentration of 100 μM and the fluorescence unit (FU). It is a characteristic diagram showing the relationship between the amplified fragment length obtained from an electrophoresis image (first time) of a DNA library amplified with sugarcane NiF8 DNA as a template at a random primer concentration of 200 μM and the fluorescence unit (FU). It is a characteristic diagram showing the relationship between the amplified fragment length obtained from an electrophoresis image (second time) of a DNA library amplified with sugarcane NiF8 DNA as a template at a random primer concentration of 200 μM and the fluorescence unit (FU). It is a characteristic diagram showing the relationship between the amplified fragment length obtained from an electrophoresis image (first time) of a DNA library amplified with sugarcane NiF8 DNA as a template at a random primer concentration of 300 μM and the fluorescence unit (FU). It is a characteristic diagram showing the relationship between the amplified fragment length obtained from an electrophoresis image (second time) of a DNA library amplified with sugarcane NiF8 DNA as a template and a random primer concentration of 300 μM and the fluorescence unit (FU). It is a characteristic diagram showing the relationship between the amplified fragment length obtained from an electrophoresis image (first time) of a DNA library amplified with sugarcane NiF8 DNA as a template and a random primer concentration of 400 μM and the fluorescence unit (FU). FIG. 3 is a characteristic diagram showing the relationship between the length of an amplified fragment obtained from an electrophoresis image (second time) of a DNA library amplified with sugarcane NiF8 DNA as a template and a random primer concentration of 400 μM and the fluorescence unit (FU). FIG. 3 is a characteristic diagram showing the relationship between the length of an amplified fragment obtained from an electrophoresis image (first time) of a DNA library amplified with sugarcane NiF8 DNA as a template and a random primer concentration of 500 μM and the fluorescence unit (FU). FIG. 3 is a characteristic diagram showing the relationship between the length of an amplified fragment obtained from an electrophoresis image (second time) of a DNA library amplified at a random primer concentration of 500 μM using sugarcane NiF8 DNA as a template and the fluorescence unit (FU). FIG. 6 is a characteristic diagram showing the relationship between the length of amplified fragments obtained from an electrophoresis image of a DNA library amplified with sugarcane NiF8 DNA as a template and a random primer concentration of 600 μM and the fluorescence unit (FU). FIG. 5 is a characteristic diagram showing the relationship between the amplified fragment length obtained from an electrophoresis image of a DNA library amplified using sugarcane NiF8 DNA as a template and a random primer concentration of 700 μM and the fluorescence unit (FU). FIG. 3 is a characteristic diagram showing the relationship between the length of amplified fragments obtained from an electrophoresis image of a DNA library amplified with a sugarcane NiF8 DNA as a template and a random primer concentration of 800 μM, and a fluorescence unit (FU). FIG. 3 is a characteristic diagram showing the relationship between the amplified fragment length obtained from an electrophoresis image of a DNA library amplified with sugarcane NiF8 DNA as a template and a random primer concentration of 900 μM and the fluorescence unit (FU). FIG. 6 is a characteristic diagram showing the relationship between the length of amplified fragments obtained from an electrophoresis image of a DNA library amplified with sugarcane NiF8 DNA as a template and a random primer concentration of 1000 μM and the fluorescence unit (FU). FIG. 6 is a characteristic diagram showing the MiSeq analysis results of a DNA library amplified with random primers using sugarcane NiF8 DNA as a template. FIG. 5 is a characteristic diagram showing the relationship between the length of an amplified fragment obtained from an electrophoresis image (first time) of a DNA library amplified with random primers using rice Nipponbare DNA as a template and the fluorescence unit (FU). FIG. 5 is a characteristic diagram showing the relationship between the length of an amplified fragment obtained from an electrophoresis image (second time) of a DNA library amplified with random primers using rice Nipponbare DNA as a template and the fluorescence unit (FU). It is a characteristic view which shows the MiSeq analysis result of the DNA library which amplified the rice Nipponbare DNA as a template with the random primer. It is a characteristic view which shows the position of the rice Nihonbare genome information of the read pattern obtained from MiSeq. It is a characteristic view which shows frequency distribution of the mismatched base number of a random primer and a rice genome. FIG. 6 is a characteristic diagram showing the number of reads of sugarcane NiF8 and Ni9 and their progeny in the marker N80521152. It is the photograph which shows the sugarcane NiF8 and Ni9 in PCR marker N80521152, and the electrophoresis image of the progeny of the hybrid. FIG. 10 is a characteristic diagram showing the number of reads of sugarcane NiF8 and Ni9 and their progeny at marker N80997192. It is the photograph which shows the sugarcane NiF8 and Ni9 in PCR marker N80997192, and the electrophoresis image of the progeny of the hybrid. FIG. 5 is a characteristic diagram showing the number of reads of sugarcane NiF8 and Ni9 and their progeny in the marker N80533142. It is the photograph which shows the sugarcane NiF8 and Ni9 in PCR marker N80533142, and the electrophoretic image of the hybrid progeny. FIG. 6 is a characteristic diagram showing the number of reads of sugarcane NiF8 and Ni9 and their progeny at marker N91552391. It is the photograph which shows the sugarcane NiF8 and Ni9 in the PCR marker N91552391, the electrophoretic image of the hybrid progeny. FIG. 10 is a characteristic diagram showing the number of reads of sugarcane NiF8 and Ni9 and their progeny at marker N91653962. It is the photograph which shows the sugarcane NiF8 and Ni9 in PCR marker N91653962, and the electrophoresis image of the progeny of the hybrid. FIG. 10 is a characteristic diagram showing the number of reads of sugarcane NiF8 and Ni9 and their progeny at marker N91124801. It is the photograph which shows the sugarcane NiF8 and Ni9 in the PCR marker N91124801, and the electrophoretic image of the progeny of the hybrid. FIG. 4 is a characteristic diagram showing the relationship between the amplified fragment length and the fluorescence unit (FU) obtained from an electrophoresis image (first time) of a DNA library amplified using a sugarcane NiF8 DNA as a template and a random primer of 9 bases in length. FIG. 4 is a characteristic diagram showing the relationship between the amplified fragment length and the fluorescence unit (FU) obtained from an electrophoresis image (second time) of a DNA library amplified using a sugarcane NiF8 DNA as a template and a 9-base long random primer. FIG. 5 is a characteristic diagram showing the relationship between the amplified fragment length and the fluorescence unit (FU) obtained from an electrophoresis image (first time) of a DNA library amplified using a sugarcane NiF8 DNA as a template and a 10-base long random primer. FIG. 5 is a characteristic diagram showing the relationship between the amplified fragment length and the fluorescence unit (FU) obtained from an electrophoresis image (second time) of a DNA library amplified using a sugarcane NiF8 DNA as a template and a 10-base long random primer. FIG. 5 is a characteristic diagram showing the relationship between the amplified fragment length and the fluorescence unit (FU) obtained from an electrophoresis image (first time) of a DNA library amplified using a sugarcane NiF8 DNA as a template and a random primer of 11 base length. FIG. 5 is a characteristic diagram showing the relationship between the amplified fragment length and the fluorescence unit (FU) obtained from an electrophoresis image (second time) of a DNA library amplified with an 11 base long random primer using sugarcane NiF8 DNA as a template. FIG. 5 is a characteristic diagram showing the relationship between the amplified fragment length and the fluorescence unit (FU) obtained from an electrophoresis image (first time) of a DNA library amplified with a sugarcane NiF8 DNA as a template and a 12-base long random primer. FIG. 5 is a characteristic diagram showing the relationship between the amplified fragment length and the fluorescence unit (FU) obtained from an electrophoretic image (second time) of a DNA library amplified using a sugarcane NiF8 DNA as a template and a 12-base long random primer. FIG. 5 is a characteristic diagram showing the relationship between the amplified fragment length and the fluorescence unit (FU) obtained from an electrophoresis image (first time) of a DNA library amplified with a 14-base long random primer using sugarcane NiF8 DNA as a template. FIG. 5 is a characteristic diagram showing the relationship between the amplified fragment length and the fluorescence unit (FU) obtained from an electrophoresis image (second time) of a DNA library amplified with a 14-base long random primer using sugarcane NiF8 DNA as a template. FIG. 3 is a characteristic diagram showing the relationship between the amplified fragment length and the fluorescence unit (FU) obtained from an electrophoresis image (first time) of a DNA library amplified using a sugarcane NiF8 DNA as a template and a 16-base long random primer. FIG. 3 is a characteristic diagram showing the relationship between the amplified fragment length and the fluorescence unit (FU) obtained from an electrophoretic image (second time) of a DNA library amplified using a sugarcane NiF8 DNA as a template and a 16-base long random primer. FIG. 5 is a characteristic diagram showing the relationship between the amplified fragment length and the fluorescence unit (FU) obtained from an electrophoresis image (first time) of a DNA library amplified with 18-base random primers using sugarcane NiF8 DNA as a template. FIG. 4 is a characteristic diagram showing the relationship between the amplified fragment length obtained from an electrophoresis image (second time) of a DNA library amplified with an 18 base long random primer using sugarcane NiF8 DNA as a template and the fluorescence unit (FU). FIG. 4 is a characteristic diagram showing the relationship between the amplified fragment length and the fluorescence unit (FU) obtained from an electrophoresis image (first time) of a DNA library amplified using a sugarcane NiF8 DNA as a template and a 20-base long random primer. FIG. 3 is a characteristic diagram showing the relationship between the amplified fragment length and the fluorescence unit (FU) obtained from an electrophoresis image (second time) of a DNA library amplified with 20 base-long random primers using sugarcane NiF8 DNA as a template. FIG. 5 is a characteristic diagram showing the results of examining the reproducibility of a DNA library amplified using sugarcane NiF8 DNA as a template and using 8- to 35-base-long random primers in a concentration range of 0.6 to 300 μM. FIG. 3 is a characteristic diagram showing the relationship between the length of an amplified fragment obtained from an electrophoresis image (first time) of a DNA library amplified with one kind of random primer using sugarcane NiF8 DNA as a template and the fluorescence unit (FU). FIG. 4 is a characteristic diagram showing the relationship between the amplified fragment length and the fluorescence unit (FU) obtained from an electrophoresis image (second time) of a DNA library amplified with one kind of random primer using sugarcane NiF8 DNA as a template. FIG. 3 is a characteristic diagram showing the relationship between the length of an amplified fragment obtained from an electrophoresis image (first time) of a DNA library amplified with two kinds of random primers using sugarcane NiF8 DNA as a template and the fluorescence unit (FU). FIG. 5 is a characteristic diagram showing the relationship between the amplified fragment length and the fluorescence unit (FU) obtained from an electrophoresis image (second time) of a DNA library amplified with two kinds of random primers using sugarcane NiF8 DNA as a template. FIG. 3 is a characteristic diagram showing the relationship between the length of an amplified fragment obtained from an electrophoresis image (first time) of a DNA library amplified with three types of random primers using sugarcane NiF8 DNA as a template and the fluorescence unit (FU). FIG. 5 is a characteristic diagram showing the relationship between the amplified fragment length and the fluorescence unit (FU) obtained from an electrophoresis image (second time) of a DNA library amplified with 3 types of random primers using sugarcane NiF8 DNA as a template. FIG. 3 is a characteristic diagram showing the relationship between the length of an amplified fragment obtained from an electrophoresis image (first time) of a DNA library amplified with 12 kinds of random primers using sugarcane NiF8 DNA as a template and the fluorescence unit (FU). It is a characteristic view showing the relationship between the amplified fragment length obtained from an electrophoresis image (second time) of a DNA library amplified with 12 kinds of random primers using sugarcane NiF8 DNA as a template and the fluorescence unit (FU). FIG. 4 is a characteristic diagram showing the relationship between the length of an amplified fragment obtained from an electrophoresis image (first time) of a DNA library amplified with 24 kinds of random primers using sugarcane NiF8 DNA as a template and the fluorescence unit (FU). FIG. 4 is a characteristic diagram showing the relationship between the length of an amplified fragment obtained from an electrophoresis image (second time) of a DNA library amplified with 24 kinds of random primers using sugarcane NiF8 DNA as a template and the fluorescence unit (FU). It is a characteristic view showing the relationship between the amplified fragment length and the fluorescence unit (FU) obtained from the electrophoresis image (first time) of a DNA library amplified with 48 kinds of random primers using sugarcane NiF8 DNA as a template. It is a characteristic view showing the relationship between the amplified fragment length obtained from the electrophoresis image (second time) of a DNA library amplified with 48 kinds of random primers using sugarcane NiF8 DNA as a template and the fluorescence unit (FU). FIG. 3 is a characteristic diagram showing the relationship between the length of an amplified fragment obtained from an electrophoresis image (first time) of a DNA library amplified with 10 base B of random primer using sugarcane NiF8 DNA as a template and the fluorescence unit (FU). It is a characteristic view showing the relationship between the amplified fragment length obtained from an electrophoresis image (second time) of a DNA library amplified with 10 base B of random primer using sugarcane NiF8 DNA as a template and the fluorescence unit (FU). FIG. 3 is a characteristic diagram showing the relationship between the length of an amplified fragment obtained from an electrophoretic image (first time) of a DNA library amplified with 10 bases C of random primer using sugarcane NiF8 DNA as a template and the fluorescence unit (FU). It is a characteristic view showing the relationship between the amplified fragment length obtained from an electrophoresis image (second time) of a DNA library amplified with 10 base C of random primer using sugarcane NiF8 DNA as a template and the fluorescence unit (FU). FIG. 3 is a characteristic diagram showing the relationship between the length of an amplified fragment obtained from an electrophoresis image (first time) of a DNA library amplified with 10 base D of random primer using sugarcane NiF8 DNA as a template and the fluorescence unit (FU). It is a characteristic view showing the relationship between the amplified fragment length obtained from an electrophoresis image (second time) of a DNA library amplified with 10 base D of random primer using sugarcane NiF8 DNA as a template and the fluorescence unit (FU). FIG. 3 is a characteristic diagram showing the relationship between the length of an amplified fragment obtained from an electrophoresis image (first time) of a DNA library amplified with 10 base E of random primer using sugarcane NiF8 DNA as a template and the fluorescence unit (FU). FIG. 5 is a characteristic diagram showing the relationship between the amplified fragment length and the fluorescence unit (FU) obtained from an electrophoretic image (second time) of a DNA library amplified using sugarcane NiF8 DNA as a template and random primer 10 base E. It is a characteristic view showing the relationship between the amplified fragment length obtained from an electrophoresis image (first time) of a DNA library amplified with 10 base F of random primer using sugarcane NiF8 DNA as a template and the fluorescence unit (FU). It is a characteristic view showing the relationship between the amplified fragment length and the fluorescence unit (FU) obtained from an electrophoresis image (second time) of a DNA library amplified with 10 base F of random primer using sugarcane NiF8 DNA as a template. FIG. 3 is a characteristic diagram showing the relationship between the length of an amplified fragment obtained from an electrophoretic image (first time) of a DNA library amplified with 10 base A of random primer using human genomic DNA as a template and the fluorescence unit (FU). FIG. 5 is a characteristic diagram showing the relationship between the length of an amplified fragment obtained from an electrophoresis image (second time) of a DNA library amplified with 10 base A of random primer using human genomic DNA as a template and the fluorescence unit (FU). It is a characteristic view which shows typically the preparation method of the DNA library with which it uses for a next-generation sequencing apparatus. It is a characteristic view which shows typically the preparation method of the DNA library with which it uses for a next-generation sequencing apparatus. FIG. 3 is a characteristic diagram showing the relationship between the length of an amplified fragment obtained from an electrophoresis image (first time) of a DNA library amplified with 10 base G of random primer using sugarcane NiF8 DNA as a template and the fluorescence unit (FU). FIG. 3 is a characteristic diagram showing the relationship between the length of an amplified fragment obtained from an electrophoresis image (second time) of a DNA library amplified with 10 base G of random primer using sugarcane NiF8 DNA as a template and the fluorescence unit (FU). Amplified fragment length and fluorescence unit (FU) obtained from the electrophoresis image (first time) of a DNA library amplified with a primer for next-generation sequencer using a DNA library prepared with 10 base G of random primer for sugarcane NiF8 as a template FIG. Amplified fragment length and fluorescence unit (FU) obtained from the electrophoresis image (second time) of a DNA library amplified with the primer for the next-generation sequencer using a DNA library prepared with 10 base G of random primer for sugarcane NiF8 as a template FIG. It is a characteristic view which shows the MiSeq analysis result of the DNA library which amplified the DNA of sugarcane NiF8 by the random primer 10 base G. It is a characteristic view showing the relationship between the amplified fragment length and the fluorescence unit (FU) obtained from the electrophoresis image (first time) of the DNA library amplified using the rice Nipponbare DNA as a template and random primer 12 base B. FIG. 5 is a characteristic diagram showing the relationship between the amplified fragment length and the fluorescence unit (FU) obtained from the electrophoresis image (second time) of a DNA library amplified with 12 bases B of random primers using rice Nipponbare DNA as a template. Amplified fragment length and fluorescence unit (FU) obtained from the electrophoresis image (first time) of DNA library amplified with next-generation sequencer primer using DNA library prepared using random primer 12base B as template for rice Nipponbare FIG. Amplified fragment length and fluorescence unit (FU) obtained from the electrophoresis image (second) of DNA library amplified with next-generation sequencer primer using DNA library prepared using random primer 12 base B as a template for rice Nipponbare FIG. It is a characteristic diagram showing the distribution of the lead pattern obtained by analyzing with MiSeq a DNA library amplified with rice base DNA of rice Nipponbare as a template and random primer sequence and rice Nipponharu reference sequence. . It is a characteristic view which shows the MiSeq analysis result of the DNA library which amplified the rice Nipponbare DNA as a template with the random primer 12 base B.

Hereinafter, the present invention will be described in detail.

In the method for producing a DNA library according to the present invention, a nucleic acid amplification reaction is performed with a reaction solution prepared by adjusting a primer having an arbitrary base sequence (hereinafter referred to as a random primer) to a high concentration, and the amplified nucleic acid fragment is subjected to DNA live. Rally. Here, the high concentration means that the concentration is higher than the primer concentration in a normal nucleic acid amplification reaction. That is, the DNA library preparation method according to the present invention is characterized in that a higher concentration of random primers is used compared to the primer concentration in a normal nucleic acid amplification reaction. Here, as a template contained in the reaction solution, genomic DNA prepared from a target organism for preparing a DNA library can be used.

In the method for preparing a DNA library according to the present invention, the target species is not limited at all, and any species such as animals, plants, microorganisms, viruses including humans can be targeted. That is, according to the DNA library production method of the present invention, a DNA library can be produced from any species.

In this method for preparing a DNA library, nucleic acid fragments (nucleic acid fragment groups) can be amplified with high reproducibility by defining the concentration of random primers as described above. Here, reproducibility refers to the degree to which nucleic acid fragments that are amplified match between multiple nucleic acid amplification reactions when multiple nucleic acid amplification reactions are performed using the same template and the same random primer. means. That is, high reproducibility (high reproducibility) is a nucleic acid that is amplified between multiple nucleic acid amplification reactions when the same template and the same random primer are used for multiple nucleic acid amplification reactions. This means that the degree of matching of fragments is high.

Regarding the level of reproducibility, for example, a fluorescence unit (Fluorescence Unit: FU) obtained by performing a plurality of nucleic acid amplification reactions using the same template and the same random primer and electrophoresis of the amplified fragments obtained each time. ) For Spearman's rank correlation coefficient, and can be evaluated based on the coefficient. Spearman's rank correlation coefficient is generally represented by ρ. As an example, ρ> 0.9 can be evaluated as having reproducibility.

[Random primer]
The sequence of the random primer that can be used in the method for producing a DNA library according to the present invention is not limited at all. For example, nucleotides having a length of 9 to 30 bases can be used. In particular, a random primer is a nucleotide having an arbitrary sequence and a length of 9 to 30 bases, and the type of nucleotide (type of sequence) is not particularly limited, but one or more nucleotides, preferably 1 to 10000 types More preferably 1 to 1000 nucleotides, more preferably 1 to 100 nucleotides, and most preferably 1 to 96 nucleotides. By using nucleotides (nucleotide groups) in the above-mentioned range as random primers, amplified nucleic acid fragments can be obtained with higher reproducibility. When the random primer includes a plurality of nucleotides, all the nucleotides do not have to have the same base length (9 to 30 bases), and may include a plurality of nucleotides having different base lengths.

Usually, in order to obtain a specific amplicon using a nucleic acid amplification reaction, the base sequence of a primer is designed according to the amplicon. For example, a pair of primers is designed so as to sandwich a position corresponding to an amplicon in template DNA such as genomic DNA. In this case, since the primer is designed to hybridize to a specific region contained in the template, it can be referred to as a “specific primer”.

On the other hand, unlike a primer designed for the purpose of obtaining a specific amplicon, the random primer is not designed to hybridize to a specific region in the template DNA, but obtains a random amplicon. Designed for. The random primer may have any nucleotide sequence, and can participate in random amplicon amplification by accidentally hybridizing to a complementary region contained in the template DNA.

That is, the random primer can be a nucleotide having an arbitrary sequence involved in random amplicon amplification as described above. Here, the arbitrary sequence is not limited in any way. For example, it may be designed as a base sequence randomly selected from the group of adenine, guanine, cytosine and thymine, or may be designed as a specific base sequence. . Examples of the specific base sequence include a base sequence including a restriction enzyme recognition sequence and a base sequence having an adapter sequence used for a next-generation sequencer.

When designing multiple types of nucleotides as random primers, a method of selecting a plurality of base sequences of a predetermined length by randomly selecting from the group of adenine, guanine, cytosine and thymine can be applied. Moreover, when designing multiple types of nucleotides as random primers, a method of designing a plurality of base sequences composed of a common part consisting of a specific base sequence and a non-common part consisting of an arbitrary base sequence can also be applied. Here, the non-common part may be a base sequence randomly selected from the group of adenine, guanine, cytosine and thymine, or a combination of all four types of bases consisting of adenine, guanine, cytosine and thymine, or It can be a partial combination selected from all these combinations. The common part is not particularly limited and may be any base sequence.For example, a base sequence including a restriction enzyme recognition sequence, a base sequence having an adapter sequence used for a next-generation sequencer, a base sequence common to a specific gene family and can do.

When a plurality of base sequences of a predetermined length are selected by randomly selecting from 4 types of bases as a plurality of random primers, 30% or more of the whole, preferably 50% or more, more preferably 70% or more, Preferably, 90% or more is designed to have 70% or less identity, preferably 60% or less identity, more preferably 50% or less identity, and most preferably 40% or less identity. preferable. In the case of designing a plurality of base sequences of a predetermined length by randomly selecting from four types of bases as a plurality of random primers, the nucleotides in the above range are designed to have the same identity in the above range. Thus, an amplified fragment can be obtained over the entire genomic DNA of the target species. That is, the uniformity of the amplified fragment can be improved.

When designing multiple base sequences consisting of a common part consisting of a specific base sequence and a non-common part consisting of an arbitrary base sequence as a plurality of random primers, for example, several bases on the 3 ′ end side are set as non-common parts. The remaining 5 ′ end side can be designed as a common part. If n bases on the 3 ′ end side are non-common parts, 4 ⁿ types of random primers can be designed. Here, n may be 1 to 5, preferably 2 to 4, more preferably 2 to 3.

For example, as a random primer composed of a common part and a non-common part, the 5 ′ end side is an adapter sequence (common part) used for the next-generation sequencer, and the 3 ′ end side is 2 bases (non-common part). A total of 16 types of random primers can be designed. In addition, if 3 'terminal side is 3 bases (non-common part), a total of 64 types of random primers can be designed. As the types of random primers increase, amplified fragments can be obtained more comprehensively over the entire genomic DNA of the target species. Therefore, when designing a random primer composed of a common part and a non-common part, it is preferable that the base on the 3 'end side is 3 bases.

However, for example, after designing 64 types of base sequences consisting of a common portion and a non-common portion of 3 bases, 63 or less types of random primers selected from these 64 types of base sequences may be used. In other words, compared to using all 64 types of random primers, using 63 or less types of random primers gives better results in nucleic acid amplification reactions and analyzes using next-generation sequencers. There is. Specifically, when 64 types of random primers are used, the number of reads of a specific nucleic acid amplified fragment may be remarkably increased. In this case, it is better to use the remaining 63 or less random primers except for one or a plurality of random primers involved in amplification of the specific nucleic acid amplification fragment among 64 types of random primers. Will be obtained.

Similarly, when 16 types of random primers composed of a common portion and a non-common portion of 2 bases are designed, when 15 or less types of random primers selected from 16 types of random primers are used, nucleic acid amplification reaction In some cases, excellent results are shown in analyzes using next-generation sequencers.

On the other hand, the nucleotide used as the random primer is particularly preferably designed so that the GC content is in the range of 5 to 95%, more preferably in the range of 10 to 90%. It is more preferable to design so as to be in the range of 80%, and most preferable to design so as to be in the range of 20 to 70%. By using a set of nucleotides having a GC content in the above range as a random primer, an amplified nucleic acid fragment can be obtained with higher reproducibility. The GC content is the ratio of guanine and cytosine contained in the entire nucleotide chain.

Further, the nucleotide used as the random primer is particularly preferably designed so that the continuous base is 80% or less, more preferably 70% or less, based on the total length, It is more preferable to design so as to be not more than%, and it is most preferable to design so as to be not more than 50%. Alternatively, the nucleotide used as a random primer is particularly preferably designed so that the number of consecutive bases is 8 or less, more preferably designed to be 7 or less, and 6 or less. More preferably, it is more preferable to design so as to be 5 or less. An amplified nucleic acid fragment can be obtained with higher reproducibility by using a set of nucleotides having the number of consecutive bases in the above range as a random primer.

Furthermore, the nucleotide used as a random primer is particularly preferably designed so that it does not have a complementary region of 6 bases or more, preferably 5 bases or more, more preferably 4 bases or more in the molecule. By designing the nucleotide so that it does not have a complementary region in the above range, double strand formation in the molecule can be prevented, and an amplified nucleic acid fragment can be obtained with higher reproducibility.

Furthermore, when designing multiple types of nucleotides as random primers, it should be designed not to have a complementary region with a length of 6 bases or more, preferably 5 bases or more, more preferably 4 bases or more, especially between the plurality of nucleotides. Is preferred. By designing so as not to have a complementary region in the above range between a plurality of nucleotides, double strand formation between nucleotides can be prevented, and amplified nucleic acid fragments can be obtained with higher reproducibility.

Furthermore, when designing multiple types of nucleotides as a random primer, it should be designed in particular so that 6 'or more, preferably 5 or more, more preferably 4 or more on the 3' end side do not form a complementary sequence. Is preferred. By designing so as not to have a complementary sequence in the above-mentioned range on the 3 'terminal side of a plurality of nucleotides, double-stranded formation between nucleotides can be prevented, and amplified nucleic acid fragments can be obtained with higher reproducibility.

The complementary region and the complementary sequence are, for example, a region and sequence having 80 to 100% identity (for example, a region and sequence in which 4 bases or 5 bases are complementary if the region is 5 bases long). Alternatively, it means a region and sequence having 90 to 100% identity (for example, a region and sequence in which 5 bases are complementary in the case of a 5-base long region).

Furthermore, it is preferable that the nucleotide used as the random primer is designed to have a Tm value suitable for the temperature cycle conditions (particularly, annealing temperature) in the nucleic acid amplification reaction. Although not particularly limited, the Tm value can be calculated by a known calculation method such as the closest base pair method, the Wallace method, and the GC% method. Specifically, nucleotides used as random primers should be designed so that the Tm value is 10 to 85 ° C., preferably 12 to 75 ° C., more preferably 14 to 70 ° C., and most preferably 16 to 65 ° C. Is preferred. By designing the Tm value of the nucleotide to be in the above range, an amplified nucleic acid fragment can be obtained with higher reproducibility under a predetermined temperature cycle condition (particularly, a predetermined annealing temperature) in the nucleic acid amplification reaction.

Furthermore, when a plurality of nucleotides are designed as random primers, the variation in Tm value of each nucleotide among a plurality of nucleotides is 50 ° C. or less, preferably 45 ° C. or less, more preferably 40 ° C. or less, most preferably It is preferable to design so that it may be 35 degrees C or less. By designing the variation in Tm value between multiple nucleotides to be within the above range, amplified nucleic acid fragments can be generated with higher reproducibility under a predetermined temperature cycle condition (particularly, a predetermined annealing temperature) in a nucleic acid amplification reaction. Obtainable.

[Nucleic acid amplification reaction]
In the method for producing a DNA library according to the present invention, a large number of amplified fragments are obtained by a nucleic acid amplification reaction using the above-described random primer and genomic DNA as a template. In particular, in the nucleic acid amplification reaction, the concentration of the random primer in the reaction solution is set higher than the primer concentration in the normal nucleic acid amplification reaction. Thereby, a large number of amplified fragments can be obtained using genomic DNA as a template while achieving high reproducibility. As a result, the large number of amplified fragments obtained can be used as a DNA library that can be used for genotyping and the like.

Here, the nucleic acid amplification reaction is a reaction comprising genomic DNA as a template, the above-described random primer, DNA polymerase, deoxynucleotide triphosphate (dNTP, that is, a mixture of dATP, dCTP, dTTP and dGTP) and a buffer as a substrate. This is a reaction for synthesizing an amplified fragment by adding a predetermined temperature cycle condition with a liquid. The nucleic acid amplification reaction requires a predetermined concentration of Mg ²⁺ in the reaction solution, and the buffer contains MgCl ₂ in the above-described composition. If the buffer does not contain MgCl _2, so that the MgCl ₂ is included in addition to the above composition.

Particularly in the nucleic acid amplification reaction, the concentration of the random primer is preferably set as appropriate according to the base length of the random primer. Here, the base length of the random primer is an average value (a simple average or a weighted average considering the amount of nucleotides) when a plurality of types of nucleotides having different base lengths are used as a random primer. be able to.

Specifically, a nucleic acid amplification reaction is performed using a random primer having a length of 9 to 30 bases and a condition in which the concentration of the random primer is 4 to 200 μM, preferably 4 to 100 μM. Under these conditions, a large number of amplified fragments, particularly a large number of amplified fragments having a length of 100 to 500 bases, can be obtained by nucleic acid amplification reaction while achieving high reproducibility.

More specifically, the concentration of the random primer is preferably 40 to 60 μM when the random primer has a length of 9 to 10 bases. When the random primer has a length of 10 to 14 bases and the random primer has a base length y and the random primer concentration is x, y> 3E + 08x ^−6.974 and 100 μM or less are satisfied. It is preferable. The concentration of the random primer is preferably 4 to 100 μM when the random primer has a length of 14 to 18 bases. The concentration of the random primer is preferably 4 μM or more when the random primer has a length of 18 to 28 bases, and preferably satisfies y <8E + 08x− ^5.533 . The concentration of the random primer is preferably 6 to 10 μM when the random primer has a length of 28 to 29 bases. By setting the concentration of the random primer as described above according to the base length of the random primer, a large number of amplified fragments can be obtained more reliably while achieving high reproducibility.

The inequalities of y> 3E + 08x− ^6.974 and y <8E + 08x− ^5.533 described above were obtained as a result of detailed examination of the relationship between the length and the concentration of the random primer, as will be described in Examples described later. This is calculated as a range in which a large number of DNA fragments having a length of 500 bases can be amplified with good reproducibility.

The genomic DNA used as a template in the nucleic acid amplification reaction is not particularly limited, but is preferably 0.1 to 1000 ng, more preferably 1 to 500 ng, when the amount of the reaction solution is 50 μl. It is more preferably 5 to 200 ng, and most preferably 10 to 100 ng. By setting the amount of genomic DNA as a template within this range, a large number of amplified fragments can be obtained while achieving high reproducibility without inhibiting the amplification reaction from random primers.

The method for preparing genomic DNA is not particularly limited, and a conventionally known method can be applied. Further, genomic DNA can be easily prepared from the target species by using a commercially available kit. In addition, as genomic DNA, those extracted from living organisms by a conventionally known method or a commercially available kit may be used as they are, or those extracted from living organisms may be purified, restriction enzyme treatment or ultrasonic waves may be used. You may use the thing after processing.

In the nucleic acid amplification reaction, the DNA polymerase is not particularly limited, and an enzyme having DNA polymerase activity under the temperature cycle conditions for the nucleic acid amplification reaction can be used. Specifically, a heat-resistant DNA polymerase that is used in a normal nucleic acid amplification reaction can be used. For example, examples of the DNA polymerase include DNA polymerases derived from thermophilic bacteria such as Taq DNA polymerase, and hyperthermophilic Archaea DNA polymerases such as KOD DNA polymerase and Pfu DNA polymerase. In particular, in the nucleic acid amplification reaction, it is preferable to use Pfu DNA polymerase as the DNA polymerase together with the random primer described above. By using these DNA polymerases, a large number of amplified fragments can be obtained more reliably while achieving high reproducibility.

Further, in the nucleic acid amplification reaction, the concentration of deoxynucleotide triphosphate (dNTP, that is, a mixture of dATP, dCTP, dTTP and dGTP) serving as a substrate is not particularly limited, and can be 5 μM to 0.6 mM. It is preferably set to ˜0.4 mM, more preferably 20 μM to 0.2 mM. By setting the concentration of dNTP as a substrate within this range, it is possible to prevent an error due to erroneous incorporation by DNA polymerase and to obtain a large number of amplified fragments while achieving high reproducibility.

Furthermore, in the nucleic acid amplification reaction, the buffer is not particularly limited, and examples thereof include a solution containing MgCl ₂ as described above, such as a solution containing Tris-HCl (pH 8.3) and KCl. Here, the concentration of Mg ²⁺ is not particularly limited, but may be, for example, 0.1 to 4.0 mM, preferably 0.2 to 3.0 mM, and preferably 0.3 to 2.0 mM. More preferred is 0.5 to 1.5 mM. By setting the Mg ²⁺ concentration in the reaction solution within this range, a large number of amplified fragments can be obtained while achieving high reproducibility.

Furthermore, the temperature cycle condition in the nucleic acid amplification reaction is not particularly limited, and a normal temperature cycle can be adopted. Specifically, the temperature cycle means first the first heat denaturation temperature for separating the template genomic DNA into single strands, and then “thermal denaturation temperature → annealing temperature → extension reaction temperature” a plurality of times (for example, 20 -40 times) and, if necessary, a cycle for setting the extension reaction temperature for a predetermined time and finally setting the temperature for storage can be exemplified.

The heat denaturation temperature may be, for example, 93 to 99 ° C, preferably 95 to 98 ° C, more preferably 97 to 98 ° C. The annealing temperature may be, for example, 30 to 70 ° C., preferably 35 to 68 ° C., more preferably 37 to 65 ° C., depending on the Tm value of the random primer described above. The elongation reaction temperature can be, for example, 70 to 76 ° C, preferably 71 to 75 ° C, more preferably 72 to 74 ° C. Moreover, as temperature for a preservation | save, it can be set as 4 degreeC, for example.

In addition, the first heat denaturation can be, for example, 5 seconds to 10 minutes, preferably 10 seconds to 5 minutes, more preferably 30 seconds to 2 minutes in the temperature range described above. The thermal denaturation in the cycle of “thermal denaturation temperature → annealing temperature → extension reaction temperature” is, for example, 2 seconds to 5 minutes, preferably 5 seconds to 2 minutes, more preferably 10 seconds to 1 minute in the temperature range described above. be able to. The annealing in the cycle of “thermal denaturation temperature → annealing temperature → extension reaction temperature” should be, for example, 1 second to 3 minutes, preferably 3 seconds to 2 minutes, more preferably 5 seconds to 1 minute in the above-mentioned temperature range. Can do. The extension reaction in the cycle of “thermal denaturation temperature → annealing temperature → extension reaction temperature” is, for example, 1 second to 3 minutes, preferably 3 seconds to 2 minutes, more preferably 5 seconds to 1 minute in the temperature range described above. be able to.

Further, as a method for producing a DNA library according to the present invention, an amplified fragment may be obtained by a nucleic acid amplification reaction employing a hot start method. The hot start method is a method for preventing mispriming before the cycle of “thermal denaturation temperature → annealing temperature → extension reaction temperature” and nonspecific amplification derived from primer dimers. In the hot start method, an enzyme in which the DNA polymerase activity is suppressed by binding an anti-DNA polymerase antibody or performing chemical modification is used. In this state, DNA polymerase activity is suppressed, and nonspecific reaction before the temperature cycle can be prevented. In the hot start method, the DNA polymerase activity is recovered by setting the temperature higher during the first temperature cycle, and the subsequent nucleic acid amplification reaction proceeds.

As described above, by using the above-described random primer having a length of 9 to 30 bases and performing a nucleic acid amplification reaction with the random primer concentration in the reaction solution being 4 to 200 μM, a large number of random primers can be obtained using genomic DNA as a template. Amplified fragments can be obtained. When the 9 to 30 base long random primer described above is used and the concentration of the random primer in the reaction solution is 4 to 200 μM, the nucleic acid amplification reaction is very reproducible. That is, according to the nucleic acid amplification reaction described above, a large number of amplified fragments can be obtained while achieving very high reproducibility. Therefore, the obtained many amplified fragments can be used as a DNA library in gene analysis targeting genomic DNA.

In addition, by using the above-mentioned 9-30 base length random primer and performing a nucleic acid amplification reaction with the random primer concentration in the reaction solution being 4 to 200 μM, in particular, about 100 to 500 base length using genomic DNA as a template. A large number of amplified fragments can be obtained. The large number of amplified fragments having a length of about 100 to 500 bases have a size suitable for large-scale analysis of base sequences by a next-generation sequencer, for example, and highly accurate sequence information can be obtained. That is, according to the present invention, a DNA library containing a DNA fragment having a length of about 100 to 500 bases can be prepared.

Furthermore, by using the above-mentioned 9-30 base long random primer and performing a nucleic acid amplification reaction with the random primer concentration in the reaction solution being 4-200 μM, it is possible to uniformly amplify the entire genomic DNA. Can be obtained. In other words, in the nucleic acid amplification reaction using the random primer, the DNA fragment is not amplified in a predetermined region of the genomic DNA but is amplified throughout the genome. That is, according to the present invention, a uniform DNA library can be prepared for the entire genome.

In addition, after performing nucleic acid amplification reaction using the random primer mentioned above, a restriction enzyme process, a size selection process, a sequence capture process, etc. can be performed with respect to the obtained amplified fragment. By performing these restriction enzyme treatments, size selection treatments, and sequence capture treatments on the amplified fragments, specific amplified fragments (fragments having a specific restriction enzyme site, amplification in a specific size range) are obtained from the obtained amplified fragments. Fragment, an amplified fragment having a specific sequence). The specific amplified fragments obtained by these various treatments can be used as a DNA library.

[Genomic DNA analysis method]
Genomic DNA analysis such as genotype analysis can be performed by using the DNA library prepared as described above. As described above, the DNA library has very high reproducibility, has a size suitable for a next-generation sequencer, and has uniformity throughout the entire genome. Therefore, the DNA library can be used as a DNA marker (also referred to as a genetic marker or genetic marker). Here, the DNA marker broadly means a characteristic base sequence existing in genomic DNA. In addition, the DNA marker can be a base sequence on the genome that serves as a marker related to genetic traits. DNA markers such as genotyping, linkage maps, gene mapping, breeding including selection steps using markers, backcrossing using markers, quantitative trait locus mapping, bulk segregant analysis, breed identification, or linkage It can be used for imbalance mapping.

For example, the base sequence of the DNA library prepared as described above can be determined using a next-generation sequencer or the like, and the presence or absence of a DNA marker can be confirmed based on the obtained base sequence.

As an example, the presence or absence of a DNA marker can be confirmed from the number of reads in the obtained base sequence. Here, the next-generation sequencer is not particularly limited, but is also referred to as a second-generation sequencer, and means a base sequence determination device capable of determining the base sequences of tens of millions of DNA fragments in parallel. The sequencing principle in the next-generation sequencer is not particularly limited. For example, the principle is that the target DNA is amplified on the flow cell by the bridge PCR method and the sequencing-by-synthesis method, and sequencing is performed while synthesizing. One can mention the principle of sequencing by the PCR method and the pyrosequencing method for sequencing by measuring the amount of pyrophosphate released during DNA synthesis. More specifically, examples of next-generation sequencers include Illumina's MiniSeq, MiSeq, NextSeq, HiSeq and HiSeq X series, Roche's Roche 454 GS FLX sequencer, and the like.

As another example, the presence or absence of a DNA marker can be confirmed by comparing a base sequence obtained for a DNA library prepared as described above with a base sequence for reference. Here, the base sequence for reference means a known sequence as a standard, and can be a known sequence stored in a database, for example. That is, for a given organism, a DNA library is prepared as described above, its base sequence is determined, and the base sequence of the DNA library is compared with a base sequence for reference. Then, a base sequence different from the reference base sequence can be used as a DNA marker (characteristic base sequence present in genomic DNA) related to the predetermined organism. Further, the identified DNA marker can be further analyzed according to a standard method to determine the relevance to the genetic trait (phenotype). That is, a DNA marker related to a phenotype (sometimes referred to as a selection marker) can be specified from the DNA markers specified as described above.

Furthermore, as another example, the above DNA library prepared by using the base sequence obtained for the DNA library prepared as described above using genomic DNA derived from another organism or genomic DNA derived from another tissue The presence or absence of a DNA marker can be confirmed by comparing with the base sequence. That is, for two or more organisms or two different tissues, DNA libraries are prepared as described above, their base sequences are determined, and the base sequences of the DNA libraries are compared with each other. And the base sequence which is different between DNA libraries can be used as a DNA marker (characteristic base sequence existing in genomic DNA) related to the tested organism or tissue. Further, the identified DNA marker can be further analyzed according to a standard method to determine the relevance to the genetic trait (phenotype). That is, a DNA marker related to a phenotype (sometimes referred to as a selection marker) can be specified from the DNA markers specified as described above.

Incidentally, a pair of primers that specifically amplify the DNA marker can also be designed based on the obtained base sequence. The presence or absence of a DNA marker in the extracted genomic DNA can also be confirmed by performing a nucleic acid amplification reaction using the designed pair of primers and genomic DNA extracted from the target organism as a template.

Alternatively, the DNA library prepared as described above can be used for metagenomic analysis for examining the diversity of microorganisms, somatic genome mutation analysis such as tumor tissue, genotype analysis using microarrays, ploidy determination analysis, chromosome analysis It can be used for analysis such as number calculation analysis, chromosome increase / decrease analysis, chromosome partial insertion / deletion / replication / translocation analysis, foreign genome contamination analysis, parent-child discrimination analysis, and mating seed purity test analysis.

[Application to next-generation sequencing technology]
As described above, by performing the nucleic acid amplification reaction with the random primer in the reaction solution at a high concentration, a large number of amplified fragments can be obtained with high reproducibility using genomic DNA as a template. Since the obtained amplified fragment has the same base sequence as that of the random primer at both ends, it can be easily used for next-generation sequencing technology by using the base sequence.

Specifically, first, as described above, a nucleic acid amplification reaction is performed in a reaction solution (first reaction solution) containing genomic DNA and a high concentration of random primers, and a large number of nucleic acid amplification reactions are performed using genomic DNA as a template. The amplified fragment (first DNA fragment) is obtained. Next, a reaction solution (second reaction solution) containing a large number of obtained amplified fragments (first DNA fragments) and a primer (referred to as a next-generation sequencer primer) designed based on the base sequence of the random primer. ) To carry out a nucleic acid amplification reaction. Here, the next-generation sequencer primer is a nucleotide containing a region used for the base sequence determination reaction. More specifically, for example, the primer for the next-generation sequencer has a base sequence that matches 70% or more of the base sequence at the 3 ′ end with the base sequence at the 5 ′ end side of the first DNA fragment, preferably 80%. More than 90% matching base sequence, more preferably more than 95% matching base sequence, more preferably more than 95% matching base sequence, more preferably more than 97% matching base sequence, most preferably more than 100% matching base sequence It can be a nucleotide having a region necessary for a base sequence determination reaction (sequence reaction) by a next-generation sequencing apparatus.

Here, the “region used for the base sequence determination reaction” included in the next-generation sequencer primer is not particularly limited because it varies depending on the type of next-generation sequencer. For example, the next-generation sequencer uses a sequencing primer. When the base sequence determination reaction is performed, the base sequence can be complementary to the base sequence of the sequencing primer. In addition, when a next-generation sequencer performs a base sequencing reaction using capture beads to which a predetermined DNA is bound, the “region used for the base sequencing reaction” refers to the base of the DNA bound to the capture beads. The base sequence can be complementary to the sequence. Furthermore, if the next-generation sequencer reads a sequence from the current change when a DNA strand having a paired-pin loop passes through a protein having a nano-sized pore, the "region used for base sequencing" Can be a base sequence complementary to the base sequence that forms the hairpin loop.

By designing the base sequence at the 3 ′ end of the next-generation sequencer primer as described above, the next-generation sequencer primer can hybridize to the 3 ′ end of the first DNA fragment under stringent conditions. The second DNA fragment can be amplified using the first DNA fragment as a template. Here, the stringent condition means a condition in which a so-called specific hybrid is formed and a non-specific hybrid is not formed.For example, referring to Molecular Cloning: A Laboratory Manual (Third Edition) Can be determined. Specifically, the stringency can be set according to the temperature at the time of Southern hybridization and the salt concentration contained in the solution, and the temperature at the time of the washing step of Southern hybridization and the salt concentration contained in the solution. More specifically, as stringent conditions, for example, the sodium concentration is 25 to 500 mM, preferably 25 to 300 mM, and the temperature is 42 to 68 ° C., preferably 42 to 65 ° C. More specifically, it is 5 × SSC (83 mM NaCl, 83 mM sodium citrate), and the temperature is 42 ° C.

In particular, when the first DNA fragment is obtained using multiple types of random primers, next-generation sequencer primers may be prepared to handle all random primers, or some random primers are supported. You may prepare the primer for the next-generation sequencer.

For example, as a plurality of types of random primers, a set of random primers (several bases at the 3 ′ end is an arbitrary sequence) consisting of a common base sequence other than a few bases at the 3 ′ end (for example, about 1 to 3 bases) is used. In this case, the 5 ′ ends of the obtained first DNA fragments all have a common sequence. Therefore, the base sequence at the 3 'end of the next-generation sequencer primer is set to a base sequence that matches 70% or more with the common base sequence at the 5' end of the first DNA fragment. By designing the next-generation sequencer primer in this way, it becomes a next-generation sequencer primer corresponding to all random primers. By using this next-generation sequencer primer, the second DNA fragment can be amplified using all of the first DNA fragment as a template.

Similarly, as a plurality of types of random primers, a set of random primers (several bases at the 3 ′ end is an arbitrary sequence) consisting of a common base sequence other than a few bases at the 3 ′ end (for example, about 1 to 3 bases). Even in the case of using the second DNA fragment, a second DNA fragment can be obtained using a part of the obtained first DNA fragments as a template. Specifically, the base sequence at the 3 ′ end of the primer for the next-generation sequencer is the base sequence common to the 5 ′ end of the first DNA fragment, followed by a sequence of several bases (the number of the 3 ′ end of the random primer). The base sequence (corresponding to the base sequence (arbitrary sequence)) is 70% or more, so that the second DNA fragment can be amplified using a part of the first DNA fragment as a template.

On the other hand, when the first DNA fragment is obtained using multiple types of random primers consisting of all arbitrary base sequences, multiple types of next-generation sequencer primers are used to support all of the first DNA fragments. The second DNA fragment may be obtained by using a plurality of next-generation sequencer primers so as to correspond to a part of the first DNA fragment. .

As described above, the second DNA fragment amplified using the next-generation sequencer primer has a region necessary for the base sequence determination reaction (sequence reaction) by the next-generation sequencer included in the next-generation sequencer primer. is doing. The region necessary for the sequence reaction is not particularly limited because it differs depending on the next-generation sequence device. For example, when used in a next-generation sequencing device based on the principle that the target DNA is amplified on the flow cell by the bridge PCR method and sequencing-by-synthesis method and synthesized while being synthesized, the primer for the next-generation sequencer is It includes the region necessary for PCR and the region necessary for sequencing-by-synthesis. The region necessary for bridge PCR is a region that hybridizes to the oligonucleotide immobilized on the flow cell, and is a 9-base long region including the 5 'end of the primer for the next-generation sequencer. The region necessary for the sequencing-by-synthesis method is a region where the sequence primer used for the sequencing reaction hybridizes, and is a region in the middle of the next-generation sequencer primer.

Also, as a next-generation sequence device, an Ion Torrent sequence device can be mentioned. When using the Ion Torrent sequencing apparatus, the primer for the next generation sequencer has a so-called ion adapter on the 5 ′ end side, and binds to the particles to be subjected to emulsion PCR. In the Ion-Torrent sequencer, particles coated with a template amplified by emulsion PCR are placed on an ion chip and subjected to a sequence reaction.

Note that the nucleic acid amplification reaction using the second reaction solution containing the next-generation sequencer primer and the first DNA is not particularly limited, and normal nucleic acid amplification reaction conditions can be applied. That is, the conditions described in the column of [Nucleic acid amplification reaction] described above can be employed. For example, the second reaction solution is a template of the first DNA fragment, the above-mentioned primer for the next-generation sequencer, DNA polymerase, and the substrate deoxynucleotide triphosphate (dNTP, that is, a mixture of dATP, dCTP, dTTP and dGTP) ) And buffer.

In particular, the concentration of the primer for the next-generation sequencer can be 0.01 to 5.0 μM, preferably 0.1 to 2.5 μM, and most preferably 0.3 to 0.7 μM. .

The first DNA fragment used as a template in the nucleic acid amplification reaction is not particularly limited, but is preferably 0.1 to 1000 ng and preferably 1 to 500 ng when the reaction volume is 50 μl. More preferred is 5 to 200 ng, still more preferred is 10 to 100 ng.

The method for preparing the first DNA fragment used as a template is not particularly limited, and the reaction solution after completion of the nucleic acid amplification reaction using the random primer described above may be used as it is, or the first solution from the reaction solution may be used. A purified DNA fragment may be used.

In addition, the type of DNA polymerase used in the nucleic acid amplification reaction, the concentration of deoxynucleotide triphosphate (dNTP, that is, a mixture of dATP, dCTP, dTTP, and dGTP), the buffer composition, and the temperature cycle conditions described above are described above [ The conditions can be as described in the column of “Nucleic acid amplification reaction”. Moreover, also in the nucleic acid amplification reaction using the next-generation sequencer primer, the hot start method may be employed, or an amplified fragment may be obtained by the nucleic acid amplification reaction.

As described above, the first DNA fragment obtained using random primers can be used as a template, and the second DNA fragment amplified using the next-generation sequencer primer can be used to apply to next-generation sequencing equipment. A DNA library can be easily prepared.

In the above-described example, the first DNA fragment obtained using a random primer is used as a template, and the second DNA fragment amplified using a next-generation sequencer primer is used as a DNA library. The range is not limited to this example. For example, the DNA library according to the present invention amplifies the second DNA fragment using the first DNA fragment obtained using a random primer as a template, and further uses the second DNA fragment as a template for a next-generation sequencer. A third DNA fragment may be obtained using a primer, and the third DNA fragment may be used as a DNA library applicable to a next-generation sequencing apparatus.

To create a DNA library that can be applied to next-generation sequencing equipment, similarly, repeat the nucleic acid amplification reaction using the obtained DNA fragment as a template after the nucleic acid amplification reaction using the second DNA fragment as a template. It can be prepared by using a primer for next-generation sequencer in the nucleic acid amplification reaction. At this time, the number of repeated nucleic acid amplification reactions is not particularly limited, but is 2 to 10 times, preferably 2 to 5 times, more preferably 2 to 3 times.

EXAMPLES Hereinafter, although this invention is demonstrated further in detail using an Example, the technical scope of this invention is not limited to a following example.
[Example 1]
1. Flowchart In this example, according to the flowchart shown in FIG. 1, a DNA library was prepared by PCR using genomic DNA extracted from various biological species as a template and various random primer sets. In addition, the prepared DNA library was used to perform sequence analysis using a so-called next-generation sequencer, and the genotype was analyzed based on the obtained read data.

2. Materials In this example, genomic DNA was extracted and purified from sugarcane varieties NiF8, Ni9 and their progeny 22 lines, and rice cultivar Nipponbare using DNeasy Plant Mini kit (QIAGEN), respectively. , Genomic DNA derived from 22 progenies of the hybrid, and genomic DNA derived from Nipponbare. In this example, human genomic DNA was purchased from Takara Bio and used as human-derived genomic DNA.

3. Method 3.1 Relationship between PCR conditions and DNA fragment size 3.1.1 Random primer design When designing a random primer, the GC content should be in the range of 20-70% and the number of consecutive bases should be 5 or less. Set. The base length is 8 base length, 9 base length, 10 base length, 11 base length, 12 base length, 14 base length, 16 base length, 18 base length, 20 base length, 22 base length, 24 base length. 16 types, 26 base length, 28 base length, 29 base length, 30 base length and 35 base length, were set. For each base length, 96 types of base sequences were designed, and a set consisting of 96 types of random primers was prepared. For the 10 base length, 6 sets (each set includes 96 types of random primers) were designed (these 6 sets are referred to as 10 bases A to 10 bases F). That is, in this example, 21 types of random primer sets were produced.

The base sequences of random primers contained in these 21 types of random primer sets are shown in Tables 1 to 21.

3.1.2 Standard PCR
2. Add the final concentration of 0.6 μM random primer (10 base A), 0.2 mM dNTP mixture, 1.0 mM MgCl ₂ and 1.25 unit DNA Polymerase (TAKARA, PrimeSTAR) to the genomic DNA described in (NiF8-derived genomic DNA: 30 ng). The reaction solution was adjusted with a volume of 50 μl. PCR temperature cycle conditions are as follows: 98 ° C for 2 minutes, then 30 cycles of 98 ° C for 10 seconds, 50 ° C for 15 seconds and 72 ° C for 20 seconds, then store at 4 ° C Condition. In this example, a number of nucleic acid fragments obtained by PCR using random primers, including the standard PCR described here, are referred to as a DNA library.

3.1.3 Purification and Electrophoresis of DNA Library After purifying the DNA library obtained in 3.1.2 with MinElute PCR Purification Kit (QIAGEN), electrophoresis with Agilent 2100 Bioanalyzer (Agient Technologies) A fluorescence unit (Fluorescence Unit: FU) was obtained.

3.1.4 Examination of annealing temperature Add the final concentration of 0.6 μM random primer (10 base A), 0.2 mM dNTP mixture, 1.0 mM MgCl ₂ and 1.25 unit DNA Polymerase (TAKARA, PrimeSTAR) to the genomic DNA described in (NiF8-derived genomic DNA: 30 ng). The reaction solution was adjusted with a volume of 50 μl. The temperature cycle conditions for PCR were: 98 ° C for 2 minutes, then 30 cycles of 98 ° C for 10 seconds, various annealing temperatures for 15 seconds and 72 ° C for 20 seconds, followed by 4 ° C The conditions for storage were taken. In this example, 37 ° C., 40 ° C., and 45 ° C. were examined as the annealing temperature. The DNA library obtained in this experiment was purified and electrophoresed in the same manner as in 3.1.3.

3.1.5 Examination of enzyme amount DNA DNA Polymerase (TAKARA, PrimeSTAR) with a final concentration of 0.6 μM random primer (10 base A), 0.2 mM dNTP mixture, 1.0 mM MgCl ₂ and 2.5 unit or 12.5 unit And the reaction solution was adjusted to a final reaction volume of 50 μl. PCR temperature cycle conditions are as follows: 98 ° C for 2 minutes, then 30 cycles of 98 ° C for 10 seconds, 50 ° C for 15 seconds and 72 ° C for 20 seconds, then store at 4 ° C Condition. The DNA library obtained in this experiment was purified and electrophoresed in the same manner as in 3.1.3.

3.1.6 Examination of MgCl ₂ concentration Add the final concentration 0.6 μM random primer (10 base A), 0.2 mM dNTP mixture, predetermined concentration MgCl ₂ and 1.25 unit DNA Polymerase (TAKARA, PrimeSTAR) to the genomic DNA described in (NiF8-derived genomic DNA: 30 ng) The reaction solution was adjusted with a reaction volume of 50 μl. PCR temperature cycle conditions are as follows: 98 ° C for 2 minutes, then 30 cycles of 98 ° C for 10 seconds, 50 ° C for 15 seconds and 72 ° C for 20 seconds, then store at 4 ° C Condition. In this example, the usual MgCl ₂ concentration was examined as 2 times (2.0 mM), 3 times (3.0 mM) and 4 times (4.0 mM). The DNA library obtained in this experiment was purified and electrophoresed in the same manner as in 3.1.3.

3.1.7 Examination of base length of random primer Add the final concentration of 0.6 μM random primer, 0.2 mM dNTP mixture, 1.0 mM MgCl ₂ and 1.25 unit DNA Polymerase (TAKARA, PrimeSTAR) to the genomic DNA described in (NiF8-derived genomic DNA: 30 ng). Adjusted. PCR temperature cycle conditions are as follows: 98 ° C for 2 minutes, then 30 cycles of 98 ° C for 10 seconds, 50 ° C for 15 seconds and 72 ° C for 20 seconds, then store at 4 ° C Condition. In this example, as the random primer, the 8 base length (Table 7), 9 base length (Table 8), 11 base length (Table 9), 12 base length (Table 10), 14 base length (described above) Table 11), 16 base length (Table 12), 18 base length (Table 13) and 20 base length (Table 14) were examined. The DNA library obtained in this experiment was purified and electrophoresed in the same manner as in 3.1.3.

3.1.8 Examination of random primer concentration Add the random primer (10 base A), 0.2 mM dNTP mixture, 1.0 mM MgCl ₂ and 1.25 unit DNA Polymerase (TAKARA, PrimeSTAR) at the specified concentration to the genomic DNA described in (NiF8-derived genomic DNA: 30 ng) The reaction solution was adjusted with 50 μl. PCR temperature cycle conditions are as follows: 98 ° C for 2 minutes, then 30 cycles of 98 ° C for 10 seconds, 50 ° C for 15 seconds and 72 ° C for 20 seconds, then store at 4 ° C Condition. In this example, 2, 4, 6, 8, 10, 20, 40, 60, 100, 200, 300, 400, 500, 600, 700, 800, 900, and 1000 μM were examined as the random primer concentration. did. The DNA library obtained in this experiment was purified and electrophoresed in the same manner as in 3.1.3. In this experiment, the reproducibility of repeated data was evaluated by Spearman's rank correlation (ρ> 0.9).

3.2 Confirmation of reproducibility by MiSeq 3.2.1 Preparation of DNA library The final concentration of 60 μM random primer (10 base A), 0.2 mM dNTP mixture, 1.0 mM MgCl ₂ and 1.25 unit DNA Polymerase (TAKARA, PrimeSTAR) is added to the genomic DNA described in (NiF8-derived genomic DNA: 30 ng). The reaction solution was adjusted with 50 μl. PCR temperature cycle conditions are as follows: 98 ° C for 2 minutes, then 30 cycles of 98 ° C for 10 seconds, 50 ° C for 15 seconds and 72 ° C for 20 seconds, then store at 4 ° C Condition. The DNA library obtained in this experiment was purified and electrophoresed in the same manner as in 3.1.3.

3.2.2 Preparation of Sequence Library A sequence library for MiSeq analysis was prepared from the DNA library obtained in 3.2.1 using the KAPA Library Preparation Kit (Roche).

3.2.3 MiSeq analysis Using the MiSeq Reagent Kit V2 500 Cycle (Illumina), the sequence library for MiSeq analysis obtained in 3.2.2 was analyzed under a pair-end condition with a read length of 100 bases.

3.2.4 Read Data Analysis Random primer sequence information was deleted from the read data obtained in 3.2.3, and the lead pattern was specified. Then, the number of leads was counted for each lead pattern, the number of leads between repetitions was compared, and reproducibility was evaluated using a correlation coefficient.

3.3 Analysis of rice cultivar Nipponbare 3.3.1 Preparation of DNA library The final concentration of 60 μM random primer (10 base A), 0.2 mM dNTP mixture, 1.0 mM MgCl ₂ and 1.25 unit DNA Polymerase (TAKARA, PrimeSTAR) was added to the genomic DNA described in (Nipponbare-derived genomic DNA: 30 ng). The reaction solution was adjusted with 50 μl. PCR temperature cycle conditions are as follows: 98 ° C for 2 minutes, then 30 cycles of 98 ° C for 10 seconds, 50 ° C for 15 seconds and 72 ° C for 20 seconds, then store at 4 ° C Condition. The DNA library obtained in this experiment was purified and electrophoresed in the same manner as in 3.1.3.

3.3.2 Sequence library preparation, MiSeq analysis and read data analysis Sequence library preparation, MiSeq analysis and read data analysis using a DNA library prepared from Nipponbare-derived genomic DNA are described in 3.2.2, The methods described in 3.2.3 and 3.2.4 were followed.

3.3.3 Evaluation of genome uniformity The read pattern obtained in 3.3.2 is mapped to Nipponbare genome information (NC_008394 to NC_008405) with bowtie2, and the genome of each read pattern Identified the location.

3.3.4 Non-specific amplification Based on the position information of each lead pattern specified in 3.3.3, the sequence of the random primer is compared with the sequence on the genome where the random primer anneals, and the number of mismatches is counted. did.

3.4 Polymorphism detection and genotyping 3.4.1 Preparation of DNA library Genomic DNA (NiF8-derived genomic DNA, Ni9-derived genomic DNA, hybrid progeny-derived genomic DNA or Nipponbare-derived genomic DNA: 30 ng) with a final concentration of 60 μM random primer (10 base A), 0.2 mM dNTP mixture, 1.0 mM MgCl ₂ And 1.25 unit DNA Polymerase (TAKARA, PrimeSTAR) were added, and the reaction solution was adjusted with a final reaction volume of 50 μl. PCR temperature cycle conditions are as follows: 98 ° C for 2 minutes, then 30 cycles of 98 ° C for 10 seconds, 50 ° C for 15 seconds and 72 ° C for 20 seconds, then store at 4 ° C Condition. The DNA library obtained in this experiment was purified and electrophoresed in the same manner as in 3.1.3.

3.4.2 HiSeq analysis Each DNA library prepared in 3.4.1 was commissioned for Takara Biohe analysis under the pair-end condition of 16 samples per lane and 100 bases of read length, and read data was obtained from each. .

3.4.3 Read data analysis Random primer sequence information was deleted from the read data obtained in 3.4.2 to identify the lead pattern. The number of leads was counted for each lead pattern.

3.4.4 Polymorphism detection and genotype discrimination NiF8 and Ni9-specific polymorphisms were detected from the number of leads in the lead pattern obtained as a result of the analysis in 3.4.3, and the lead pattern was used as a marker. In addition, the genotypes of 22 progeny lines were determined based on the number of reads. The accuracy of genotyping was evaluated based on the reproducibility of repeated data of two progeny lines.

3.5 Confirmation experiment using PCR markers 3.5.1 Primer design Among the markers specified in 3.4.4, a total of 6 markers, NiF8 type 3 marker and Ni9 type 3 marker, are used to determine the primer from the marker sequence information of the paired ends. Each was designed (Table 22).

3.5.2 PCR and electrophoresis Using TaKaRa Multiplex PCR Assay Kit Ver.2 (TAKARA) Designed with 1.25 μl Multiplex PCR Enzyme Mix, 2 X Multiplex PCR Buffer 12.5 μl, 3.5.1 using the genomic DNA described in 1 (NiF8-derived genomic DNA, Ni9-derived genomic DNA or hybrid progeny-derived genomic DNA: 15 ng) as a template 0.4 μM primer was added, and the reaction solution was adjusted with a final reaction volume of 25 μl. PCR temperature cycle conditions were 94 ° C for 1 minute, then 30 cycles of 94 ° C for 30 seconds, 60 ° C for 30 seconds and 72 ° C for 30 seconds, then 72 ° C for 10 minutes. The condition was maintained at 4 ° C. after that. The amplified DNA fragment was electrophoresed on TapeStation (Agilent Technologies).

3.5.3 Genotype data comparison From the result of electrophoresis obtained in 3.5.2, the genotype of the marker was determined based on the presence or absence of a band, and compared with the number of reads of the marker.

3.6 Relationship between random primer concentration and length 3.6.1 Effect of random primer length under high concentration conditions To the genomic DNA described in (NiF8-derived genomic DNA: 30 ng), add a random primer of a predetermined length of 10 μM, 0.2 mM dNTP mixture, 1.0 mM MgCl ₂ and 1.25 unit DNA Polymerase (TAKARA, PrimeSTAR) The reaction solution was adjusted with a volume of 50 μl. In this experiment, the length of the random primer is 9 bases (Table 8), 10 bases (Table 1, 10 bases A), 11 bases (Table 9), 12 bases (Table 10), 14 bases. The length (Table 11), 16 base length (Table 12), 18 base length (Table 13) and 20 base length (Table 14) were examined. As for the PCR temperature cycle conditions, in a reaction system using a 9-base long random primer, first, 98 ° C was set to 2 minutes, and then 98 ° C for 10 seconds, 37 ° C for 15 seconds and 72 ° C for 20 seconds. After performing 30 cycles as a cycle, the conditions were to store at 4 ° C. As for PCR temperature cycle conditions, in a reaction system using a random primer of 10 bases or more in length, first, 98 ° C is set to 2 minutes, and then 98 ° C for 10 seconds, 50 ° C for 15 seconds and 72 ° C. After performing 30 cycles with 20 seconds as one cycle, the conditions were to store at 4 ° C. The DNA library obtained in this experiment was purified and electrophoresed in the same manner as in 3.1.3.

3.6.2 Relationship between random primer concentration and length To the genomic DNA described in (NiF8-derived genomic DNA: 30 ng), a random primer of a predetermined length is added to a predetermined concentration, and 0.2 mM dNTP mixture, 1.0 mM MgCl ₂ and 1.25 unit DNA Polymerase (TAKARA, PrimeSTAR ) Was added to prepare a reaction solution with a final reaction volume of 50 μl. In this experiment, a random primer having a length of 8 to 35 bases shown in Tables 1 to 21 was verified as the length of the random primer, and a range of 0.6 to 300 μM was verified as the concentration of the random primer.

As for PCR temperature cycle conditions, in the reaction system using 8 base length and 9 base length random primers, first set 98 ° C. for 2 minutes, then 98 ° C. for 10 seconds, 37 ° C. for 15 seconds and 72 ° C. After performing 30 cycles with 20 seconds as one cycle, the conditions were to store at 4 ° C. As for PCR temperature cycle conditions, in a reaction system using a random primer of 10 bases or more in length, first, 98 ° C is set to 2 minutes, and then 98 ° C for 10 seconds, 50 ° C for 15 seconds and 72 ° C. After performing 30 cycles with 20 seconds as one cycle, the conditions were to store at 4 ° C. The DNA library obtained in this experiment was purified and electrophoresed in the same manner as in 3.1.3. In addition, the reproducibility of repeated data was evaluated by Spearman's rank correlation (ρ> 0.9).

3.7 Number of random primers 1 type, 2 types, 3 types, 12 types, selected from 96 types of random primers (10 bases A) of 10 bases shown in Table 1 in the genomic DNA described in (NiF8-derived genomic DNA: 30 ng) 24 or 48 random primers were added to a final concentration of 60 μM, 0.2 mM dNTP mixture, 1.0 mM MgCl ₂ and 1.25 unit DNA Polymerase (TAKARA, PrimeSTAR) were added, and the reaction solution was adjusted with a final reaction volume of 50 μl. . In this experiment, random primers were selected in order from No. 1 in Table 1 as 1 type, 2 types, 3 types, 12 types, 24 types, or 48 types of random primers and verified. PCR temperature cycle conditions are as follows: 98 ° C for 2 minutes, then 30 cycles of 98 ° C for 10 seconds, 50 ° C for 15 seconds and 72 ° C for 20 seconds, then store at 4 ° C Condition. The DNA library obtained in this experiment was purified and electrophoresed in the same manner as in 3.1.3. In addition, the reproducibility of repeated data was evaluated by Spearman's rank correlation (ρ> 0.9).

3.8 Random primer sequence To the genomic DNA described in (NiF8-derived genomic DNA: 30 ng), 1 set selected from 5 sets of random primers shown in Tables 2 to 6 is added to a final concentration of 60 μM, 0.2 mM dNTP mixture, 1.0 mM MgCl ₂ and 1.25 unit DNA Polymerase (TAKARA, PrimeSTAR) were added, and the reaction solution was adjusted with a final reaction volume of 50 μl. PCR temperature cycle conditions are as follows: 98 ° C for 2 minutes, then 30 cycles of 98 ° C for 10 seconds, 50 ° C for 15 seconds and 72 ° C for 20 seconds, then store at 4 ° C Condition. Purification and electrophoresis of the DNA library obtained in this experiment
The same method as in 3.1.3 was performed. In addition, the reproducibility of repeated data was evaluated by Spearman's rank correlation (ρ> 0.9).

3.9 DNA library using human-derived genomic DNA To the genomic DNA described in (Human-derived genomic DNA: 30 ng), add a final primer of 60 μM random primer (10 base A), 0.2 mM dNTP mixture, 1.0 mM MgCl ₂ and 1.25 unit DNA Polymerase (TAKARA, PrimeSTAR) The reaction solution was adjusted with a reaction volume of 50 μl. PCR temperature cycle conditions are as follows: 98 ° C for 2 minutes, then 30 cycles of 98 ° C for 10 seconds, 50 ° C for 15 seconds and 72 ° C for 20 seconds, then store at 4 ° C Condition. The DNA library obtained in this experiment was purified and electrophoresed in the same manner as in 3.1.3. In addition, the reproducibility of repeated data was evaluated by Spearman's rank correlation (ρ> 0.9).

4). Results and Discussion 4.1 Relationship between PCR conditions and DNA library size In PCR using random primers (3.1.2 above) following normal PCR conditions, the size of the amplified DNA library is 2 kbp. As described above, it was a polymer, and no amplification was observed at the desired size of 100 bp to 500 bp (FIG. 2). The reason why a DNA library of 100 bp to 500 bp could not be obtained was thought to be because the probability that the Langem primer functions as a primer in a region of 500 bp or less is low. It was considered necessary to induce non-specific amplification with good reproducibility in order to produce a DNA library of the desired size of 100 bp to 500 bp.

Therefore, the annealing temperature (3.1.4 above), the amount of enzyme (3.1.5 above), the MgCl ₂ concentration (3.1.6 above), the primer length (3. 1.7) and the primer concentration (3.1.8 above) and the DNA library size were examined.

In the experiment described in 3.1.4 above, FIG. 3 shows the result when the annealing temperature is 45 ° C., FIG. 4 shows the result when the annealing temperature is 40 ° C., and the result when the annealing temperature is 37 ° C. Is shown in FIG. As shown in FIGS. 3 to 5, when the annealing temperature is lowered to 45 ° C., 40 ° C. and 37 ° C., the amount of amplification of the high molecular weight DNA library increases, but the amplification of the low molecular weight DNA library is not observed. It was.

In the experiment described in 3.1.5 above, FIG. 6 shows the result when the amount of enzyme is doubled, and FIG. 7 shows the result when the amount of enzyme is 10 times. As shown in FIGS. 6 and 7, even though the amount of the enzyme was increased to 2 times or 10 times the normal amount, the high molecular DNA library was increased, but the amplification of the DNA library was not observed in the low molecule.

In the experiments described above 3.1.6, Figure 9, MgCl ₂ concentration results when the FIG. 8, MgCl ₂ concentration results when the MgCl ₂ concentration and twice the normal and the normal triple FIG. 10 shows the result when the ratio is 4 times the normal value. As shown in FIGS. 8 to 10, although the amplification amount of the high molecular weight DNA library is changed even when the MgCl ₂ concentration is increased to 2 times, 3 times, or 4 times the normal concentration, amplification of the low molecular weight DNA library is observed. There wasn't.

In the experiment described in 3.1.7 above, when the random primer length is 8 base length, 9 base length, 11 base length, 12 base length, 14 base length, 16 base length, 18 base length and 20 base length The results are shown in FIGS. 11 to 18, respectively. As shown in FIGS. 11 to 18, no matter how long random primers were used, no significant change was observed compared to the results shown in FIG. 2 (10 base random random primers).

The results of the experiments described in 3.1.8 above are summarized in Table 23.

As shown in FIGS. 19 to 47, when a 10-base-long random primer is used, amplification is observed for a 1 kbp DNA fragment at a random primer concentration of 6 μM, and the DNA fragment decreases as the concentration increases. Became clear. In addition, as a result of examining reproducibility when the concentration of the random primer is 6 to 500 μM, ρ is slightly low at 0.889 at 6 μM, which is 10 times normal, but when it is 8 μM or more, which is equivalent to 13.3 times the normal, it is further 833.3 times In the case of corresponding 500 μM, ρ showed a high value of 0.9 or more. From this result, it was revealed that a DNA fragment of 1 kbp or less can be amplified while achieving high reproducibility by significantly increasing the concentration of the random primer from that under normal PCR conditions. However, when the concentration of the random primer exceeds 500μ and is too high, amplification of a DNA fragment of a desired size cannot be seen. Therefore, in order to amplify low-molecular-weight DNA fragments with excellent reproducibility, it has become clear that there is an optimum range in which the concentration of random primers is higher than that in normal PCR and is below a predetermined value. It was.

4.2 Confirmation of reproducibility by MiSeq As described in 3.2 above, in order to confirm the reproducibility of DNA library production, a DNA library amplified with random primers using genomic DNA extracted from NiF8 as a template. The result of analysis by the next-generation sequencer MiSeq is shown in FIG. As a result of the above 3.2.4, 47,484 lead patterns were obtained. As a result of comparing the number of reads between the repetitions, the correlation coefficient r = 0.991 was shown to be highly correlated between the repetitions as in the electrophoresis results. From the above results, it was considered that a DNA library can be produced with good reproducibility using random primers.

4.3 Analysis of rice cultivar Nipponbare As described in 3.3 above, the result of electrophoresis using a genomic DNA extracted from rice Nipponbare, whose genome information has been made public, as a template and random primers These are shown in FIGS. 49 and 50. From the results shown in FIGS. 49 and 50, ρ showed a very high value of 0.979. Moreover, the result of analyzing the read data by MiSeq is shown in FIG. From the results shown in FIG. 51, the correlation coefficient r was as high as 0.992. From these results, it became clear that a DNA library can be prepared with very high reproducibility by using random primers even in rice.

In addition, as described in 3.3.3 above, the resulting read pattern was mapped to the Nipponbare Genome Information, and as a result, DNA fragments were amplified evenly and evenly in the genome at a rate of 1 at 6.2 kbp. (Fig. 52). In addition, as a result of comparing the sequence of the random primer and the genome information, there was an average of 3.6 mismatches, and one or more mismatches occurred in 99.0% of the primer pairs (FIG. 53). From the above results, it was revealed that a DNA library using random primers was prepared by nonspecific amplification uniformly and reproducibly over the entire genome.

4.4 Sugarcane polymorphism detection and genotyping As described in 3.4 above, a DNA library was prepared with random primers using sugarcane NiF8, Ni9, and 22 progenies of the hybrid, and the next-generation sequencer HiSeq Based on the read data, polymorphism detection of parents and genotypes of progeny were determined. The results are shown in Table 24.

As shown in Table 24, NiF8 type was able to produce 8,683 markers, Ni9 type was 11,655 markers, a total of 20,338 markers. In addition, the reproducibility of genotyping in the progeny of the hybrid was as high as 99.97%, and the genotyping accuracy was considered to be extremely high. In particular, sugarcane is polyploid (8x + n), has an extremely large number of chromosomes of 100 to 130, and has a genome size of 10 Gbp, which is three times more than humans. Therefore, the current situation is that genotyping is extremely difficult over the entire genomic DNA. As described above, a very large number of markers can be prepared by using random primers, and sugarcane can be genotyped with high accuracy.

4.5 Confirmation Experiment with PCR Marker As described in 3.5 above, PCR was performed on the NiF8 and Ni9, and 22 progenies of the hybrids using the primers shown in Table 22, and the genotype was determined by electrophoresis. Compared with the number. The number of leads and the electrophoretic image of the NiF8 type marker N80521152 are shown in FIGS. 54 and 55, respectively. The number of leads and electrophoretic image of the NiF8 type marker N80997192 are shown in FIGS. 56 and 57, respectively. The number of leads and electrophoretic images of the NiF8 type marker N80533142 are shown in FIGS. 58 and 59, respectively. The number of leads and electrophoretic image of Ni9 type marker N91552391 are shown in FIGS. 60 and 61, respectively. The number of leads and electrophoretic image of Ni9 type marker N91653962 are shown in FIGS. 62 and 63, respectively. The number of leads and electrophoretic image of the Ni9 type marker N91124801 are shown in FIGS. 64 and 65, respectively.

As shown in Figs. 54 to 65, the results of all PCR markers designed in 3.5 above were consistent with the analysis results of the next-generation sequencer, so genotyping using the next-generation sequencer was used as a marker technology. It was considered possible.

4.6 Relationship between Random Primer Concentration and Length As explained in 3.6.1 above, 9 base length (Table 8), 10 base length (Table 1, 10 base A), 11 base length (Table 9) DNA library using random primers of 12 base length (Table 10), 14 base length (Table 11), 16 base length (Table 12), 18 base length (Table 13) and 20 base length (Table 14) The fabricated results are shown in FIGS. Table 25 summarizes these results.

As shown in FIGS. 66 to 81, when a high concentration random primer of 10.OμM corresponding to the normal 13.3 times is used, in the range of 9 base length to 20 base length, while achieving very high reproducibility, It was revealed that small DNA fragments can be amplified. In particular, the longer the base length of the random primer (especially 12 bases or more), the tendency of the amplified fragment to decrease in molecular weight was observed. When a 9-base long random primer was used, the amplification amount of the DNA fragment could be increased by setting the annealing temperature to 37 ° C.

Also, as explained in 3.6.2 above, in order to clarify the relationship between the length and the length of the random primer, PCR was performed with the random primer in the range of 8 to 35 bases in length and the random primer concentration in the range of 0.6 to 300 μM. And tried to create a DNA library. The results are shown in Table 26.

As shown in Table 26, DNA fragments of small molecules (100 to 500 bases) can be reproduced with high reproducibility by setting the length of random primers to 9 to 30 bases and the concentration of random primers to 4.0 to 200 μM. It became clear that it could be amplified. In particular, when the length of the random primer is 9 to 30 bases and the concentration of the random primer is 4.0 to 100 μM, it is possible to reliably amplify a low molecular weight DNA fragment (100 to 500 bases) with high reproducibility. It became clear.

Further, when the results shown in Table 26 are examined in more detail, it is found that the length and concentration of the random primer are preferably adjusted within a region surrounded by a frame as shown in FIG. More specifically, the concentration of the random primer is preferably 40 to 60 μM when the random primer is 9 to 10 bases long. The concentration of the random primer satisfies y> 3E + 08x ^-6.974 and 100 μM or less when the random primer base length is y and the random primer concentration is x when the random primer has a length of 10 to 14 bases. It is preferable. The concentration of the random primer is preferably 4 to 100 mM when the random primer has a length of 14 to 18 bases. The concentration of the random primer is preferably 4 μM or more when the random primer has a length of 18 to 28 bases, and preferably satisfies y <8E + 08x− ^5.533 . The concentration of the random primer is preferably 4 to 10 μM when the random primer has a length of 28 to 29 bases. These y> 3E + 08x− ^6.974 and y <8E + 08x− ^5.533 are equations calculated based on the power approximation of Microsoft Excel.

As described above, it was revealed that a low molecular weight DNA fragment (100 to 500 bases) can be amplified with high reproducibility by defining the base length and concentration of the random primer within a predetermined range. For example, in the next-generation sequencer, it is known that the data accuracy is remarkably lowered when a polymer DNA fragment is analyzed. As shown in this example, by defining the base length and concentration of random primers within a predetermined range, it is possible to create a DNA library with a molecular size suitable for next-generation sequencer analysis with good reproducibility. It can be said that it is suitable for next-generation sequencer marker analysis.

4.7 Number of Random Primers As explained in 3.7 above, a DNA library was prepared using 1, 2, 3, 12, 24, or 48 random primers (concentration: 60 μM). The results are shown in FIGS. Table 27 summarizes these results.

As shown in FIGS. 83 to 94, a low molecular weight DNA fragment can be obtained while achieving very high reproducibility in any of 1, 2, 3, 12, 24 or 48 random primers. It became clear that it could be amplified. In particular, it can be seen that as the type of random primer increases, the peak of the electrophoretic image becomes smaller and the bias tends to be eliminated.

4.8 Random Primer Sequence As explained in 3.8 above, each of the random primer sets (10 base B, 10 base C, 10 base D, 10 base E and 10 base F) shown in Tables 2 to 6 is used. The results of preparing a DNA library using these are shown in FIGS. Table 28 summarizes these results.

As shown in FIGS. 95 to 104, when using any set of 10 base B, 10 base C, 10 base D, 10 base E, and 10 base F, the low molecular weight is achieved while achieving very high reproducibility. It became clear that DNA fragments could be amplified.

4.9 Human DNA library preparation As described in 3.9 above, the results of preparing a DNA library using human-derived genomic DNA and a random primer (10 base A) at a final concentration of 60 μM are shown in FIGS. Indicated. FIG. 105 shows the results of the first iteration and FIG. 106 shows the results of the second iteration. As shown in FIGS. 105 and 106, it was revealed that even when human-derived genomic DNA was used, a low-molecular-weight DNA fragment could be amplified while achieving very high reproducibility.

[Example 2]
1. Flowchart In this example, according to the schematic diagram shown in FIGS. 107 and 108, a first DNA fragment was prepared by PCR using genomic DNA as a template and random primers, and then the prepared first DNA fragment was A second DNA fragment is prepared by PCR using a next-generation sequencer primer as a template, and a sequence analysis using a so-called next-generation sequencer is performed using the prepared second DNA fragment as a sequencer library. Genotypes were analyzed based on the read data obtained.

2. Materials In this example, genomic DNA was extracted and purified from sugarcane variety NiF8 and rice variety Nipponbare using DNeasy Plant Mini kit (QIAGEN), and used as genomic DNA derived from NiF8 and genomic DNA derived from Nihonbare, respectively.

3. Method 3.1 Study with Sugarcane NiF8 3.1.1 Design of Random Primer and Primer for Next Generation Sequencer In this example, random primer is 10 bases at the 3 ′ end in Nextera adapter for Illumina next generation sequencer. Designed based on That is, in this example, GTTACACACG (SEQ ID NO: 2041, 10 base G) was used as a random primer. Similarly, primers for next-generation sequencers were designed based on the sequence information of Illumina's Nextera adaptor (Table 29).

3.1.2 Preparation of DNA library Add final concentration of 0.2 mM dNTP mixture, 1.0 mM MgCl ₂ and 1.25 unit DNA Polymerase (TAKARA, PrimeSTAR) to 60 μM random primer (10 base G) to the genomic DNA (30 ng) derived from NiF8 as described in 4. DNA library (first DNA fragment) by PCR (98 ° C for 2 minutes, 98 ° C for 10 seconds, 50 ° C for 15 seconds, 72 ° C for 20 seconds for 30 cycles, and stored at 4 ° C) in 50 µl volume ) Was produced.

3.1.3 Purification and Electrophoresis After purifying the DNA library of 3.1.2 with the MinElute PCR Purification Kit (QIAGEN), it was electrophoresed with the Agilent 2100 Bioanalyzer (Agilent Technologies), and the fluorescence unit (Fluorescence Unit: FU) ) In addition, the reproducibility of repeated data was evaluated by Spearman's rank correlation (ρ> 0.9).

3.1.4 Preparation of DNA library for next-generation sequencer 3. Final concentration 0.2 mM dNTP mixture, 1.0 mM MgCl ₂ and 1.25 unit DNA Polymerase (100 ng) were added to the first DNA fragment (100 ng) purified in 3.1.3. Next, add 0.5μM primer for next generation sequencer to PCR (95 ℃ for 2 minutes, 98 ℃ for 15 seconds, 55 ℃ for 15 seconds, 72 ℃ for 20 seconds) After the 25-cycle reaction, a DNA library (second DNA fragment) for the next-generation sequencer was prepared by 72 minutes at 72 ° C and then stored at 4 ° C. Purification and electrophoresis of the DNA library for the next-generation sequencer was performed in the same manner as in 3.1.3.

3.1.5 MiSeq analysis 3. Use the MiSeq Reagent Kit V2 500 Cycle (Illumina) for the next-generation sequencer DNA library (1.4) as described in 3.1.4. And analyzed with MiSeq.

3.1.6 Read data analysis A lead pattern was identified from the read data of 3.1.5. Then, the number of leads was counted for each lead pattern, the number of leads between repetitions was compared, and reproducibility was evaluated using a correlation coefficient.

3.2 Examination in Rice Variety Nipponbare 3.2.1 Design of Random Primer and Next Generation Sequencer Primer In this example, the random primer is 10 bases at the 3 ′ end of Nextera adapter for Illumina next generation sequencer. Designed based on That is, in this example, as a random primer, 16 types of base sequences consisting of 10 bases located at the 3 ′ end of the Nextera adapter and 12 bases in total with an arbitrary sequence of 2 bases added to the 3 ′ end of the 10 bases. (Table 30, 12 bases B).

In this example, next-generation sequencer primers designed based on the sequence information of Illumina Nextera adapters as in 3.1.1 were used.

3.2.2 Preparation of DNA library Add the final concentration of 0.2 mM dNTP mixture, 1.0 mM MgCl ₂ and 1.25 unit DNA Polymerase (TAKARA, PrimeSTAR) to the genomic DNA (30 ng) derived from Nipponbare explained in 4. DNA library (first DNA fragment) by PCR (98 ° C for 2 minutes, 98 ° C for 10 seconds, 50 ° C for 15 seconds, 72 ° C for 20 seconds for 30 cycles, and stored at 4 ° C) in 50 µl volume ) Was produced.

3.2.3 Purification and electrophoresis After purifying the DNA library of 3.2.2 using the MinElute PCR Purification Kit (QIAGEN), electrophoresis is performed using the Agilent 2100 Bioanalyzer (Agilent Technologies) and the fluorescence unit (Fluorescence Unit: FU). Got. In addition, the reproducibility of repeated data was evaluated by Spearman's rank correlation (ρ> 0.9).

3.2.4 Preparation of DNA library for next-generation sequencer 3. Final concentration 0.2 mM dNTP mixture, 1.0 mM MgCl ₂ and 1.25 unit DNA Polymerase (TAKARA) were added to the first DNA fragment (100 ng) purified in 3.2.3. , PrimeSTAR) with 0.5 μM next-generation sequencer primers, and PCR with a final reaction volume of 50 μl (95 ° C for 2 minutes, 98 ° C for 15 seconds, 55 ° C for 15 seconds, 72 ° C for 20 seconds 25 After the cycle reaction, a DNA library (second DNA fragment) for the next-generation sequencer was prepared by storing at 72 ° C. for 1 minute and then storing at 4 ° C.). Purification and electrophoresis of the DNA library for the next-generation sequencer was performed in the same manner as in 3.1.3.

3.2.5 MiSeq analysis 3. Using the MiSeq Reagent Kit V2 500 Cycle (Illumina) for the DNA library (second DNA fragment) for the next-generation sequencer in 2.4. Analyzed with MiSeq.

3.2.6 Read data analysis The lead pattern of 3.2.5 was mapped to Nipponbare genome information (NC_008394 to NC_008405) with bowtie2, and the coincidence rate between the random primer sequence and the genomic DNA was confirmed. In addition, a lead pattern was identified from the lead data of 3.2.5, the number of leads was counted for each lead pattern, the number of leads between repetitions was compared, and reproducibility was evaluated using a correlation coefficient.

4). Results and Discussion 4.1 Results of Sugarcane NiF8 PCR Using a random primer (10 base G) consisting of 10 bases at the 3 'end in Nextera adaptor for next-generation sequencers from Illumina, PCR under high concentration conditions of 60 μl The results of electrophoresis at this time are shown in FIGS. As shown in FIGS. 109 and 110, amplification was observed in a wide region including 100 bp to 500 bp (first DNA fragment). The reason why the amplification could be confirmed in a wide region was thought to be because the amplification was also observed in a region other than the genomic DNA region corresponding to the random primer. Moreover, since the rank correlation coefficient between repeated data was 0.957 and 0.9 or more, high reproducibility was recognized in the amplification pattern.

Next, as described in 3.1.4, the results of electrophoresis when PCR was performed using the next-generation sequencer primers are shown in FIGS. In other words, in order to create a DNA library (second DNA fragment) linked to the next-generation sequencer adapter Nextera adaptor, a primer for the next-generation sequencer consisting of Illumina's Nextera adaptor sequence using the first DNA fragment as a template. PCR was performed. Illumina's next-generation sequencers have extremely low analysis accuracy if the DNA library contains many short fragments of 100 bp or less and long fragments of 1 kbp or more. As shown in FIGS. 111 and 112, the DNA library for the next-generation sequencer prepared in this example (second DNA fragment) was distributed mainly in the range of 150 bp to 1 kbp with a peak at around 500 bp. Therefore, it was considered suitable as a DNA library for next-generation sequencers. Moreover, since the rank correlation coefficient between repeated data was 0.989 and 0.9 or more, high reproducibility was recognized in the amplification pattern.

Also, as a result of analyzing the obtained DNA library (second DNA fragment) with the next-generation sequencer MiSeq, read data of 3.5 Gbp and 3.6 Gbp were obtained. Further, the values of> 30 indicating the data accuracy of MiSeq were 93.3% and 93.1%. The manufacturer recommended values were lead data 3.0 Gbp or more and> = Q30 85.0% or more, so the next-generation sequencer DNA library (second DNA fragment) prepared in this example was used for next-generation sequencer analysis. It was considered possible. In order to confirm the reproducibility, the number of reads between the repetitions of 34,613 lead patterns obtained by MiSeq was compared. The results are shown in FIG. As shown in FIG. 113, the number of reads showed a high correlation with r = 0.996 between the repeats as in the electrophoresis results.

As described above, a DNA library (first DNA fragment) was obtained by PCR using a high concentration of random primers consisting of 10 bases at the 3 ′ end of Nextera Adaptor adapter for Illumina next-generation sequencers. After that, the next-generation sequencer DNA library (second DNA fragment) consisting of many fragments can be easily and reproducibly by PCR using the Nextera-Adaptor sequence next-generation sequencer primer. did it.

4.2 Examination Results at Rice Nipponbare Total length 12 of Illumina's Nextera adapter Nextera adapter, 10 bases located at the 3 'end, and an arbitrary sequence of 2 bases added to the 3' end of the 10 bases FIGS. 114 and 115 show the results of electrophoresis when PCR was carried out under high concentration conditions of 40 μl using 16 kinds of random primers (12 base B) consisting of bases. As shown in FIGS. 114 and 115, amplification was observed in a wide region including 100 bp to 500 bp (first DNA fragment). The reason why amplification in a wide region could be confirmed was considered to be because amplification was performed in a region other than the genomic DNA region corresponding to the random primer as in 4.1. Also in this example, since the rank correlation coefficients were 0.950 and 0.9 or more, high reproducibility was recognized in the amplification pattern.

Next, as described in 3.2.4, the results of electrophoresis when PCR was performed using primers for the next-generation sequencer are shown in FIGS. 116 and 117. In other words, in order to create a DNA library (second DNA fragment) linked to the next-generation sequencer adapter Nextera adaptor, a primer for the next-generation sequencer consisting of Illumina's Nextera adaptor sequence using the first DNA fragment as a template. PCR was performed. As a result, the DNA library for the next-generation sequencer (second DNA fragment) prepared in this example was distributed mainly in the range of 150 bp to 1 kbp with a peak around 300 bp as shown in FIGS. 116 and 117. Therefore, it was considered suitable for a DNA library for next-generation sequencers. Moreover, since the rank correlation coefficient between repeated data was 0.992 and 0.9 or more, high reproducibility was recognized in the amplification pattern.

Moreover, as a result of analyzing the obtained DNA library (second DNA fragment) with the next-generation sequencer MiSeq, read data of 4.0 Gbp and 3.8 Gbp were obtained. Moreover, the values of> 30 indicating the data accuracy of MiSeq were 94.0% and 95.3%. From this result, it was considered that the DNA library (second DNA fragment) of the next-generation sequencer prepared in this example can be used for the analysis of the next-generation sequencer, as in 4.1.1. FIG. 118 shows the result of comparing the random primer sequence and the Nipponbare reference sequence for evaluating the matching rate between the random primer sequence and the genome for 19,849 read patterns obtained by MiSeq. As shown in FIG. 118, the average coincidence ratio between the random primer sequence and the Nipponbare reference sequence was 34.5%. In particular, since there was no lead pattern that completely matched the random primer sequence and the Nipponbare reference sequence, it was considered that the random primer was bound to the sequence that did not match the random primer and amplified. This was considered to be consistent with the bioanalyzer results. In order to confirm the reproducibility of the lead pattern, the number of leads was compared between the repetitions. The results are shown in FIG. As shown in FIG. 119, similar to the results of electrophoresis, r 反復 = 0.999 was highly correlated between the repetitions.

As explained above, from the next 12 bases in total of 10 bases located at the 3 ′ end in the Nextera Adaptor for Illumina's next-generation sequencer, and any 2 base sequences added to the 3 ′ end of the 10 bases After obtaining a DNA library (first DNA fragment) by PCR using 16 kinds of random primers at high concentrations, PCR using primers comprising the sequence of Nextera Adaptor was performed easily and with good reproducibility. A DNA library (second DNA fragment) for next-generation sequencers consisting of fragments could be produced.

Example 3
1. Materials and Methods 1.1 Materials In this example, genomic DNA was extracted and purified from rice cultivar Nipponbare using DNeasy Plant Mini kit (QIAGEN) and used as genomic DNA derived from Nipponbare.

1.2 Preparation of DNA library Genomic DNA described in 1.1 (Nipponbare-derived genomic DNA: 30 ng), final concentration 60 μM random primer (10 base A), 0.2 mM dNTP mixture, 1.0 mM MgCl ₂ and 1.25 unit DNA Polymerase (TAKARA, PrimeSTAR) was added, and the reaction solution was adjusted with a final reaction volume of 50 μl. PCR temperature cycle conditions are as follows: 98 ° C for 2 minutes, then 30 cycles of 98 ° C for 10 seconds, 50 ° C for 15 seconds and 72 ° C for 20 seconds, then store at 4 ° C Condition. The DNA library obtained in this experiment was purified with the MinElute PCR Purification Kit (QIAGEN).

1.3 Preparation of Sequence Library A sequence library for MiSeq analysis was prepared from the DNA library obtained in 1.2 using the KAPA Library Preparation Kit (Roche).

1.4 MiSeq Analysis Using the MiSeq Reagent Kit V2 500 Cycle (Illumina), the MiSeq analysis sequence library obtained in 1.3 was analyzed under a pair-end condition with a read length of 100 bases.

1.5 Analysis of Base Sequence Information Random primer sequence information was deleted from the read data obtained in 1.4, and base sequence information of each lead was specified. The nucleotide sequence information of each read was mapped to rice genome information (kasalath_genome) with bowtie2, and single nucleotide polymorphism (SNP) and insertion deletion mutation (inDel) were identified as markers for each chromosome.

2. Results and Discussion Table 31 shows the results of mapping the nucleotide sequence information of the DNA library prepared from the genomic DNA derived from rice Nipponbare using random primers to the genome information of rice casalas.

As shown in Table 31, 2,694 to 5,579 SNPs (average 3,812.6, 45,751 in total) could be identified in each chromosome. Further, as shown in Table 31, 227-569 (average 349.3, total 4,191) InDel (insertion / deletion) could be identified in each chromosome. From the above results, as shown in the present example, by comparing the base sequence information of a DNA library prepared with random primers with the known base sequence information, the characteristics present in the genome of the tested organism It became clear that a DNA marker as a basic nucleotide sequence can be specified.

All publications, patents and patent applications cited in this specification shall be incorporated into the present specification as they are.

Claims (23)

A method for preparing a DNA library, in which a nucleic acid amplification reaction is performed in a reaction solution containing genomic DNA and a high concentration of random primers, and a DNA fragment obtained by the nucleic acid amplification reaction is obtained using genomic DNA as a template. 2. The method for producing a DNA library according to claim 1, wherein the reaction solution contains 4 to 200 μM of the random primer. The method for producing a DNA library according to claim 1, wherein the reaction solution contains 4 to 100 µM of the random primer. The method for preparing a DNA library according to claim 1, wherein the random primer is a nucleotide having a length of 9 to 30 bases. The method for preparing a DNA library according to claim 1, wherein the DNA fragment has a length of 100 to 500 bases. A genomic DNA analysis method using the DNA library prepared by the DNA library preparation method according to any one of claims 1 to 5 as a DNA marker. Claims including the step of determining the base sequence of a DNA library prepared by the method for preparing a DNA library according to any one of claims 1 to 5 and confirming the presence or absence of the DNA marker based on the base sequence Item 7. The genomic DNA analysis method according to Item 6. The genomic DNA analysis method according to claim 7, wherein in the step of confirming the presence or absence of the DNA marker, the presence or absence of the DNA marker is confirmed from the number of reads in the base sequence of the DNA library. Compare the base sequence of the above DNA library with the base sequence of the above DNA library prepared using known sequence information or genomic DNA derived from other organisms or other tissues, and based on the difference in base sequence, a DNA marker The genomic DNA analysis method according to claim 7, wherein the presence or absence of the DNA is confirmed. A step of preparing a pair of primers that specifically amplify the DNA marker based on the base sequence of the DNA marker, and a nucleic acid amplification reaction using the pair of primers with the genomic DNA extracted from the target organism as a template The genomic DNA analysis method according to claim 6, further comprising a step of confirming the presence or absence of the DNA marker in the genomic DNA from the result of the nucleic acid amplification reaction. Performing a nucleic acid amplification reaction in a first reaction solution containing genomic DNA and a high concentration of random primers, obtaining a first DNA fragment obtained by the nucleic acid amplification reaction using genomic DNA as a template;
Nucleic acid amplification in the second reaction solution containing, as a primer, the first DNA fragment obtained and a nucleotide sequence containing at least 3% of the nucleotide sequence at the 5 'end of the random primer at the 3' end Carrying out a reaction to obtain a second DNA fragment in which the nucleotide is linked to the first DNA fragment. The method for producing a DNA library according to claim 11, wherein the first reaction solution contains 4 to 200 µM of the random primer. The method for preparing a DNA library according to claim 11, wherein the first reaction solution contains 4 to 100 µM of the random primer. The method for preparing a DNA library according to claim 11, wherein the random primer is a nucleotide having a length of 9 to 30 bases. The method for preparing a DNA library according to claim 11, wherein the first DNA fragment has a length of 100 to 500 bases. The primer for amplifying the second DNA fragment includes a region used for the base sequence determination reaction, or the primer used for the nucleic acid amplification reaction using the second DNA fragment as a template or a repeated nucleic acid amplification reaction is a base. The method for producing a DNA library according to claim 11, comprising a region used for a sequencing reaction. The second DNA fragment obtained by the method for producing a DNA library according to any one of claims 11 to 15, or a primer for a sequencer used for a base sequence determination reaction by the method for producing a DNA library according to claim 16. A method for analyzing a DNA library, comprising a step of determining a base sequence of a DNA fragment obtained using a primer containing a complementary region to. A genomic DNA analysis method using the DNA library prepared by the DNA library preparation method according to any one of claims 11 to 17 as a DNA marker. A process comprising: determining a base sequence of a DNA library produced by the method for producing a DNA library according to any one of claims 11 to 17, and confirming the presence or absence of the DNA marker based on the base sequence. Item 19. The genomic DNA analysis method according to Item 18. The genomic DNA analysis method according to claim 19, wherein in the step of confirming the presence or absence of the DNA marker, the presence or absence of the DNA marker is confirmed from the number of reads in the base sequence of the DNA library. Compare the base sequence of the above DNA library with the base sequence of the above DNA library prepared using known sequence information or genomic DNA derived from other organisms or other tissues, and based on the difference in base sequence, a DNA marker The genomic DNA analysis method according to claim 19, wherein the presence or absence of the DNA is confirmed. A step of preparing a pair of primers that specifically amplify the DNA marker based on the base sequence of the DNA marker, and a nucleic acid amplification reaction using the pair of primers with the genomic DNA extracted from the target organism as a template The genomic DNA analysis method according to claim 18, further comprising a step of confirming the presence or absence of the DNA marker in the genomic DNA from the result of the nucleic acid amplification reaction. A DNA library produced by the method for producing a DNA library according to any one of claims 1 to 5 and claims 11 to 16.

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