WO2006059386A1

WO2006059386A1 - Method of isolating double-stranded dna containing simple sequence repeats

Info

Publication number: WO2006059386A1
Application number: PCT/JP2004/017938
Authority: WO
Inventors: Takashi Matsumoto; Yuichi Katayose; Jianzhong Wu; Hiroko Yamane; Nobukazu Namiki; Hiroyuki Kanamori
Original assignee: National Institute Of Agrobiological Sciences; Society For Technoinnovation Of Agriculture, Forestry And Fisheries
Priority date: 2004-12-02
Filing date: 2004-12-02
Publication date: 2006-06-08
Also published as: JPWO2006059386A1

Abstract

It is intended to provide a method for isolating a DNA containing simple sequence repeats (SSRs). Namely, a method of isolating a double-stranded DNA containing SSRs which comprises (1) the step of incubating a double-stranded DNA plasmid library together with a probe containing the simple sequence repeats to be isolated in the presence of a homologous recombinant protein and (2) the step of isolating a plasmid hybridized with the probe. A kit and a probe which are useful in this method and a method of producing the probe are also provided. This probe is composed of a region comprising the simple sequence repeats and base sequence(s)other than the simple sequence repeats which are attached to one of the 5’-side and the 3’-side thereof or both of these sides. This probe may be modified in the region attached to the 3’-side with a bonding ligand such as biotin.

Description

Specification

Method for isolating double-stranded DNA containing simple repetitive sequences

Technical field

[0001] The present invention relates to a method for isolating double-stranded DNA containing a repetitive sequence.

Background art

[0002] Simple Sequence Repeats (SSRs) are short! Base sequences that are simple sequences repeated twice or more. The base sequence constituting the simple repeat sequence is composed of a relatively short base sequence repeat such as gagagaga .... A repeat sequence of 2 base units as in this example is called a tandem repeat. Having a repeat sequence in the genome is a common feature of eukaryotes. It has been clarified that DNA containing simple repetitive sequences includes a family called satellite DNA, minisatellite DNA, and microsatellite DNA.

[0003] When genomic DNA is centrifuged in a salt gradient of cesium chloride, satellite DNA is separated into bands (satellite) that correspond to the average G + C content of the genomic DNA. It is a repetitive sequence identified as DNA. On the chromosome, it has been revealed that it exists mainly in the centrum or telomere region. The base sequence composing satellite DNA is tandem repeat. The base sequence of satellite DNA has been confirmed to be highly diverse not only among species, but also within the same species. Satellite DNA is useful as a marker for confirming genetic differences between cultivars.

[0004] Tandem repeat sequences are scattered throughout the entire genome only in specific regions such as centromeres and telomeres. Power mini-satellite DNA identified as one of the SSRs scattered throughout the genome. Minisatellite DNA is used for DNA fingerprinting because it shows polymorphism within species. For example, minisatellite DNA is used in the forensic field as an indicator for identifying individuals and identifying parent-child relationships. In animals and plants, minisatellite DNA can also be used to identify varieties.

[0005] Similar to minisatellite DNA, microsatellite DNA is DNA composed of SSRs found in the entire genome. In mammals, (dA.dT) n repeat sequences are common. Hi (DCA.dTG) n or (dCT.dAG) n repeats are present at 0.5% and 0.2% of the genome, respectively. There are microsatellite DNAs with repeating unit strengths of 3 bases or more, but the frequency is usually not high. In fact, the existence of microsatellite sequences composed of repetitive sequences of about 2-5 bases has been clarified.

[0006] Since microsatellite DNA is widely distributed throughout the genome, it is particularly useful as a genetic marker. For example, if a microsatellite DNA having a genetic association with a specific trait can be identified, a gene associated with the specific trait (or a transcriptional regulatory region) exists near the microsatellite DNA. Likely to be. In order to proceed with such genetic analysis, it is necessary to collect information on a large number of microsatellite DNA and cover the entire genome as densely as possible.

[0007] Various repetitive sequences can be basically isolated by the following procedure. First, a genomic library is prepared by randomly cutting the genome. Hybridize a probe containing a repetitive sequence to the fragment sequence constituting the library, and isolate a positive clone. By determining the base sequence of the isolated positive clone, the base sequence of the repetitive sequence can be determined (Karagyozov et al, Nucleic Acid Research, 1993, Vol. 21, No. 16 3911-3912; Zane et al, Mol Ecol. 2002 Jan; ll (l): l-16). In practice, however, various constraints have prevented efficient acquisition of repetitive sequences.

[0008] One of the limitations is the use of the PCR method. For example, when a fragmented genomic library is screened with a probe containing a repetitive sequence, the hybridized genomic fragment is amplified and cloned by the PCR method (Figure 2). At this time, it is difficult to amplify all the genomic fragments evenly with the current technology. For example, as a result of a relatively short base sequence being preferentially amplified, it is likely that the repetitive sequences to be cloned are biased toward short ones. That is, information on longer repetitive sequences may be lost.

Non-Patent Document 1: Karagyozov et al, Nucleic Acid Research, 1993, Vol.21, No.16 3911— 3912, Construction of random small-insert genomic libraries highly enriched for simple sesquence repeats

Non-Patent Document 2: Zane et al, Mol Ecol. 2002 Jan; ll (l): l— 16., Strategies for microsatellite isolation: a review. Disclosure of the invention

Problems to be solved by the invention

[0009] An object of the present invention is to provide a technique for isolating DNA containing SSRs.

Means for solving the problem

[0010] DNA containing SSRs is isolated mainly by hybridization with a probe and cloning of the hybridized genomic fragment. In these steps, if repetitive sequences can be isolated without bias, efficient repetitive sequence isolation can be realized as a result. Specifically, DNA containing SSRs needs to be comprehensively screened at the stage of hybridization with the probe. In other words, it is necessary to provide conditions that can sufficiently hybridize to the target genome fragment under the stringency given the probe power used for screening. In addition, as described above, for example, when PCR was applied, it was predicted that there might be a bias in the repetitive sequences isolated in the course of the reaction. Therefore, for example, it would be useful if a method capable of isolating the target DNA without depending on the PCR method could be provided.

[0011] In order to satisfy these conditions, the present inventors have found that positive clones can be efficiently isolated by directly screening plasmid vectors carrying genomic fragments, and completed the present invention. did. Furthermore, the present inventors have repeatedly studied the design of probes for screening. As a result, the present invention was completed by clarifying that a probe having a specific structure is useful in isolation of DNA containing SSRs. That is, the present invention relates to a method for isolating DNA containing the following SSRs, a kit therefor, a probe for isolating DNA containing SSRs, and a method for producing the same.

[1] A method for isolating double-stranded DNA containing a simple repetitive sequence, comprising the following steps.

(1) incubating a double-stranded DNA plasmid library with a probe containing a simple repetitive sequence to be isolated in the presence of a homologous recombinant protein;

(2) Isolating the plasmid hybridized with the probe

[2] The method according to [1], wherein the plasmid library is a plasmid library containing genomic fragments.

[3] The method according to [2], wherein the step of isolating the plasmid includes the following steps (2a) to (2c). (2a) recovering the plasmid hybridized with the probe,

(2b) transforming the recovered plasmid into a host cell into which the plasmid can be introduced, and

(2c) recovering the amplified plasmid from the host cell

[4] The method according to [1], wherein the probe comprises the following regions (a) and (b) force.

(a) a region consisting of simple repeat sequences, and

(b) Region located on both the 3 'side and / or 5' side of region (a), and consisting of base sequences other than simple repeat sequences

[5] The method according to [4], wherein the base force of the region (b) a, t, c, g, and u force are selected.

[6] The method according to [4], wherein the probe has a binding ligand in the region (b).

[7] The method according to [6], comprising a step of capturing the plasmid on a solid phase that binds to the binding ligand before or after incubating the plasmid and the probe.

[8] The method according to [4], wherein the length of region (a) is 25-100 bases.

[9] The method according to [4], wherein the length of region (b) is 30 to 60 bases.

[10] The method according to [4], wherein the probe includes a plurality of kinds of probes having different numbers of repeats of the simple repeat sequence constituting the region (a).

[11] Homologous recombination protein strength The method according to [1], wherein the group strength that also has RecA protein, Rad51 protein, and Rad52 protein strength is selected.

[12] A kit for the isolation of simple repetitive sequences, comprising the following elements:

A zero-order region (a) and (b) force, and a probe having a binding ligand in region (b),

(a) a region consisting of simple repeat sequences, and

(b) a region that is located on the 3 ′ side and / or the 5 ′ side of region (a) and is composed of a base sequence other than a simple repetitive sequence,

ii) homologous thread and reversible protein, and

iii) Solid phase that binds to the binding ligand

[13] A method for producing a probe that hybridizes to a repetitive sequence including the following steps.

A) Any base other than the repeat sequence placed on the 3 'side and the repeat sequence placed on the 5' side A step of synthesizing a complementary strand using an oligonucleotide consisting of a sequence as a cage and using as a primer an oligonucleotide containing a base sequence complementary to the cage repeat at least at the 3 ′ end; and

B) Step of recovering the complementary strand synthesized in step A)

[14] Next region (a) and (b) Force probe.

(a) a region consisting of simple repeat sequences, and

(b) a region that is located on both the 3 ′ side and / or 5 ′ side of region (a) and is composed of a base sequence other than a simple repeat sequence,

[15] The probe according to [14], wherein the probe is a mixture comprising a plurality of types of probes having different numbers of repeats of the simple repetitive sequence constituting the region (a).

[16] The probe according to [14], wherein the probe has one or both of a binding ligand and a signal generating molecule in the region (b).

Alternatively, the present invention relates to a probe that can be obtained by the production method described in [13] above. The present invention also relates to the use of the element (a) and (b) potent probe described in [14] in the isolation of double-stranded DNA containing a simple repetitive sequence.

Brief Description of Drawings

FIG. 1 is a diagram showing a preferred embodiment of the method for isolating simple repetitive sequences according to the present invention. (A) A simple repetitive sequence obtained by complementary strand synthesis and a probe having other sequence power. Piotin (B) is introduced into the extended part by complementary strand synthesis. (B) The l-stranded probe and the circled plasmid form triple strands by hybridization. (C) The probe is captured by the magnetic particles having streptavidin, and the hybridized plasmid is recovered.

[Fig. 2] A diagram showing a method for isolating SSRs in the genome based on the prior art. The fragmented genomic DNA is first blunted by adding an adapter. It is then hybridized with a biotinized probe. (D) The hybridized genomic fragment is recovered by magnetic particles having streptavidin. (E) The recovered genomic fragment is amplified with PCR. (The D amplification product is incorporated into a plasmid and cloned.

FIG. 3 shows a method for synthesizing the probe of the present invention by complementary strand synthesis. FIG. 4 shows the number and type of DNA containing simple repeats isolated from the rice genome according to the present invention. In the figure, the left vertical axis indicates the number of isolated DNA clones or the number of their clusters. The left column indicates the number of isolated DNA clones, and the right column indicates the number of clusters. The horizontal axis shows the number of plates used for isolation.

BEST MODE FOR CARRYING OUT THE INVENTION

[0013] The present invention relates to a method for isolating double-stranded DNA containing simple repetitive sequences (SSRs), comprising the following steps.

(1) incubating a double-stranded DNA plasmid library with a probe containing a simple repetitive sequence to be isolated in the presence of a homologous recombinant protein; and

(2) Isolating the plasmid hybridized with the probe

[0014] In the present invention, a plasmid library is an assembly of a plurality of types of plasmids holding the DNA to be screened. The plasmid in the present invention consists of double-stranded DNA carrying DNA that may contain SSRs. The plasmid constituting the library is preferably DNA that can be amplified by transformation into an appropriate host cell. The shape of the plasmid is not limited. Usually, the plasmid is a circular double-stranded DNA. Alternatively, a plasmid that can be amplified even by linear double-stranded DNA by transformation into a host cell is preferred as the plasmid in the present invention.

[0015] The amplification of the plasmid in the host cell includes, for example, an increase in the copy number in the cell. A plasmid is also amplified when the plasmid is transferred to a cell that has divided as the cell divides, regardless of the number of copies per cell. For example, even if you have only one plasmid per cell, the number of plasmids will increase if the number of cells increases due to division. Cell division includes budding in yeast.

[0016] In the present invention, DNA to be screened is DNA that may contain SSRs. The origin of DNA is not limited. Usually, DNA derived from the cell genome is used. By targeting DNA derived from the genome of a cell, DNA containing SSRs can be isolated from the entire genome. Alternatively, DNA derived from a BAC clone, a YAC clone, or the like can be used. From specific BAC clone or YAC clone DNA containing SSRs can be isolated from a specific region of the genome.

[0017] Hereinafter, unless otherwise specified, when "genomic DNA" is described, the origin of the DNA is not limited. Thus, “genomic DNA” includes cell-derived DNA and DNA derived from isolated genomic fragments, such as BAC, YAC, or cosmid vectors.

[0018] Genomic DNA can be fragmented and inserted into a plasmid as necessary. The size of the fragment can be selected as appropriate according to the expected length of the SSRs to be screened. That is, a fragment having a size longer than the base sequence to be isolated can be selected. On the other hand, based on the principle of the present invention, a plasmid having an insert size of about 100-7000 can be isolated. Therefore, for example, for the purpose of isolating microsatellite DNA, a plasmid carrying a fragment usually in the range of 100 to 5000 bp, for example, 100 to 2000 bp, preferably 800 to 1500 bp can be used. In practice, the method of the present invention can be applied by selecting a plasmid retaining a fragment of about lOOOObp.

[0019] In the present invention, in preparing a plasmid library, any method for fragmenting genomic DNA may be used. In general, a method of fragmenting a genome by a physical method or a biochemical method is used. As a physical method, DNA can be cleaved by ultrasound. Biochemical methods include digestion with restriction enzymes. A plasmid library can be obtained by ligating the fragmented genomic DNA to a plasmid vector. The fragmented DNA can be selected in advance of the desired size before ligation. Alternatively, a plasmid in which DNA having a desired size is inserted may be selected after ligation. Furthermore, a commercially available genomic library can be used if an appropriate genomic library is commercially available.

[0020] The plasmid vector for inserting the genomic fragment is not limited. In general,

For the purpose of SSRS isolation, pBluescript vector or pUC vector is used. These vectors can be amplified by cloning into E. coli. [0021] In the isolation method of the present invention, one plasmid library is incubated with a probe containing a simple repeat sequence to be isolated in the presence of a homologous recombinant protein. Probes containing simple repetitive sequences to be isolated can be prepared by any method. Methods for synthesizing DNA consisting of the target nucleotide sequence are known. For example, DNA having an arbitrary base sequence can be synthesized by chemically linking bases. Also, DNA consisting of complementary sequences can be synthesized by annealing a primer to the DNA that is in the shape of a cage and performing a complementary strand synthesis reaction. Furthermore, a region having the desired base sequence can be excised from the DNA retained in the vector with a restriction enzyme.

[0022] Various derivatives can also be used as bases used for DNA synthesis. For example, chromophore, fluorophore, or luminescent molecule

Nucleotide derivatives labeled with (luminophore) or the like are known. By utilizing nucleotide derivatives modified with these signal generating molecules, the probe can be labeled. Alternatively, a base modified with a binding ligand can be used. For example, nucleotide derivatives modified with binding ligands such as piotin and digoxin are known. Probes modified with binding ligands are useful for physical separation of plasmids that are not hybridized to hybridized plasmids.

[0023] The simple repetitive sequence constituting the probe is often high in frequency for each species, and the sequence may be clarified. By using such a known simple repetitive sequence, DNA containing a simple repetitive sequence having a high frequency of appearance can be isolated. Alternatively, a novel simple repetitive sequence can be identified by using an unknown simple repetitive sequence as the probe base sequence. For example, the presence of simple repeat sequences of 3 or more bases in humans is rare. Therefore, if a simple repeat sequence containing a repeat unit of 3 bases or more is used as a probe, an unknown simple repeat sequence may be isolated. One type of probe may be used, or a plurality of types of probes may be mixed and used. The following are examples of typical probe base sequences that can be used for SSRS in higher plants such as rice. In the base sequences exemplified below, the number of n is 1 to 100, for example, 10 to 50, usually 20 to 40.

(CA) n (TA) n

(GC) n

(GGA) n

(TTA) n

[0024] The base sequence of the probe used in the present invention includes a base sequence constituting a simple repetitive sequence or a complementary sequence thereof. In the present invention, a double-stranded DNA plasmid is targeted for hybridization. Needless to say, double-stranded DNA has a complementary sequence. Therefore, the base sequence of the probe may be the simple repetitive sequence itself to be isolated.

[0025] The homologous recombination protein can be used in the present invention regardless of its origin as long as it has homologous recombination activity. In the present invention, the homologous recombination activity refers to the activity of recombining a portion consisting of a base sequence homologous to a single-stranded DNA contained in a double-stranded DNA with the single-stranded DNA. This activity can also be said to be an activity of hybridizing a single-stranded DNA to a complementary base sequence present in a double-stranded DNA. Many proteins having such activity are known. For example, RecA is a homologous recombinant protein derived from E. coli. A refined product of RecA produced by genetic recombination is commercially available (〃RecA〃; trade name of New England Biolabs / NEB, Product Code M0249S-200 g included). In addition, the amino acid sequence of RecA such as E. coli has also been determined as follows.

E.coli; V00328, J01672

Shigella flexnen; X55553

Salmonella; NP.457222

[0026] As homologues of RecA in eukaryotic organisms, Rad51 and Rad52 have been identified in human yeast (GenBank Accession Number: X64270, S38937) (; G Basils, M Aker, and RK Mortimer, Mol Cell Biol 1992 July; 12 (7): 3235-3246. "Nucleotide sequence and transcriptional regulation of the yeast recombinational repair gene RAD51.". These homologous recombination proteins can be used in the present invention. The replacement protein may be not only a naturally-derived protein, but also a protein obtained as a gene recombinant, and as long as it has the homologous recombination activity necessary for the present invention, an amino acid may be used. Mutants containing acid sequence mutations can also be used. For example, a Rad51 fragment containing a region necessary for maintaining homologous recombination activity, or a mutant protein containing the fragment as a partial structure is useful as a homologous recombination protein in the present invention. Domains required for the homologous recombination activity of Rad51 have already been identified (Miller KA, Sawicka D, Barsky D, Albala JS., Nucleic Acids Res. 2004 Jan 02; 32 (1): 169—78. Domain mapping of the Rad51 paralog protein complexes.). Similarly, as long as it has the homologous recombination activity necessary for the present invention, it is possible to use a mutant containing an amino acid sequence mutation.

In the present invention, a double-stranded DNA plasmid library is incubated with a probe containing a simple repetitive sequence to be isolated in the presence of a homologous recombinant protein. These elements are incubated under conditions that allow the sequence-specific expression and hybridization of the probe to double-stranded DNA by the homologous recombinant protein to be used. Usually, a double-stranded plasmid is mixed after a complex of a probe and a homologous recombination protein is formed in advance. Therefore, in the present invention, the step (1) of incubating a double-stranded DNA plasmid library with a probe containing a simple repeat sequence to be isolated in the presence of a homologous recombinant protein is, for example, Can be implemented in this way.

[La] (la) Incubating the homologous recombinant protein with a probe containing a simple repetitive sequence to be isolated; and

(lb) Step (2) After step (la), a double-stranded DNA plasmid library is added and incubated further

More specifically, for example, the following conditions can be shown as reaction conditions when RecA is used as a homologous recombinant protein.

[0029] First, a probe (single-stranded DNA) and RecA protein are incubated with ATP. The conditions for incubation are, for example, 20-40 ° C for 5 minutes to 30 minutes. These elements are usually incubated at near neutral pH. Specifically, pH 7-8 is suitable. Upon incubation, RecA protein forms a complex with ATP and a probe (single-stranded DNA). For the incubation, for example, a buffer solution having the following composition can be used. 25 mM Tris acetate buffer (pH 7.5)

8mM magnesium acetate

2mM CoCl

2

lmg / mL ushi serum albumin (BSA)

ATP is added at 0.1-10mM. Instead of ATP, a derivative of ATP can also be used. For example, dexiadenine triphosphate (dATP) or adenosine 5 and [y-thio] triphosphate (γ-S-ATP) can be used instead of ATP. Alternatively, ATP and γ-S-ATP can be mixed and used. By using y-S-ATP or a mixture with ATP, the triple-stranded structure formed by the action of RecA is more stably maintained. Therefore, the use of γ-S-ATP is a preferred condition in the present invention. When ATP and γ-S-ATP are mixed, the total concentration of both can be adjusted to be within the above-mentioned concentration range. The mixing ratio of ATP and γ-S-ATP can be, for example, 1: 10—10: 1, usually 1: 2—2: 1, preferably 1: 1. More specifically, ImM ATP and ImM γ-S-ATP can be included. Under the above conditions, RecA is added, for example, 0.1—10 g / mL, usually 0.2—lg / mL, more specifically 8 g / 30 L (about 0.27 g / mL). (Zhumabayava et al, BioTechniques 27: 834-845 (October 1999), RecA-Mediated Affinity Capture: A Method for FuU-Length cDNA Cloning).

[0030] Subsequently, the plasmid is added and incubated under conditions that allow hybridization of the probe to the double-stranded DNA by the homologous recombination protein. In practice, a plasmid (double-stranded DNA) may be added to the complex [RecA protein ATP probe (single-stranded DNA)] formed in advance. For example, if incubated at 20–40 ° C for 5 to 30 minutes, the probe can hybridize to complementary sequences in double-stranded DNA. When the probe is hybridized, it has a three-stranded structure with the probe inserted between the two strands.

[0031] Alternatively, in Rad51, which is a human-derived homologous recombination protein, a probe (single-stranded DNA) is incubated with ATP. ATP is added at 0.1-10mM. Also in Rad51, its derivatives can be used in place of ATP. Incubation conditions are, for example, 15-30 ° C, 1-20 minutes. The following buffer is used for the reaction. Synthetic buffers can be used.

20 mM Tris-HCl buffer (pH 7.4)

1.5 mM MgCl

2

0.1mg / mL ushi serum albumin (BSA)

[0032] After the incubation, a complex consisting of a probe (single-stranded DNA) and a recombinant protein is formed by further adding Rad54 and incubating. If a single plasmid library (double-stranded DNA) is then added, the probe can be hybridized to the plasmid by homologous recombination. The conditions for incubating the complex with the plasmid can be, for example, 5–60 minutes at 20–40 ° C. In this way, a triple-stranded structure consisting of a probe (single-stranded DNA) and a plasmid (double-stranded DNA) is formed.

[0033] Double-stranded DNA plasmid library in step (1), including a simple repeat sequence to be isolated, and a simple repeat sequence to be isolated when incubated in the presence of a homologous recombinant protein The plasmid hybridizes with the probe. By isolating the hybridized plasmid, a clone containing the desired simple repetitive sequence can be obtained. For example, if a probe that has been modified with a binding ligand in advance is used, clones that have been or hybridized can be easily recovered using the affinity for the binding ligand.

[0034] Specifically, when a pyotin probe is used, a clone hybridized with the probe can be captured on the solid phase by contact with the solid phase to which avidin is bound. The captured clone is separated from a hybridized clone. By washing the solid phase, clones that did not hybridize are removed. Subsequently, the hybridized clone can be recovered by placing the solid phase under conditions for eliminating the hybridization. A series of steps is shown in Fig. 1.

[0035] In the present invention, the recovered clones can be further amplified. It is desirable to use a method that can amplify SSRs evenly. For example, as described above, the PCR method has a possibility of imparting bias. If the degree of amplification varies depending on the structure or length of the SSRs base sequence, the final DNA isolated may be biased. Therefore, in order to amplify the clone recovered by the present invention, it is preferable to use, for example, a biological method. Specifically Can be amplified by introducing the plasmid into a suitable host cell. That is, the present invention provides a method for isolating DNA containing SSRs, comprising the steps of isolating a plasmid by the following steps (2a)-(2c).

(2a) recovering the plasmid hybridized with the probe,

(2c) recovering the amplified plasmid from the host cell

[0036] In the method for isolating DNA containing SSRs of the present invention, a plasmid is used as an isolation target. The plasmid can usually be amplified by transformation into a suitable host cell. In principle, the copy of the plasmid in the cell is independent of the type of insert carried by the plasmid. In other words, regardless of the base sequence of the insert DNA, a copy of the transformed plasmid is transmitted to the dividing cell. And preferably a copy is also produced in the cell. Thus, the collected plasmid is amplified.

[0037] For example, using a binding ligand introduced into a probe, a plasmid having DNA containing SSRs to be isolated is captured on a solid phase. Separate the solid phase by centrifugation or filtration. Alternatively, if magnetic particles are used for the solid phase, the solid phase can be recovered magnetically. The solid phase is then washed to remove any vigorous plasmids. The captured plasmid is recovered from the hybridization with the probe by placing it in denaturing conditions. The thus recovered plasmid is transformed into a host cell. For example, a plasmid that can be amplified in E. coli is transformed into E. coli. If E. coli having the plasmid is isolated, the plasmid carrying the DNA containing the target SSRs is cloned. Thus, double-stranded DNA containing SSRs is isolated according to the present invention. The cloned plasmid can be purified as necessary, and the nucleotide sequence of the insert can be determined.

[0038] In a series of processes, the amplification efficiency of each clone can be regarded as equal. For example, plasmid transformation efficiency is not affected by the base sequence composition of the insert. In addition, the transformed plasmid is copied inside the cell or transmitted as the cell divides. Even in the process, it is considered unlikely that bias will occur due to the base sequence of the insert. In particular, if the insert size is limited at the stage of synthesizing the library, the length of the insert will remain within a certain range. Therefore, the bias of amplification efficiency due to the length of the insert is suppressed, and the amplification efficiency of the plasmid is kept highly uniform. As a result, DNA containing SSRs hybridized with the probe can be isolated without bias. That is, the present invention can be expected to improve the isolation efficiency of DNA containing SSRs.

[0039] Based on the present invention, the base sequences of SSRs can be verified between the cloned plasmids. As a result of the verification, SSRs consisting of the same base sequence can be determined as the same type and grouped into clusters. The number of clusters means the type of SSRs identified. Unknown SSRs can be identified by comparing the clustered nucleotide sequences with previously reported SSRs.

[0040] By the way, the structure of the probe used in the present invention is not limited as long as it includes a simple repetitive sequence (or its complementary sequence) to be isolated! Thus, the probe can include a common base sequence. For example, a base sequence having a function other than hybridization with a plasmid can be added to the base sequence to be a probe. As such an additional base sequence, for example, an additional base sequence for introducing a binding ligand can be shown.

[0041] RecA, a typical homologous recombination protein, can search double-stranded DNA in a very short time to find a homologous sequence (Honigberg et al., Proc. Natl. Acad. Sci. USA. 9586-9590, 1986). Attempts to apply this action to homologous sequence searches are known. In other words, a method in which a probe is hybridized to double-stranded DNA by the action of a homologous recombination protein has been applied to cDNA isolation and the like (Teintze et al., Biochem. Biophys. Res. Commun "Vol. 201, No. 3, 804-811, 1995).

[0042] The application of probes incorporating binding ligands has also been reported (Rigas et al, Proc.

Natl. Acad. Sci. USA. Vol. 83, 9591-9595, 1986). For example, in these methods, double-stranded DNA hybridized with the probe can be recovered using the binding partner of the binding ligand. Using this principle, a method for isolating clones containing the desired nucleotide sequence from cDNA has been put into practical use (CloneCapture ™ cDNA Selection Kit; Clonetech). However, in the known method, a binding ligand was introduced into the region that hybridizes to the double-stranded DNA.

[0043] The molecular weight of a typical binding ligand, piotin, is about 244. In contrast, the average size of a single molecule is about 330. In other words, the portion modified with the binding ligand has a greatly swollen structure compared to the normal single-stranded oligonucleotide portion. In a hybrid between double strands and single strands, a structure in which single strand DNA is interrupted between the two strands is formed. In other words, compared to hybrids between single strands, it is considered to be more sensitive to structural changes. Therefore, the hybridization between two strands and one strand is susceptible to steric hindrance by the modifying molecule. In other words, in known methods using homologous recombination proteins, the binding ligand may cause steric hindrance when hybridizing single-stranded DNA with double-stranded DNA using homologous recombination proteins.

[0044] In order to avoid the possibility of such steric hindrance by the binding ligand, an additional sequence can be used in the probe of the present invention. Specifically, a probe having a binding ligand in an additional sequence portion that is not a base sequence for hybridizing to double-stranded DNA can be used. That is, the present invention provides a probe containing the following regions (a) and (b), and a method for isolating double-stranded DNA containing SSRs using this probe.

(a) a region consisting of simple repeat sequences, and

(b) Region located on both the 3 'side and / or 5' side of region (a) and consisting of base sequences other than simple repeat sequences

[0045] The region (a) of the probe having the above structure is a region that hybridizes to one of the strands of the DNA constituting the double-stranded DNA. Region (b) is added to region (a). Region (b) may be modified with a signal generating molecule or a binding ligand. On the other hand, it is preferable that there is no nucleotide modification in the region (a). Region (b) is a region that does not participate in hybridization. Therefore, modification of region (b) does not cause steric hindrance in the hybrid.

[0046] The region (b) of the probe in the present invention does not participate in hybridization, and therefore, any salt. It can consist of a base sequence. For example, a base sequence composed of a series of a small number of bases is easy to synthesize. Furthermore, it is a general condition required for a probe that no hybridization occurs within the same molecule. In the present invention, since the region (a) is composed of a simple repetitive sequence, a complementary base sequence or the same base sequence does not exist between the region (a) and the region (b). Like to design, so that. In order to satisfy these conditions, for example, a base sequence having a continuous force of a, t, c, g, or u is suitable as a base sequence constituting the region (b). Among them, a base sequence in which a, t, or u is continuous is preferable. These bases have a lower affinity for base pair binding than binding between c / g. For this reason, it is expected that the influence of hybridization within the same strand can be further reduced.

[0047] The probe having the structure as described above can be prepared by any method.

For example, a probe can be obtained by synthesizing region (a) and region (b) and linking them together. Alternatively, a base can be added to the end of the previously synthesized region (a) by an enzyme such as terminal transferase. It is difficult to artificially control the number of bases added by terminal transferase. Therefore, the region (b) constituting the probe obtained by this method may have a non-uniform length. In order to control the length of the region (b), it is preferable to synthesize it completely chemically or to use a complementary strand synthesis reaction which is dependent on the cage type.

[0048] In the probe having the above structure used in the present invention, the length of the region (a) can be designed to an arbitrary length depending on the length of the SSRs to be isolated. In general SSRs isolation, the length of region (a) is usually about 25-100 bases. On the other hand, the length of region (b) is not limited. When chemically synthesizing, 30-60 bases is advantageous because it can be easily synthesized.

[0049] The probe having the above-described structure can be synthesized by utilizing a cage-type-dependent complementary strand synthesis reaction. In other words, the present invention provides a method for producing a probe that can be repeated and repeated in a repetitive sequence including the following steps.

A) An oligonucleotide consisting of a repetitive sequence arranged on the 3 ′ side and an arbitrary nucleotide sequence other than the repetitive sequence arranged on the 5 ′ side is used as a cage, and at least the 3 ′ end has the above-mentioned cage-type repeat. A complementary strand is synthesized using an oligonucleotide containing a complementary base sequence as a primer. About, and

B) Step of recovering the complementary strand synthesized in step A)

Specifically, the above steps can be illustrated as follows.

Type: 3 '-[Simple repetitive sequence]-[Any base sequence other than repetitive sequence]-5'

5 '-[Primer]> -3'

Old Rigo nucleotide

[0050] The product generated by the complementary strand synthesis reaction is composed of a complementary sequence of the oligonucleotide used as a cage. When a nucleotide derivative is given as a substrate in the complementary strand synthesis, the nucleotide derivative can also be introduced into the complementary strand synthesis product. For example, a nucleotide derivative modified with a binding ligand gives a complementary strand synthesis product modified with the binding ligand. Since the portion corresponding to the simple repeat sequence of the complementary strand synthesis product is supplied as a primer, it is not modified. As a result, a simple repetitive sequence portion that hybridizes to a double-stranded DNA is not modified, and a probe in which a region that is not involved in hybridization is modified with a binding ligand can be obtained.

The region force corresponding to [primer] constituting the probe synthesized in this embodiment corresponds to the region ( _a ) in the probe, and the portion extended by the complementary strand synthesis reaction corresponds to the region (b). Region (b) is composed of a complementary sequence of [any base sequence other than repetitive sequence].

[0051] In the present invention, a structure having an arbitrary base sequence other than a repetitive sequence on the 5 'side can also be used as the primer. That is, the present invention provides a method for producing a probe that hybridizes to a repetitive sequence including the following steps.

A) a step of hybridizing a region containing the 3 ′ end with a region containing the 3 ′ end of at least one primer set having mutually complementary nucleotide sequences, wherein the primer comprises: A step of being an oligonucleotide comprising a repetitive sequence arranged on the 3 ′ side and an arbitrary base sequence other than the repetitive sequence arranged on the 5 ′ side,

B) extending the 3 ′ end of the primer by complementary strand synthesis reaction; and

C) Step of recovering the double-stranded DNA obtained in step B)

The above process can be illustrated as follows. Type: Ku--3 '-[Simple repetitive sequence A]-[Other than repetitive sequences]-5'

5 '[other than repetitive sequence]-[simple repetitive sequence B]-3'―): primer

[0052] In this embodiment, a combination of oligonucleotides each having a simple repeat sequence A and B complementary to each other and having a structure in which any base sequence other than the repeat sequence is linked to its 5 'side is used. . Complementary strand synthesis reactions proceed with simple repeat sequences A and B complementary to each other, with each other in the shape of an arrow and “arbitrary base sequences other than repeat sequences” in the direction of the arrows. As a result, DNA having a simple repetitive sequence at the center and a base sequence other than the simple repetitive sequence (or its complementary sequence) on the 5 ′ side and 3 ′ side is synthesized. If it is denatured after synthesis, the new primer or cage is annealed again to the cage or primer, and the same reaction is repeated. In other words, DNA with the same structure is exponentially amplified by the principle of PCR. The reaction in which DNAs having a base sequence complementary to each other on the 3 ′ side as described above are annealed to synthesize a complementary strand with both 3 ′ terminal forces is a known reaction also called the Overgo method. However, its application to the synthesis of probes containing repetitive sequences modified with binding ligands is novel.

The region corresponding to [simple repetitive sequence A] and [simple repetitive sequence B] constituting the DNA synthesized in this embodiment is the region (a) in the probe, and the portion extended by the complementary strand synthesis reaction It corresponds to each area (b). Region (b) is composed of a complementary sequence of [any base sequence other than repetitive sequence].

[0053] A modified nucleotide can be introduced into a complementary sequence (that is, region (b)) of the base sequence other than the simple repetitive sequence of the DNA thus synthesized. For example, DNA modified with a binding ligand is useful as a probe in the present invention. The purpose of the present invention is to isolate double-stranded DNA containing a simple repetitive sequence. Therefore, the DNA synthesized as described above can be used as a probe for any of the DNAs containing simple repeats A and B.

[0054] In the present invention, the complementary strand synthesis reaction in step A) can be performed by a DNA polymerase that catalyzes the saddle type-dependent complementary strand synthesis reaction. For example, the Klenow fragment, which is an active fragment of DNA polymerase derived from Escherichia coli, is useful in a method for synthesizing a probe based on the above reaction. In addition, Taq polymerase used in PCR, etc. These DNA polymerases can also be used in the method of the present invention.

[0055] In the complementary strand synthesis reaction for obtaining the probe of the present invention, any base sequence other than the repetitive sequence is not limited. For example, as described above, the same base sequence can be used as an arbitrary base sequence other than a repetitive sequence. Specifically, a sequence such as a stretch can be any base sequence other than a repetitive sequence. Modified oligonucleotides can be used as substrates for complementary strand synthesis reactions. For example, biotinylated uracil can be used as a binding ligand-modified nucleotide.

[0056] When a probe used in the present invention is synthesized by a complementary strand synthesis reaction, a highly uniform probe can be synthesized by keeping the stringency of the complementary strand synthesis reaction high. However, in the above method, a simple repetitive sequence such as cacacaca .... anneals to the base sequence of tgtgtgtg ..... t. In such a combination of base sequences, the possibility of deviation at the annealing position cannot be denied. The misalignment position causes the generation of multiple types of probes with different simple repeat sequences.

[0057] For example, in the following example, a probe including a simple repetitive sequence composed of ca and gt is synthesized. (Ca) is 25'-ca-3 'repeat sequence 25 times, (gt) 25 is 5'-tg-3' 2

twenty five

Each of the 5 repeat sequences is shown.

aaaaa- (ca) ₂₅ -3 '

3-(gt) ₂₅ -aaaaa

[0058] When (ca) and are annealed, aaaaa ..... on the 5 'side of each other is used as a saddle shape,

25 25

A complementary strand is also synthesized with a 3 ′ end force. Theoretically, complementary-strand synthesis produces DNA with the following structure.

aaaaa- (ca ₂₅ -uuuuu -3

3-uuuuu- (gt) ₂₅ -aaaaa

However, in reality, in the annealing between simple repeat sequences, the 25 repeat sequences do not completely match, and there is a possibility that a deviation of n units will occur. Due to the deviation of n units, the probe is synthesized with many repeating sequences of n times. In the example below, there is a deviation of 2 units (the part indicated by + — +). Figure 3 also shows that DNA probes with many repeating units are generated due to the difference in repeating units. + ― +

aaaaa-cacaca cacaca-3 '

3'-gtgtgt gtgtgt-aaaaa The resulting DNA will have a 4 unit long repeat sequence as follows.

aaaaa- a) ₂ (ca) ₂₅ (ca) ₂ -uuuuu-3 '

3-uuuuu- (gt) ₂ (gt) ₂₅ (gt) ₂ -aaaaa

[0060] In the present invention, the DNA probe thus generated with several units of annealing position deviation is rather useful in the isolation of DNA containing a simple repetitive sequence. In the isolation of unknown simple repeat sequences, it is desirable that simple repeat sequences whose length cannot be specified can also be isolated. Increasing the length variation of the probe can be an effective means for that. Therefore, the above-described method, which can obtain a mixture of probes containing simple repetitive sequences of different lengths from a single length of starting material, is preferred as a method for synthesizing probes for isolating simple repetitive sequences. That is, the present invention provides a mixture of a plurality of types of probes comprising the following regions (a) and (b) and having different numbers of repeats of the simple repetitive sequence constituting the region (a).

(a) a region consisting of simple repeat sequences, and

(b) a region composed of a base sequence other than a simple repetitive sequence, located on either or both of the 3 'side and 5' side of region (a),

[0061] The components constituting the method for isolating SSRs of the present invention can be combined in advance to form a kit. That is, the present invention provides a kit for the isolation of simple repetitive sequences comprising the following elements.

(a) a region consisting of simple repeat sequences, and

ii) homologous thread and reversible protein, and

iii) Solid phase that binds to the binding ligand

Example [0062] 1. Synthesis of DNA probe

Two types of oligonucleotides with the following base sequence capabilities were synthesized. (A) 30 is a 30-mer chain of A, (CA) 25 and (TG) 25 are 25 repetitions of ca and tg, respectively. CA-oligo: 5'- (A) 30 (CA) 25-3 '(SEQ ID NO: 1, 80mer)

TG-oligo: 5'- (A) 30 (TG) 25-3 '(SEQ ID NO: 2, 80mer)

Amersham's Megaprime DNA labeling system was used for biotinylation. The specific operation is as follows. First, 200 pmol of CA-oligo and 200 pmol of TG-oligo were annealed. Next, 20 pmol of dATP, dGTP, dCTP, and dTTP, respectively, and finally lOOOpmol of piotin 21-dUTP were obtained. Further, 2 units of DNA polymerase (Klenow fragment) was added and reacted at room temperature for 2 hours.

[0063] By this reaction, a 30-residue piotin 匕 U was linked to the 3 ′ side of the CA-oligo and TG-oligo, and DNA probes having structures as shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively. Synthesized. Therefore, the obtained DNA probe is a chimeric molecule in which U (RNA) is optionally linked to a DNA portion that functions as a probe. Furthermore, in this reaction, it is possible that DNA probes with different repeating units were synthesized by annealing with the CA repeat of the CA-oligo and the TG repeat of the TG-oligo shifted. . Hereinafter, the deviation of the repeating unit will be described with reference to FIG.

[0064] For example, when annealing is performed with the repeating unit shifted by 5 units, DNA probes having the base sequences described in SEQ ID NO: 5 and SEQ ID NO: 6 are generated. In addition, when n repeating units are displaced and annealed, a DNA probe having the base sequence represented by the general formula described in SEQ ID NO: 7 and SEQ ID NO: 8 may be generated. When the repeating unit is shifted in this way, u or t is actually introduced at the position corresponding to t of the DNA probe, and thus the probe part may contain RNA. Further, in FIG. 3, in order to help understanding, the complementary strand consisting of U with respect to A of the oligonucleotide oligonucleotide that is a saddle type is shown. In practice, however, dTTP is introduced with the same potential as dUTP for complementary strand synthesis. As a result, the synthesized complementary strand can be T or U. The ratio of both depends on the concentrations of the substrates dTTP and dUTP. The annealing conditions are usually about 10 minutes at room temperature. Common buffers can be used for annealing. Specifically, a buffer solution having the following composition can be shown.

Tris-HCl

MgCl

2

β— mercaptoethanol

It can also be annealed in a commercially available buffer. For example, Amersham Biosciences Megaprime DNA labeling system (Kit J can be used for annealing. In addition, a buffer composition suitable for complementary strand synthesis is also known. For example, when using a Klenow fragment for DNA polymerase. It is possible to use a buffer solution having the following composition.

50 mM Tris-HCl (pH 7.5)

10 mM MgCl

2

ImM DTT

[0065] 2. Purification of piotinylated probe

The synthesized probe was recovered by QIAGEN QIAquick nucleotide removal kit. Operation was in accordance with product instructions. The purified pyotinylated probe was separated by agarose electrophoresis, and a double-stranded band having the desired size (100-200 bp) was cut out. DNA was purified from the excised agarose gel with a filter (ultrafree-DA, manufactured by Amicon). The recovered DNA was further purified by ethanol precipitation.

[0066] 3. Plasmid library for screening

Rice genomic DNA was extracted and mechanically fragmented by sonication. A genomic DNA fragment of l-13 kb in size was selected and ligated into a plasmid vector that had been digested with Smal and dephosphorylated. PUC 18 was used as the plasmid vector. The vector inserted with genomic DNA was transformed into E. coli DH10B strain and plated to obtain a plasmid library of about 1.000.000 clones. DNA was collectively purified from the transformed E. coli by the alkaline method and used for subsequent screening by hybridization. [0067] 4. Triple chain formation

The biotin probe prepared in 2 was boiled for 5 minutes and then rapidly cooled on ice to form a single strand. Buffer solution is added to single-stranded piotinated probe, and 2.5 mM CoCl, ATP (ATP and

2

ATP-gumma-S mixture) and RecA protein 8 microgram were collected and incubated at 37 ° C for 20 minutes. The composition of the buffer is as follows.

25 mM Tris acetate buffer (pH 7.5)

8mM magnesium acetate

2mM CoCl

2

lmg / mL ushi serum albumin (BSA)

Next, the plasmid library lOmicrogram purified in step 3 was collected and incubated at 37 ° C for 1 hour to form triple chains. Thereafter, RecA was decomposed by proteinase K treatment in the presence of 0.1% SDS, and proteinase K was inactivated by adding Phenylmethylsulfonyl Fluoride (PMSF).

[0068] 5. Streptavidin microbeads

Four solutions containing a pyotinylated probe and a plasmid vector were mixed with Streptavidin magnetic beads equilibrated with buffer. The composition of the buffer solution for equilibration is as follows.

10 mM Tris-HCl (pH 7.5)

ImM EDTA

1M NaCl

After standing at room temperature, DNA that did not bind to streptavidin was washed. The composition of the cleaning solution is as follows.

10 mM Tris-HCl (pH 7.5)

ImM EDTA

2M NaCl

After washing, the bound DNA was eluted with an alkaline solution (0. IN NaOH, ImM EDTA). The eluted plasmid vector was purified by ethanol precipitation.

[0069] 6. Analysis of isolated plasmid

Escherichia coli was transformed with the plasmid vector recovered in 5 to construct a library. The library was further diluted and plated on 96-well microplates. For each clone, DNA was purified and the nucleotide sequence of the insert was determined. Simple repeat sequences contained in the base sequences were detected by commercially available software such as Repeat masker. The number of simple repetitive sequences detected was counted as the number of isolated SSR clones. Furthermore, a plurality of clones having the same base sequence were counted as one cluster. That is, the number of clusters means the kind of isolated simple repeat sequence.

The results of attempts to isolate simple repeat sequences consisting of (TAA) n, (GGA) n, (GC) n, and (TA) n by the same procedure are summarized below. Table 1 shows the cloning results for each simple repeat sequence.

[table 1]

Library Z motif: Indicates the motif that constitutes the base sequence of the probe and the name of the library concentrated by the probe.

Number of clones hit: Shows the number of positive clones, that is, the number of wells in which 96 positive clones were detected and the percentage (%).

SSRs: indicates the number of plasmids containing cloned simple repeats.

Type of SSRs: Number of simple repeats with different base sequences among the identified simple repeats

(Simple repetitive sequence type). The number is the same as the number of clusters.

Average length (bp): Shows the average length (bp) of identified simple repeats.

Maximum length (bp): Indicates the length (bp) of the longest simple repeat sequence identified.

The base sequences of the probes used for isolation of these SSRS are shown below. The portion of the nucleotide sequence indicated as T / U indicates that it may become T or U in probe synthesis. Furthermore, in the probes marked with *, the T in the motif part may also be U.

Probe for (TAA) n: 5 '(A) 30 (TAA) 16-33 (T / U) 30 * (SEQ ID NO: 9) (TTA) n probe: 5 '(A) 30 (TTA) 16-33 (T / U) 30 * (SEQ ID NO: 10) (GGA) n probe: 5' (A) 30 (GGA) 16_33 ( T / U) 30 (SEQ ID NO: 11) Probe for (CCT) n: 5 '(A) 30 (CCT) 16-33 (T / U) 30 * (SEQ ID NO: 12) Probe for (GC) n: 5 '(A) 30 (GC) 25-50 (T / U) 30 (SEQ ID NO: 13)

Probe for (TA) n: 5 '(A) 30 (TA) 25_50 (T / U) 30 * (SEQ ID NO: 14)

[0071] Each library after further enrichment The number of isolated SSRs motifs is summarized below. In any library, it can be appreciated that a clone containing the motif sequence used for the probe can be selectively isolated.

(Table 2) The number of pre-wells (hit clones) and the number of isolated SSRs (CA-SSR) in which clones containing CA-motifs that were isolated from each library were detected

hit clone CA-SSR

[CA library 39 47]

G then library 1 1

G JA library 3 3

TAA library 6 6

[0072] (Table 3) Number of pre-wells (hit clones) and clones containing isolated SSRs (GC-SSR)

nit clone GC-SSR

CA library 1 1

[G library 5 6]

GGA library 2 2

TAA library 0 0

[0073] (Table 4) The power of each library The number of pre-wells (hit clones) and the number of isolated SSRs (GGA-SSR) in which clones containing the isolated GGA-motif were detected

hit clone GGA-SSR

CA library 11 13

GC library 8 10

[GGA library 22 38] TAA library 12 14

[0074] (Table 5) The power of each library The number of pre-wells in which clones containing the isolated TAA-motif were detected (hit clone) and the number of isolated SSRs (TAA-SSR)

hit clone TAA-SSR

CA library 5 5

GC library 9 9

GGA library 7 7

[TAA library 41 49]

[0075] Among DNAs containing SSRs isolated by each probe, the ratio of DNAs containing a motif similar to the base sequence is summarized below. As shown in Table 6, in the present invention, high specificity for the probe base sequence can be expected.

[Table 6]

*: 24 out of 29 TC repeats.

**: 10 out of 15 STA repeats.

***: 1 out of 16 ΤΑ repeats.

(CA) n, (TAA) n, and (GGA) n, which are abundantly contained in the rice genome, were enriched to 20-40% from the obtained genomic library. The present invention has shown that plasmids containing simple repeat sequences can be efficiently enriched.

Furthermore, the base sequence constituting the (CA) n repeat of rice isolated as described above was actually mapped to the rice genome sequence. Table 7 summarizes the distribution on each chromosome.

[Table 7] chr.1 chr.2 chr.3 chr.4 chr.5 chr.6 chr.7 chr.8 chr.9 chr.10chr.11 chr.12 Unknown position Total

* SSR: The number of CA or AC or GT or TG repeats 9bp or more mapped to each chromosome.

* Number of clusters: The number of SSRs that do not have overlapping nucleotide sequences among the SSRs mapped to each chromosome. Duplication was clustered by chromosomal location. In other words, even if the number of repeats in the actually isolated DNA was different, the number of clusters was set to 1 when mapped at the same position.

An SSR with an unknown position is considered to be a base sequence that cannot be found on the genomic base sequence determined at this stage. Such a strong base sequence that could not be mapped determined the number of clusters based on the assembly results between the base sequences.

* Clone: Number of clones containing an isolated SSR from which the nucleotide sequence mapped to each chromosome is derived.

[0077] The results in Table 7 show that 503 (440) clones of (CA) n were actually obtained from 1056 clones (11 plates of 96 well plates) enriched with (CA) n. Indicates that it has been positioned. The cloned SSR was distributed over 12 chromosomes. In other words, it can be said that SSR sequences could be mapped efficiently over a wide range of chromosomes.

[0078] Further, the analysis results for 11 sheets of 96 wall plates are summarized in FIG. As is clear from Fig. 4, as the number of plates (ie, the number of clones) increases, the number of isolated SSRs (force ram left: SSR) and type (column right: cluster) increase steadily. RU That is, the SSR enriched library constructed according to the present invention shows that various SSRs are efficiently enriched.

[0079] Since the present invention does not depend on PCR, clones having long inserts can be concentrated. In addition, in the conventional method, the level of amplification by PCR differs from sequence to sequence, and the resulting SSR was heavily biased. This method creates libraries with extremely low sequence bias. Made possible. Based on the results of this experiment, theoretically, a sequence of 5000 to 10,000 clones was performed for one motif, and 1000 (for example, for GGA motif) force 2000 (for example, CA motif or TTA) It can be predicted that the SSR of the motif will be obtained.

Industrial applicability

[0080] The present invention is useful for isolation of DNA containing simple repetitive sequences (SSRs). According to the present invention, various DNAs containing SSRs as structural units, such as satellite DNA, minisatellite DNA, or microsatellite DNA, can be isolated. By analyzing the base sequence of DNA containing SSRs isolated by the present invention, a base sequence useful as a genetic marker can be identified.

[0081] For example, the minisatellite array is useful for personal identification, parent-child discrimination, and the like. Alternatively, the base sequence of microsatellite DNA can be used as a marker for knowing the physical position on the genome by mapping on the genome sequence. In order to use the base sequence information of microsatellite DNA as a marker, it is assumed that the marker is mapped to the entire genome at a high density. Specifically, the following advantages can be expected by using one-on-one mapping with high density.

[0082] First, genomic fragments cloned into an artificial chromosome such as BAC or YAC can be quickly and easily aligned using a marker. Furthermore, when used as a genetic marker, markers that are densely mapped are particularly important. For example, in linkage analysis with phenotypes, the mapping density of markers greatly affects the accuracy of map based cloning such as positional cloning. The presence of high-density markers is also an important clue in selective marker breeding.

[0083] Alternatively, it is considered that the mapping of loci that contribute to quantitative traits also contributes to the improvement of the accuracy of high-density markers. In general, multiple loci are involved in quantitatively changing traits such as rice seed weight. Thus, in normal linkage analysis, the association between loci and traits is often discontinuous. Increase the density of markers on the genetic linkage map using a large number of DNA markers and contribute to traits It is possible to identify the locus to be used.

Genome sizes range from 430 million base pairs in rice and 6 billion base pairs in humans. It can be said that the analysis of the genomes of these species has already been completed with considerable accuracy. However, identifying many genetic markers according to the size of the genome is a major challenge in the future. Word V, in other words, the importance of isolating microsatellite DNA markers that can cover the entire large genome has increased rapidly. The method of the present invention that enables efficient isolation of SSRs will greatly contribute to the progress of gene analysis in the future through the isolation of SSRs markers.

Claims

Claims [I] A method for isolating double-stranded DNA comprising a simple repetitive sequence, comprising the following steps:

(2) Isolating the plasmid hybridized with the probe

[2] The method according to claim 1, wherein the plasmid library is a plasmid library containing genomic fragments.

[3] The method according to claim 2, wherein the step of isolating the plasmid comprises the following steps (2a) — (2c).

(2a) recovering the plasmid hybridized with the probe,

(2c) recovering the amplified plasmid from the host cell

[4] The method of claim 1, wherein the probe has the following areas (a) and (b) force.

(a) a region consisting of simple repeat sequences, and

(b) Region located on the 3 'side and / or 5' side of region (a) and consisting of base sequences other than simple repeats

[5] The method according to claim 4, wherein the base sequence force a, t, c, g, and u force of the region (b) is selected, and any one of the bases is a continuous sequence.

6. The method according to claim 4, wherein the probe has a binding ligand in the region (b).

[7] The method according to claim 6, comprising a step of capturing the plasmid on a solid phase that binds to a binding ligand before or after the incubation of the plasmid and the probe.

8. The method according to claim 4, wherein the length of the region (a) is 25-100 bases.

[9] The method according to claim 4, wherein the length of the region (b) is 30 to 60 bases.

[10] The method according to claim 4, wherein the probe comprises a plurality of types of probes having different numbers of repeats of the simple repetitive sequence constituting the region (a).

[I I] The method according to claim 1, wherein the homologous recombinant protein is selected from the group consisting of RecA protein, Rad51 protein, and Rad52 protein.

[12] A kit for the isolation of simple repetitive sequences, comprising: i) the following region (a) and (b) force and a probe having a binding ligand in region (b),

(a) a region consisting of simple repeat sequences, and

ii) homologous thread and reversible protein, and

iii) Solid phase that binds to the binding ligand

A) An oligonucleotide consisting of a repetitive sequence arranged on the 3 ′ side and an arbitrary nucleotide sequence other than the repetitive sequence arranged on the 5 ′ side is used as a cage, and at least the 3 ′ end has the above-mentioned cage-type repeat. A process of synthesizing a complementary strand using an oligonucleotide containing a complementary base sequence as a primer, and

B) Step of recovering the complementary strand synthesized in step A)

[14] Probe consisting of the following regions (a) and (b).

(a) a region consisting of simple repeat sequences, and

15. The probe according to claim 14, wherein the probe is a mixture comprising a plurality of types of probes having different numbers of repeats of the simple repetitive sequence constituting the region (a).

16. The probe according to claim 14, wherein the probe has a binding ligand and / or a signal generating molecule in the region (b).