WO2006080136A1

WO2006080136A1 - Method of quickly extracting, purifying and cloning target gene by using closed circular double-stranded dna

Info

Publication number: WO2006080136A1
Application number: PCT/JP2005/021603
Authority: WO
Inventors: Taira Enomoto
Original assignee: Gene & Gene Technology Ltd.
Priority date: 2005-01-31
Filing date: 2005-11-24
Publication date: 2006-08-03
Also published as: JP2008099556A

Abstract

A method of quickly extracting, purifying and cloning a target gene by using, as a target, a sample containing a completely closed circular double-stranded DNA (a super coiled circular DNA) free from any DNA cleavage (nick) site from among samples containing the target gene such as a cDNA plasmid library which may occur in three states, which is characterized by comprising hybridizing a probe DNA complex particle having a probe DNA sequence and a solid layer particle with the closed circular double-stranded DNA, recovering the resulting closed circular double-stranded DNA having the DNA sequence of the target gene integrated thereinto, which is in the form allowing direct transfection into host cells, in the solid layer particle, eluting it with an eluting solution, transferring it into a host cell and thus quickly extracting, purifying and cloning the target gene.

Description

Specification

Rapid extraction, purification, and cloning of target genes using closed circular double-stranded DNA

Technical field

[0001] The present invention relates to a method mainly for rapid extraction, separation and purification, detection, cloning, etc. of a target gene from a genomic gene library or cDNA library. Background art

[0002] In order to isolate the target gene, the hybridization method of the probe DNA bound to the solid particle and the target gene (Patent Document 1) or the probe bound to the solid particle A method of extracting the target gene into a solid layer using a bridge nucleotide having a sequence complementary to both the DNA and the target gene (Patent Document 2), linking the biotin to one end of the oligonucleotide complementary to the target gene, and A method (Non-patent Document 1) for extracting a target gene from avidin-binding particles using a specific binding reaction between avidin and avidin has been developed and used. However, in both of these methods, there is a single-strand DNA sample that identifies the physical and physical properties of the target DNA molecule, that is, the target gene to be detected! Single-stranded linear DNA (open circle DNA) force closed circular double-stranded DNA (super

coiled or closed circular DNA) is not specified, and as a result, the extracted target gene DNA is almost linear DNA as a result of these methods. It is almost impossible to extract DNA in the form of DNA (closed circular DNA) that can be introduced into a host and rapidly cloned in large quantities.

[0003] These shortcomings are expressed as follows: "Escherichia coli, which has the property of producing only single-stranded circular DNA, was synthesized and extracted as single-stranded circular DNA, and the other complementary strand using the target gene primer. `` To make a form that can be introduced into E. coli by synthesizing the enzyme in vitro and making it into double-stranded DNA, '' a method that can be introduced into E. coli and mass-purified and cloned can be developed and commercialized. (Patent Document 3). However, this method uses single-stranded circular DNA Therefore, it involves a complicated extraction process and enzymatic synthesis process of re-double-stranded DNA, and there are drawbacks in terms of speed and accuracy such as causing a synthesis error.

Patent Document 1: Japanese Patent Laid-Open No. 8-89294

Patent Document 2: International Publication WO2004-101785

Patent Document 3: US Patent No. 5500356

Non-patent document 1: “Biomagnetic Techniques in Molecular Biology”, Dynal, 1995

Disclosure of the invention

Problems to be solved by the invention

[0004] In the above-mentioned "oligonucleotide probe method bound to a solid layer particle", "avidin 'biotin probe method" and "bridge nucleotide probe method", the shape of the probe for extracting the target gene is specified. The shape of the DNA to be extracted is not specified. However, DNA libraries such as the extracted plasmid (plasmid) cDNA library, plasmid genome DNA, and cosmid (cosmid) genomic library are characterized by the nature of their DNA: 1) super coiled DNA, It exists in the form of 2) open circle DNA and 3) linear DNA. Therefore, if the target gene is isolated and extracted using DNA that is a mixture of these three DNA shapes without specifying the physical and physical shape of the target DNA, the properties of the DNA hybridization In the above three DNA forms, almost only 2) and 3) open circular DNA, linear single-stranded DNA derived from linear DNA or single-stranded circular DNA is separated and extracted. However, the linear single-stranded DNA or single-stranded circular DNA isolated and extracted in this way is suitable for verifying the presence of the target gene. It is impossible or much more labor and time to introduce into a cell and clone the gene of interest from here. In addition, the “gene cloning method based on double-stranded circular synthesis from single-stranded circular DNA” (Patent Document 3) requires a complicated in vitro enzyme synthesis process after extraction, which is complicated, inaccurate, and expensive. Cost is also a problem.

[0005] In addition, in the conventional bridged nucleotide probe method, the necessity of “difference between two Tm values” is ignored, but this difference in Tm value greatly affects the extraction and purification efficiency of the target gene. This is a very important factor in gene extraction / purification 'cloning'.

In addition, in these conventional methods, the base point is 0 to 7 bases upstream in the upstream region adjacent to the base sequence in the target gene corresponding to the base sequence complementary to the target gene in the probe DNA-binding particle or bridge nucleotide. 50-base single-stranded DNA (upstream blocking sequence; upstream

Blocking sequence, Up-BS) and 20-50 base sequence single-stranded DNA (downstream blocking sequence, Down-BS) starting from 0-7 base downstream downstream Hybridization force between the denatured DNA sample containing the target gene and the probe DNA is hindered by the rehybridization of the DNA in the sample, resulting in a drastic decrease in the recovery efficiency of the target gene DNA.

[0006] Furthermore, in these conventional methods, the physical and physical properties of the target DNA must be specified, and therefore, the conditions for denaturation of double-stranded DNA prior to hybridization with a DNA probe are completely eliminated. Therefore, DNA denaturation while maintaining the properties of a closed circular double-stranded DNA that can be cloned into E. coli has not been realized. Therefore, the conventional method can extract and detect a part of the target gene, but it is almost impossible to introduce it into E. coli and to purify and clone it in large quantities.

[0007] In addition, detection of target genes by hybridization between linear DNAs 'Extraction' purification usually uses 400-600 mM as the optimum salt concentration, but this salt concentration is denatured and partially single-stranded. Closed circular DNA (two-leaf clover-like DNA; two_leaf

clover DNA) rapidly undergoes self-pairing (association) and closes again in a closed circle before hybridization with the probe DNA, and cannot be captured by the probe DNA particle, resulting in the target gene cloning. Yield reduction · There is a problem that causes a drastic decrease.

Means for solving the problem

[0008] The present invention relates to DNA cleavage (nick) of any of the samples containing the gene of interest in the cDNA plasmid 'library or the genomic' plasmid 'library or the cosmid' library in the three existing forms. Rapid extraction and purification of target genes using completely closed double-stranded DNA without a site as a sample target. The target gene DNA sequence is obtained by hybridizing the closed circular double-stranded DNA to the probe DNA complex particle containing a DNA sequence (probe DNA sequence) complementary to at least a part of the base sequence and a solid particle. The closed circular double-stranded DNA in which DNA is incorporated is collected into solid-layer particles in a form that can be directly transferred to the host cell, eluted with an elution solution, and introduced into a host cell such as Escherichia coli (transformed). Thus, the above-mentioned problem has been solved by a method characterized in that a target gene is rapidly extracted and purified and cloned.

[0009] The present invention is a novel cycle of 5 cycles or more in the range of 50 ° C to 70 ° C. Single-stranded bridge nucleotide probe for probes prepared by the hybridization method DNA binding particle Single-stranded DNA sequence that binds to the probe Single-stranded DNA sequence that includes the binding sequence Complementary probe DNA binding particle force Complementary DNA sequence that is complementary to at least a part of the base sequence of the target gene ( Probe DNA sequence) and a single-stranded bridge nucleotide for probes comprising a single-stranded bridge nucleotide complementary binding sequence for probes of the probe DNA-binding particle, The bridge nucleotide complementary binding sequence has a complementarity of 80% or more and is firmly complementary, or a single-stranded chain bound to a solid particle. Probe DNA binding particle force consisting of partial column force Complementary sequence (Tm value A) complementary to at least part of the base sequence of the target gene and complementary to at least part of single-stranded DNA sequence part of probe DNA binding particle A sequence (Tm value B), and the difference between the two Tm values is at least 8 ° C or more (Tm value B—Tm value A> 8 ° C). One or a single-stranded bridge nucleotide probe for probe DNA sharing particle consisting of a single-stranded DNA sequence part of the probe DNA-binding particle, or at least a part of the base sequence of the target gene A nucleotide containing a complementary DNA sequence (probe DNA sequence) is formed by direct covalent bonding, and as a result, a probe single-stranded bridge nucleotide is directly bonded to a solid particle. Used as composite particles By the method to solve the above problems.

[0010] The present invention also provides, among the single-stranded bridged nucleotide probe DNA-binding particles for probes, a sequence complementary to a part of the base sequence of the target gene (Tm value A) and one of the probe DNA-binding particles. A sequence complementary to at least part of the base sequence of the double-stranded DNA sequence ( Created by hybridizing a single-stranded bridge nucleotide for probe having Tm value B) to the single-stranded DNA sequence part of the probe DNA-binding particle for 5 cycles or more within the range of 50 ° C to 70 ° C The above problem was solved by the above method using the obtained product as probe DNA complex particles.

[0011] The present invention also provides the single-stranded DNA of the probe DNA-binding particle having a partial force of the single-stranded DNA sequence bound to the solid layer particle, among the single-stranded bridged nucleotide probe DNA covalent bond particles for the probe. A single-stranded bridge nucleotide having a sequence complementary to at least a portion of the sequence portion and a sequence of at least a portion of the base sequence of the target gene, and a single-stranded DNA sequence portion of the probe DNA-binding particle; Using a DNA synthesizing enzyme, a nucleotide containing a sequence (probe DNA sequence) complementary to at least a part of the base sequence of the target gene of the single-stranded bridge nucleotide is bound to the probe DNA. The above-mentioned problem was solved by the above method using a product prepared by covalently binding to the single-stranded DNA sequence portion of the particle enzymatically and chemically.

[0012] Further, the present invention relates to a 20-50 base sequence single-stranded DNA based on 0 to 7 bases upstream of the upstream region adjacent to the base sequence of the target gene corresponding to the probe DNA sequence in the probe DNA complex particle. (Upstream blocking sequence, Up-BS) and 20-50 base sequence single-stranded DNA (downstream blocking sequence, Down-BS) starting from 0-7 bases downstream of the adjacent downstream region (more than lOuM, BS The above problem was solved by the extraction, purification, and cloning method (BS method) of the target gene performed in the presence of at least 1000 times the ratio of the number of molecules to the number of target gene molecules.

[0013] Further, in the present invention, a closed circular double-stranded DNA sample containing a target gene is denatured in a 0.1N to 0.4N alkali solution at a low temperature of 0 ° C to 10 ° C for 20 minutes to 1 hour. The above problem was solved by the extraction, purification, and cloning methods of the target gene.

The present invention also provides that a closed circular double-stranded DNA sample containing the gene of interest is treated in the presence of the blocking sequence for 10 to 60 seconds under a temperature condition of 94 ° C to 98 ° C, and then immediately at 0 ° C. The above problems were solved by a method of extracting, purifying, and cloning the target gene, which was rapidly cooled to ˜4 ° C. and denatured.

Furthermore, the present invention relates to a closed circular double-stranded DNA sample containing a target gene as a probe DNA complex. The above problems have been solved by the above-described extraction, purification, and cloning methods that hybridize with body particles at a low salt concentration (50 to 350 mM).

[0014] The present invention also provides a closed circular double-stranded DNA contained in a recovered product from a host cell containing the target gene concentrated by the extraction, purification, and cloning method of the present invention as a target DNA molecule. Extraction, purification, and cloning by the above-described method of the present invention. By the multi-site cloning method, the conventional method is almost impossible or requires a great deal of time and labor. One millionth (0.0001%), 10 million It enabled rapid and reliable cloning of a very small amount of a target gene that exists only with a probability of 1 / (0.00001%).

In this specification, “in a form that can be directly transformed into a host cell” means a DNA form that can be introduced into a host and rapidly cleave the target gene DNA in large quantities, that is, at least fixed to a solid particle. Means that the shape of the closed circular DNA is maintained up to the point of time.

[0015] In this specification, the single-stranded bridge nucleotide complementary binding sequence for a probe consists of a partial force of a single-stranded sequence bound to a solid particle as a probe DNA complex particle for recovering a target gene. Probe DNA binding particle force For probes containing a DNA sequence (probe sequence) complementary to at least a part of the base sequence of the target gene and a sequence complementary to a part of the single-stranded sequence part of the probe DNA binding particle In order to achieve complementary binding to the single-stranded bridge nucleotide for the probe in the single-stranded sequence portion of the probe DNA-binding particle when using the one complementary to the single-stranded bridge nucleotide. Only an artificially created array is meant.

In the present specification, the term “complementation rate” refers to a sequence that has achieved complementary binding to the corresponding single-stranded bridge nucleotide for probes in the single-stranded bridge nucleotide complementary binding sequence for probes. It means the ratio to the entire probe single-stranded bridge nucleotide complementary binding sequence.

[0016] About closed circular double-stranded DNA samples

The closed circular double-stranded DNA sample containing the target gene DNA used in the present invention desirably contains 60% or more, particularly preferably 80% or more of the closed circular double-stranded DNA. Linear single-stranded or double-stranded DNA, open circular double-stranded with at least one nick If the DNA content exceeds 40%, it will be difficult to extract and purify the target gene in a closed circular double strand. As long as the DNA sample satisfies the above conditions, it may be derived from any of cDNA plasmid 'library, genomic DNA library, cDNA or genomic' cosmid 'library. Any method may be used for preparing a DNA sample containing 60% or more of closed circular double-stranded DNA. For example, CsCl method, gel cutting method, DNA adsorption particle membrane (Takara Bio mimi-prep kit) Depending on the method of DNA purification using particles, it is relatively easy to prepare a sample containing more than 80% closed circular double-stranded DNA. In addition, if the ratio of the closed circular double-stranded DNA exceeds 80%, it can be used as a sample for cloning the target gene DNA even if it is contaminated with RNA.

[0017] Probe DNA complex particles

The probe DNA complex particle used in the present invention is not limited as long as it contains a DNA sequence (probe DNA sequence) complementary to at least a part of the sequence of the target gene bound to the solid particle. The following two types are exemplified. One has a sequence complementary to a part of the base sequence of the target gene (Tm value A) and a sequence complementary to at least a part of the single-stranded DNA sequence part of the probe DNA binding particle (Tm value B). However, the difference between the two Tm values is at least 8 ° C (Tm value B—Tm value A> 8 ° C). Probe single nucleotide bridge nucleotide and probe DNA binding particle hybridized probe nucleotide probe Using a DNA synthesizing enzyme such as DNA binding particle (Figure 6) and the other, Taleno 'fragment' DNA 'polymerase (Klenow fragment DNA polymerase), probe the single-stranded DNA sequence portion of the probe DNA binding particle. This is a bridged nucleotide probe DNA covalent particle for probes capable of forming any arbitrary DNA sequence by enzymatically linking nucleotides containing the DNA sequence (Fig. 7). In this specification, the Tm value refers to the temperature at which half of a DNA molecule having a single-stranded complementary base sequence of about 1 to 50 base pairs is complementary and hybridized! / .

[0018] Bridge nucleotide probe DNA-binding particle for probes in which difference between two Tm values is at least 8 ° C or more

For the probe bridge nucleotide probe DNA binding particle, probe D The NA-bonded particles are probed for the target gene bridge nucleotide probe, and the probe is bound to the probe-DNA-bound particle so that it does not dissociate in the extraction process such as hybridization with the DNA-bound particle and subsequent washing. The single-stranded bridge nucleotide complementary binding sequence for complementary binding to the DNA sequence portion of the probe single-stranded bridge nucleotide prepared to bind to the DNA sequence in a complementary manner with a complementation ratio of 80% or more. It is preferable. Alternatively, the complementary binding of the single-stranded DNA sequence portion bound to the surface of the probe DNA-binding particle and the sequence portion complementary to the single-stranded DNA sequence portion of the single-stranded bridge nucleotide for probe (Tm Since value B) is a hydrogen bond that is vulnerable to heat, low salt concentration, and pH fluctuations as an individual chemical bond type, it is complementary to at least part of the base sequence of the target gene in the single-stranded bridge nucleotide. It must have a DNA sequence that forms a value that is at least 8 ° C greater than the Tm value A of the sequence (probe DNA sequence). As long as this condition is met, the probe DNA sequence portion located on the surface of the bridging nucleotide probe DNA-binding particle for the probe is capable of being infinitely long in sequence and length. Hybridization with a single-stranded bridging nucleotide To increase the efficiency of the determination as much as possible, and to minimize the occurrence of non-specific hybridization between the target gene and the single-stranded DNA sequence located on the surface of the DNA binding particle. In this case, it is desirable that the nucleotide sequence is 10 base sequences or more and 100 base sequences or less. Therefore, the single-stranded DNA sequence portion of the probe DNA-binding particle that is necessary to create the bridge nucleotide probe DNA-binding particle for the probe that sufficiently satisfies “Tm value B—Tm value A> 8 ° C” is complementary binding. It is most desirable that the sequence has more dG or dC base sequences with three hydrogen-bonding arms when formed. Ideally, the probe in the single-stranded bridge nucleotide for the probe.

The complementary sequence has a length of 20 bases or 40 bases composed of dG or dC, while being complementary to at least part of the base sequence of the target gene of the single-stranded bridge nucleotide for the probe. It is desirable that the base structure of dGdC content = 50% (± 20%) found in most gene sequences is composed of 20-50 bases. However, depending on the nature of the target gene DNA, it is not bound by these constraints. As a result, two sequences with high specificity to the target gene and “Tm value B—Tm value A> 8 ° C” are obtained. It is possible to satisfy the above conditions sufficiently. The single-stranded bridge nucleotide for a probe used in the present invention has a sequence complementary to at least a part of a single-stranded DNA sequence portion bound to a solid particle on one side, and at least a part of the base sequence of the target gene. And the other sequence (probe DNA sequence). These complementary sequences do not necessarily have to be located at the ends, but are desirably located near the ends to minimize steric hindrance during hybrid formation.

[0019] Bridge nucleotide probe probe prepared by a covalent binding method DNA covalently bonded particles

The probe bridging nucleotide probe DNA covalently bonded particle is obtained by neutralizing the single-stranded DNA sequence portion of the probe DNA-binding particle and the single-stranded bridge nucleotide for the probe, and then synthesizing DNA such as talenow, fragment, DNA, polymerase, etc. Using an enzyme, a nucleotide containing a sequence (probe DNA sequence) that is complementary to at least part of the DNA sequence of the target gene at the 3 'end of the DNA of the single-stranded DNA sequence of the probe DNA-binding particle is chemically directly Enzymatic synthesis' created by covalent linkage. In this probe nucleotide probe DNA covalently bonded particle, the nucleotide containing the probe DNA sequence is covalently bound to the surface of the solid particle, so that any temperature below 100 ° C, any salt concentration, 1N It is extremely resistant to (alternative) alkali and acid solutions, and is an optimal probe for gene DNA extraction 'purification' crawling via hybridization. Probe bridge nucleotide probe Probe in the DNA covalent bond particle Single-stranded DNA sequence of the DNA-bonded particle The base sequence of the DNA sequence is also complementary to at least part of the base sequence of the target gene in the probe single-stranded bridge nucleotide Any base sequence of the probe sequence is effective. In order to avoid the non-specific binding of DNA and the efficiency of enzyme synthesis, the bridge nucleotide probe for the probe Probe of the DNA covalently bonded particle The base sequence of the DNA sequence is free The length is 5 to several hundred bases An array is preferred.

[0020] The solid particle to which the probe DNA sequence is bound is an organic polymer such as a latex bead in both the bridge nucleotide probe DNA binding particle for probe and the bridge nucleotide probe DNA covalent bond particle for probe. Molecular particles and organic Polymer sheets, particles that have a magnetic material inside and can be separated magnetically, metal particles such as gold, glass particles and plates, and colored particles or fluorescent particles may be used.

[0021] Cycle 'Noise hybridization method

Complementary binding of a single-stranded bridging nucleotide for a probe having a difference between two Tm values used in the present invention of at least 8 ° C or more to a probe DNA-binding particle through a hydrogen bond is a cycle hybridizer. Use the Chillon method. Specifically, a sequence complementary to at least a part of the base sequence of the target gene (probe DNA sequence) and a sequence complementary to at least a part of the base sequence of the single-stranded DNA sequence part of the probe DNA binding particle A single-stranded bridge nucleotide for a probe having a cycle of 5 cycles or more within a temperature range of 50 ° C to 70 ° C in a single-stranded DNA sequence portion of a probe DNA-binding particle. Examples of hybrids:

50 ° C (20 seconds or more) 55 ° C (20 seconds or more) → 60 ° C (20 seconds or more) → 65 ° C (20 seconds or more) → 70 ° C (20 seconds or more) → 65 ° C (20 seconds) ) → 60 ° C (20 seconds or longer) → 55 ° C (20 seconds or longer) → 50 ° C (20 seconds or longer)), and the nucleotide sequence of the single-stranded DNA sequence portion of the probe DNA binding particle Probes in single-stranded bridge nucleotides for probes Can form stronger complementary hydrogen bonds between sequences complementary to at least part of the single-stranded DNA sequence portion of the DNA-binding particle. . Of course, between the single-stranded DNA sequence portion of the probe DNA-binding particle and the base sequence complementary to at least part of the single-stranded DNA sequence of the probe DNA-binding particle in the single-stranded bridge nucleotide for the probe. Complementary hydrogen bond formation is a reaction temperature of 50. Force achieved even by keeping constant temperature such as C, 60 ° C, 70 ° C, etc. The theoretically strongest complementary conjugate among the molecular conjugates produced by keeping these temperatures constant There are molecular conjugates that are hyperpided in the following conditions. This is due to the non-specific binding molecules under the severe temperature and salt concentration conditions during hyperplasia with closed circular double-stranded DNA containing the target gene. The presence of such incomplete conjugates considerably reduces the efficiency of the 'extraction' cloning of the target gene, as it is destined to be dissociated and lost together during washing. So it is better to avoid it.

[0022] Alkaline low-temperature denaturation method and high-temperature short-time denaturation method of closed circular double-stranded DNA

The DNA sample used in the present invention is simply used for the purpose of extraction and purification or detection of the target gene. In addition, the extracted and purified target gene DNA can be immediately introduced (transfected) into Escherichia coli in physicochemical form, i.e., at least until it is recovered into solid particles. It is necessary to maintain the form of the strand DNA.

Prior to the hybridization between the probe DNA complex particle and the closed circular double-stranded DNA sample containing the target gene DNA, the DNA sample must be denatured with heat or alkali to become single-stranded DNA Three DNA types generated during the sample extraction process (1) closed circular DNA,

2) Open circular DNA, 3) Stranded DNA], as shown in Fig. 1 in the process of denaturation, closed circular DNA takes the form of a partial force S like a _two- leaf clover. Are not completely disjointed, but open circular DNA and strand DNA-and when denatured, the original complementary molecules are completely separated and have many long complementary sequences in part. In the presence of a similar molecule, it will never re-hypli (re-pair) with the same complementary molecule. Therefore, it is impossible to amplify and clone the target gene DNA from the DNA extracted and recovered from the denatured open circular DNA or strand DNA, and introduced directly into the colon. Closed circular double-stranded DNA can also be nicked by high-temperature, long-term treatment (over 2 minutes) such as 70 ° C or higher, or alkaline treatment at room temperature or high temperature, resulting in open circular double-stranded DNA or linear Through the same process as the fate of double-stranded DNA, it dissociates apart. Therefore, mild denaturation conditions are essential for denaturing closed circular double-stranded DNA. In the present invention, in order to realize this, closed circular double-stranded DNA is denatured slowly and mildly at 0 ° C (ice water) in a 0.1 to 0.4 N alkaline solution to thereby open circular double-stranded DNA. Almost 100% denaturation of closed circular double-stranded DNA was achieved while suppressing mechanical, physical, and chemical cleavage of DNA to a negligible level. Actually, almost the same denaturation results can be obtained in the range of 0 ° C to 10 ° C.

In the present invention, a closed circular double-stranded DNA sample containing the gene of interest is also treated in the presence of the blocking sequence for 10 to 60 seconds under a temperature condition of 94 ° C to 98 ° C, and then immediately. The DNA was rapidly cooled to 0 ° C to 4 ° C and denatured to achieve single-stranded DNA in a closed circular double-stranded DNA. This method makes it possible to single-stranded closed circular double-stranded DNA without nicks even at high temperature treatment, and more rapid (time required for all steps including high temperature treatment and cooling; 1-2 minutes ) Closed circular double stranded DNA—allows it to be stranded. The double-leaf clover-type single-stranded DNA derived from the high-temperature treatment of the closed circular double-stranded DNA obtained in the present invention in the presence of the low-temperature alkaline denaturation / blocking sequence is Rapidly self-associates and returns to the original closed circular double-stranded DNA.

[0024] Adjacent upper / downstream region 20-100 bases single stranded DNA (upstream, downstream blocking sequence, Up-BS, Down-BS)

In the extraction, purification, and cloning methods in the present invention, the efficiency can be further increased by using the BS method described in this specification together. In other words, with respect to the BS method, single-stranded DNA of 20 to several hundred base sequences (upstream) from 0 to 7 bases upstream and downstream of the upstream and downstream regions adjacent to the target sequence of the target gene. , Downstream blocking sequence, Up-BS, Down-BS) force Dependent on the salt concentration and temperature of the double-leaf clover-type single-stranded DNA derived from low-temperature alkaline denaturation of the closed circular double-stranded DNA obtained in the present invention It has a physicochemical property that slows the rate of self-association. Closed circular double-stranded DNA (two-leaf clover-type single-stranded DNA) denatured at low temperature with the salt concentration and temperature of the hybridization solution is the original closed Utilizes the property of slowing the speed of returning to circular double-stranded DNA, resulting in increased binding between the target DNA and the probe DNA immobilized on the solid layer. Here, the length of the base sequence of 20 to several hundred base sequences of single-stranded DNA in the upstream and downstream regions is effective by about 20 to 100 base sequences, but if it is too long, it itself becomes nonspecific binding Therefore, it is desirable to have a 20-50 base sequence. In addition, the concentration of 20-50 base sequence single-stranded DNA in the adjacent upstream and downstream regions should be at least 1000 times the ratio of the number of BS molecules to the number of target gene molecules, usually at least lOuM or more. A sufficiently large increase in cloning can be obtained.

[0025] As shown in FIG. 2, 20 to several hundred base sequences based on 0 to 7 bases upstream (base sequence space between probe DNA and BS) upstream of the adjacent upstream region of the target gene sequence corresponding to the probe DNA Single-stranded DNA (upstream blocking sequence, Up-BS) and 0 to 7 bases downstream of the adjacent downstream region (base sequence space between probe DNA and BS) 20 to several hundred base sequences single-stranded DNA ( Two types of downstream blocking sequences (Down-BS) are possible, and combinations of the force applied to the sample are Up-BS-1 alone, Up-BS-2 alone, Down-BS-1 alone, Down-BS -2 alone, mixed Up-BS-1 and Down-BS-1, mixed Up-BS-1 and Down-BS-2, up-BS-2 and Down-BS-1 Eight combinations of mixed, Up-BS-2 and Down-BS-2 are possible, both of which are effective in increasing the efficiency of 'purification' clawing and extracting target genes and DNA. The combination can be selected depending on the base sequence that is the target of the cloning, but a preferred example is a mixture of Up-BS-1 and Down-BS-2.

[0026] Hybridization under low salt concentration

In the present invention, when the BS method is used in combination, if the salt concentration of the hybridization solution is set to 300 mM or more, the effect of increasing cloning by 20-50 base sequence single-stranded DNA in the adjacent upstream and downstream regions Drops considerably. On the other hand, if the salt concentration of the hybridization solution is kept between 100 and 200 mM, the effect of increasing the cloning efficiency by 20 to 50 base sequence single-stranded DNA in the adjacent upstream and downstream regions can be obtained. Therefore, the salt concentration of the hybridization solution used in the present invention is a force of 50 to 350 mM, preferably 100 to 250 mM, which is possible even at 400 to 600 mM commonly used in ordinary Sarzanno and hybridization. It is desirable to be between.

[0027] Multi-cycle cloning method

In the multi-cycle cloning method of the present invention, the closed circular double-stranded DNA contained in the recovered product from the host cell containing the target gene is concentrated by the extraction, purification and cloning method of the present invention. By repeating the extraction, purification, and cloning system of the present invention as a molecule in a short period (2-4 days), the target gene DNA that exists only in a very small amount can be rapidly and reliably cloned.

The invention's effect

[0028] By the separation, purification, and cloning method of the present invention, closed circular double-stranded DNAs of all origins, sizes, sequences, etc. including the target gene can be separated, purified, and cloned quickly, accurately, and efficiently. -It becomes possible to In addition, by using the method of the present invention, it is possible to complete a clawing operation that conventionally required several days of power for several weeks in only one day (within 24 hours).

Brief Description of Drawings

[0029] FIG. 1 is a diagram showing three types of DNA generated in the process of extracting a DNA sample. FIG. 2 is a diagram showing the positional relationship between a target gene sequence and Up-BS and Down-BS sequences. Taking double-stranded DNA as an example, the base position is the position of the target gene base sequence complementary to the probe DNA of the target gene DNA, and 0 to 7 bases upstream of the adjacent upstream region of the target gene sequence complementary to the probe DNA. 20 to several hundred base sequence single-stranded DNA (upstream blocking sequence, Up-BS) and 20 to several hundred base sequence single-stranded DNA (downstream blocking sequence, Dn- BS) position and probe Position of DNA binding particles.

FIG. 3 is a diagram showing the results of analysis of DNA samples (pCY and pGBM) purified by the SDS Al-CsCl method.

Sample 1 shows the analysis results of the plasmid pCY DNA sample obtained by the SDS-Al force method (contamination of open circular (op) DNA in addition to closed circular (sp) DNA). Sample 2 shows the analysis results of the plasmid pGBM DNA sample obtained by the SDS-Al force method (in addition to closed circular (sp) DNA, open circular (op) DNA contamination is observed). Sample 3 shows the analysis result of the DNA sample of plasmid pCY obtained by SDS / Al force / CsCl method (consisting only of 98% or more of closed circular (sp) DNA). Sample 4 shows the analysis result of the DNA sample of plasmid pGBM obtained by SDS · Al force · CsCl method (more than 98% is composed of closed circular (sp) DNA only). Sample pCY-R shows the analysis result of non-RNA-removed DNA sample of plasmid pCY obtained by SDS-Al force method (in addition to closed circular (sp) DNA and open circular (op) DNA contamination. The amount is less than 10%, and a large amount of total RNA is seen). Sample pGBM-R shows the analysis result of non-RNA-removed DNA sample of plasmid pGBM obtained by SDS-Al force method (in addition to closed circular (sp) DNA and open circular (op) DNA contamination. The amount is less than 10%, and a large amount of total RNA is seen).

[FIG. 4] A diagram showing the results of analysis of a DNA sample (mouse brain cDNA plasmid library) purified by the SDS Al force method and SDS Al force method CsCl method.

Sample 5 shows the analysis results of mouse brain cDNA plasmid library obtained by SDS-alkali method (the ratio of open circle (op) is higher than closed circle (sp)). Sample 6 shows the analysis result of mouse brain cDNA plasmid library obtained by SDS-alkali method (the ratio of closed circle (sp) is higher than open circle (op)). Sample 7 shows the analysis results of mouse brain cDNA plasmid library obtained by SDS-alkaline. CsCl method (closed circular (sp) is 95% or more and open circular (op) is very small). Sample 8 is a mouse brain cDNA plus replacement kit (Rule 26) obtained with the Takarabio Mini ^A prepDNA extraction kit. The analysis results of the Mid 'library are shown (the ratio of closed ring (sp) (approximately 80%) is higher than open ring (op) (approximately 20%)).

[FIG. 5] A diagram showing the results of detection of a cilitestin cDNA fragment (270 base pairs) by PCR.

The top shows the PCR results for white colonies and the bottom shows the PCR results for blue colonies. [(Ciriestine 'Primer 1; 5'-ACCCAGATAGGCAGTGTACAGA-3' (1508-1529) (Sequence No. 3 2) / Ciritestine 'Primer 2;

5'-TGTCATCTCTAGTCCCAGTCTG-3 '(1777-1756) (SEQ ID NO: 3 3)]

FIG. 6 is a diagram showing the relationship between Tm value A and Tm value B in a probe single-stranded bridge nucleotide.

The figure shows examples of DNA of DNA particles (Tm value B) and single-stranded bridged nucleotide (BN-CY) DNA (Tm value A) for pCY probes. (DNA sequence on the DNA particle side (30mer); SEQ ID NO: 3 4 BN-CY DNA sequence; SEQ ID NO: 3 5)

FIG. 7 is a diagram showing a method for producing a bridge nucleotide DNA covalent bond particle for a probe.

Single-stranded bridge nucleotides for probes Enzymatic synthesis of DNA particles to the 3 'end of DNA particles Covalent synthetic synthesis DNA probes (ridge nucleotides DNA covalently bonded particles) One example of preparation is a probe for pCY Single-stranded bridge nucleotides (BN -CY) Take DNA as an example. Talenou fragment Bridging nucleotide (BN-CY-2) by DNA synthesizing enzyme such as DNA polymerase After synthesizing bridge nucleotide complementary to DNA, DNA is denatured and washed with 0.2N NaOH to form hydrogen bond single strand The probe nucleotide is removed to obtain a bridge nucleotide DNA covalent bond particle for probe. (DNA sequence (30 mer) on the DNA particle side from the top of the sequence; SEQ ID NO: 3 6 / BN-CY-2 DNA; SEQ ID NO: 3 7 / Enzymatically synthesized bead-bound single-stranded synthetic complementary bridge nucleotide; SEQ ID NO: 3 8 / BN-CY-2 DNA; SEQ ID NO: 39 / Enzymatically synthesized bead covalently linked bridged nucleotide DNA; SEQ ID NO: 40)

FIG. 8 is a diagram showing the results of cloning of actin gene cDNA plasmid clones according to the method of the present invention.

Lane 1 shows the electrophoresis marker (Eh / Hind ΠΙ), Lane 2 shows the sample obtained by cleaving the actin gene clone 1 DNA cloned by BS method with restriction enzymes EcoRl and Notl, and Lane 3 shows A sample obtained by PCR of DNAO.lng of actin gene clone 1 with a lactin primer is shown. Lane 4 is a replacement paper for the BS method (Rule 26). A sample obtained by cleaving the DNA of the actin gene clone 2 with restriction enzymes EcoRl and Notl is shown.Lane 5 shows a sample obtained by PCR of the actin gene clone 2 DNAO.lng with the actin primer. Shown below is a sample of the crude extract obtained by PCR with actin primers. In the figure, the black arrow shows the actin gene cDNA of about 1.26k base pairs cloned by the BS method of the present invention, and the white arrow shows the PCR of the actin gene cDNA clone cloned by the BS method. The 730 base pair PCR product obtained is shown, and the asterisk indicates the plasmid vector 5.3 k base pair DNA of two actin gene cDNA plasmid clones cloned by BS method.

FIG. 9 is a diagram showing the results of cloning an HPRT gene cDNA plasmid clone according to the method of the present invention.

Lane 1 shows an electrophoresis marker (λ / Hind IE), Lane 2 shows a sample obtained by cleaving HPRT gene clone 1 DNA cloned by BS method with restriction enzymes EcoRl and Notl, Lane 3 Shows a sample obtained by PCR of DNAO.lng of HPRT gene clone 1 with actin primer, lane 4 shows a sample obtained by cleaving the DNA of HPRT gene clone 2 cloned by BS method with restriction enzymes EcoRl and Notl, lane 5 Shows a sample obtained by PCR of HPRT gene clone 2 DNAO.lng with actin primer, and lane 6 shows a sample obtained by PCR using a crude extract extracted by the BS method with HPRT primer. In the figure, the black arrow indicates the HPRT gene cDNA of about 1.2k base pairs cloned by the BS method of the present invention, and the white arrow indicates the HPRT gene cloned by the BS method. A PCR product of 944 base pairs obtained by PCR of the DNA clone is shown. The asterisk indicates the plasmid vector of two HPRT gene clones cloned by BS method. Show

FIG. 10 shows the results of cloning of HPRT gene cDNA plasmid by the method of the present invention.

An HPRT gene cDNA plasmid clone obtained by cloning a closed circular mouse brain cDNA plasmid sample by the method of the present invention using a probe nucleotide DNA covalent bond particle is shown. Lane 1 shows the migration target (Hind IE), Lane 2 shows a sample of the cloned HPRT gene clone 1 DNA cut with restriction enzymes EcoRl and Notl, and Lane 3 shows the HPRT gene clone 1 A sample of DNAO.l ng of PCR with aactin primer is shown, lane 4 is the replacement paper 0¾-rule 26) The cloned HPRT gene clone 2 DNA was cleaved with restriction enzymes EcoRl and Notl.Lane 5 shows the HPRT gene clone 2 DNAO.lng PCR sample with actin, and lane 6 is the cloning. Lane 7 shows a sample obtained by cleaving HPRT gene clone 3 DNA with restriction enzymes EcoRl and Notl.Lane 7 shows a sample obtained by PCR with DNAO.lng of HPRT gene clone 3 using actin primer. Lane 9 shows a sample obtained by cleaving HPRT gene clone 4 DNA with restriction enzymes EcoRl and Notl.Lane 9 shows a sample obtained by PCR of DNAO.lng of HPRT gene clone 4 with actin primer, and lane 10 shows A sample of the cloned HPRT gene clone 5 was cleaved with restriction enzymes EcoRl and Notl. Lane 11 shows the HPRT gene clone 5 DNAO.lng shows the sample PCR in § Kuching primer. In the figure, the black arrow indicates the HPRT gene cDNA of approximately 1.2k base pairs cloned by the BS method of the present invention, and the white arrow is obtained by PCR of the HPRT gene cDNA clone cloned by the BS method. The 944 base pair PCR product is shown, and the asterisk indicates the 5.3 k base pair DNA of the four HPRT gene cDNA plasmid clones cloned by BS method.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention in one example

1) Preparation of more than 80% closed circular double-stranded DNA sample containing the target gene,

2) Preparation of probe DNA or probe complex particles corresponding to at least a part of the base sequence of the target gene;

A) A sequence complementary to at least a part of the base sequence of the target gene (Tm value A) and a sequence complementary to at least a part of the base sequence of the single-stranded DNA sequence part of the probe DNA-binding particle (T

用 for difference (Rule 26) a single-stranded bridge nucleotide for a probe having a m value B) and a difference between both Tm values of at least 8 ° C (Tm value B—Tm value A> 8 ° C) with the probe DNA-binding particle. Preparation of single-stranded bridged nucleotide probe DNA-binding particles for probes by hybridization; B) Single-stranded DNA sequences of probe DNA-binding particles such as oligo (dG) latex beads (oligo (dG) latex bead) Hybridize a single-stranded bridge nucleotide that has at least a part of the sequence complementary to this part and at least a part of the base sequence of the target gene. Using a DNA-synthesizing enzyme such as DNA polymerase, a nucleotide containing a sequence (probe DNA sequence) complementary to at least a part of the base sequence of the target gene is enzymatically synthesized in the probe DNA-binding particle. Both By binding, making the direct bonded bridge nucleotide probe DNA covalently bonded particles probe to the solid layer particles through the probe DNA sequence power single-stranded DNA sequence portion,

[0031] 3) 20 to several hundreds based on 0 to 7 bases upstream (space base sequence between probe DNA and BS) upstream of the sample containing the target gene and the target gene sequence corresponding to the probe DNA sequence Single-stranded DNA (upstream blocking sequence, Up-BS) and 20 to several hundred base sequence single-stranded DNA (downstream blocking sequence, D own-BS) starting from 0 to 7 bases downstream of the adjacent downstream region A mixture of

4) Low-temperature alkali denaturation and neutralization of samples containing the target gene and Up-BS and Down-BS mixed solutions,

5) Hybridization of the sample mixture solution containing the denatured and neutralized target gene, Up-BS, Down-BS and the probe DNA complex particles under low salt concentration conditions,

6) Recovery of probe DNA complex particles bound to the target gene by washing and centrifugation

7) Elution of target gene with elution solution 'Extraction' recovery,

8) Cloning of the target gene by transfection into E. coli, etc.

9) Verification of target gene cloning by PCR or restriction enzyme digestion,

It consists of each process.

[0032] Extraction and purification of the target gene DNA of the present invention The 'cloning method' can be completed in only one day (within 24 hours). Below, an Example and its result are shown about each step.

[Example]

Hereinafter, specific examples of the present invention will be described, but the present invention is not limited thereto.

[Example 1] Relationship between the ratio of closed circular double-stranded DNA in a sample containing a target gene and the cloning efficiency of the target gene

This example was performed using a purified mixed DNA sample solution of plasmid pGBM (3035 base pairs) forming blue colonies and plasmid pCY (3768 base pairs) incorporating 734 base pair cDNAs forming white colonies. It was.

Plasmid pGBM carries the 13-galactosidase gene and forms blue colonies on agar in the presence of 13-gal (5-bromo-4-chloro-3-indolyl-13-D-galactoside) To do. On the other hand, plasmid pCY the mouse testis expressed gene Shiritesuchin _{_{_{(C yrit es tin) C D}}} NA730 bp DNA the plasmid pGBM j8 - a incorporated into Garatatoshidaze gene site recombinant plasmid, forming a white colony on agar. Because of this difference, the model system has the advantage that the efficiency of the extracted 'purified' clawing can be tested by the colony type force formed on the agar.

[0033] pCY is derived from the mouse testis cDNA library by amplifying 1044- 1777 between citristin cDNA (approximately 2650 base pairs in size) by PCR, extracting it, and inserting it into the β-galatatosidase gene of pGBM. Produced.

Extraction of the target gene from the DNA sample containing the target gene The method of 'purification' cloning and the power to indicate the result Preparation of a DNA sample containing the closed circular DNA of 80% or more (including the target gene DNA) The SDS-alkali method, SDS-alkali method-CsCl ultracentrifugation method, DNA sample gel cutting method, DNA adsorption method using DNA adsorbed particle membrane or particle, etc. can be used. Methods-Examples using CsCl ultracentrifugation, SDS-alkali method-DNA adsorption particle membrane method.

[0034] 1) Method for purifying pCY, pGBM and mouse brain cDNA plasmid library containing more than 80% closed circular DNA

SDS-alkali method, SDS-alkali method, CsCl ultracentrifugation method, SDS-alkali method, DNA adsorption membrane Law>

<SDS-alkali method>

(1) The pCY and pGBM and mouse brain cDNA plasmid libraries were transfected into E. coli, and each of the resulting plasmid colonies was cultured in large quantities.

(2) Centrifugation at 5000rpm'4 ° C 'for 15 minutes, collect the cells, and dissolve in 2ml of Solution A (25mM Tris · Η. 1 〈ρΗ = 8.0〉, 10mM EDTA, 50mM glucose) cooled to 0 ° C did.

(3) Solution B (20mg lysosome / solution A) 0.5ml was added and left on ice for 10 minutes.

[0035] (4) Solution C (1% SDS, 0.2N

NaOH) 5 ml was added, stirred gently and allowed to stand on ice for 7 minutes.

(5) Solution D (29.4 g K'acetate and 11.5 ml acetic acid in 100 ml pure water) was added to 4 ml, stirred gently, and allowed to stand on ice for 10 minutes.

(6) Centrifugation at lOOOOrpm or more '4 ° C' for 90 minutes or more, and the supernatant was collected through a funnel with 1 to 2 Kimwipes.

(7) Two times the amount of ethanol cooled to -20 ° C was added to the supernatant, and centrifuged at 10000rpM for '4 ° C' for 20 minutes to collect the precipitate.

(8) The precipitate containing the sample DNA was dissolved in 4 ml of TE (10 mM Tris pH = 8.0>, ImM EDTA).

[0036] <CsCl ultracentrifugation>

(9) 4.2 ml of CsCl and 80 ul of 10 mg / ml ethidium bromide (EtBr) solution were added to 4 ml of the sample DNA solution, stirred well, placed in a tube for CsCl ultracentrifugation, and the tube was capped.

(10) A centrifuge tube with a sample placed in a vertical rotor for a Beckman ultracentrifuge was set and centrifuged at 20 ° C.'55000 rpm for 18 hours.

(11) Of the two DNA bands formed (Azpa 'band and Lower' band), the lower band containing mainly closed circular DNA was recovered using an 18-21 gauge needle, and TE EtBr was removed with 1-butanol saturated with SS-1-butanol.

(12) A 2- to 2.5-fold volume of ethanol cooled to 20 ° C was placed on the DNA sample from which EtBr had been removed, and centrifuged at 4 ° C for 20 minutes or more at lOOOOrpm to collect the precipitate.

(13) An appropriate amount of TE solution was added to the collected precipitate to prepare a DNA sample containing closed circular DNA. [0037] <SDS-Alkaline method 'DNA adsorption membrane method>

(A) Take an appropriate amount of DNA sample extracted by SDS-alkaline method and prepare Miniprep from Takara Bio.

(B) Put the washing solution into the silica membrane column and centrifuge.

(C) Add the washing solution to the silica membrane column again and centrifuge.

(D) Add lOOul of ImM Tris lmMEDTA (pH 8.0) solution to the silica membrane column, set in a 1.5 ml tube, and centrifuge.

(E) The DNA sample eluted in the 1.5 ml tube was used as the DNA sample of the present invention hereinafter.

FIG. 3 shows pCY and pGBM purified by SDS-alkali method-CsCl ultracentrifugation. The SDS-alkali method sometimes yields DNA samples containing 90% or more of closed circular DNA, but it is not stable. If purified by the SDS-alkali method-CsCl ultracentrifugation method, more than 95% of the closed circular DNA is obtained. A sample containing is obtained. In addition, for samples in which RNA has not been removed after extraction by the SDS-alkali method, samples consisting of 90% or more of closed circular DNA (sample pCY_R, sample pGB M-R) can be stably obtained.

[0039] Figure 4 shows the SDS-alkali method (samples 5 and 6), SDS 'alkali method .CsCl method (sample 7), SDS' alkali method ', DNA adsorption membrane method, and Takara Bio Co., Ltd. mini-prep kit. > Migration result of mouse brain cDNA plasmid library purified in (Sample 8). In the sample 5 by SDS-alkali method, the closed circular DNA is 20% or less, and the open circular DNA is 80% or more, and the target circular DNA that is the target DNA molecule of the present invention is considerably small. On the other hand, in another sample 5 by the same SDS 'alkali method, the closed circular DNA was about 60%, exceeding 50% of the total DNA. Sample 7 is a sample 6 purified by SDS 'alkali method CsCl method and further purified by CsCl ultracentrifugation, and the closed circular DNA exceeds 95%. Sample 8 was purified by the SDS “alkali method” DNA adsorption membrane method using a Takara Bio Co., Ltd. mini-prep kit, and contains 80% or more of the closed circular DNA which is the target DNA of the present invention.

[0040] Extraction of target gene “Single-stranded bridge nucleotides for probes for pCY (BN-CY)” used for “purification” cloning, “oligo (dG) latex that hybridizes with polynucleotide of bridge nucleotide BN-CY” `` Bead '', `` Probe The target gene (corresponding to the DNA sequence part of the lobe) Blocking sequence upstream and downstream of the DNA sequence (BS) j and the respective preparation methods

[0041] The probe DNA sequence for pCY extraction, the blocking sequences (Up-BS and Down_BS) located above and downstream of the corresponding target gene DNA sequence, and the position and sequence of the base sequence for PCR are shown below. Show.

<Single-stranded bridge nucleotide for probe

Preparation of (BN-CY)>

[0042] [Chemical 1]

5 '-GTACTCTGATTGATGCTGCGCAGTGTGGTACCCCCCCCCCCCCCCCCCCCCCCCCCCCCC-3' (SEQ ID NO: 1)

[0043] pCY Insertion A bridge oligonucleotide sequence (BN-CY) composed of 30 bases 1286-1315 of the cDNA fragment and poly C30 base was synthesized and used as probe DNA for pCY extraction.

Oligo (dG) latex 'beads purchased from JSR Corporation were used. The BN-CY probe hybridizes with the latex 'bead oligo (dG) at the poly C portion and is fixed to the bead solid layer.

[0044] <Blocking sequence (BS) upstream and downstream of the DNA sequence of the target gene corresponding to the probe base sequence portion>

Single-stranded Up-BS (upstream blocking sequence)

[0045] [Chemical 2]

5 '-ACTCATAAAAAATGCTGTAACCCTAAAGAC-3 * (SEQ ID NO: 2)

[0046] Insertion of pCY 30 bases from 1255-1384 of the cDNA fragment were synthesized and used.

Single-stranded Down-BS (downstream blocking sequence) [0047] [Chemical 3]

5 '"CTACATGTTCTTTTATCACAGCATGGCCCT-3'

(SEQ ID NO: 3)

30 bases of 1346-1317 (minus strand) of the inserted cDNA fragment of pCY were synthesized and used.

[0048] [Chemical 4] Siritestin 'Primer 1: 5' -ACCCAGATAGGCAGTGTACAGA-3 '(1508-1529)

Siritestin 'Primer 2: 5' -TGTCATCTCTAGTCCCAGTCTG-3 '(1777-1756)

(Top; SEQ ID NO: 4 / Bottom; SEQ ID NO: 5)

[0049] <Extraction of target gene plasmid pCY>

1) lOOuM probe single-stranded bridge nucleotide (BN-CY) 2ul, lOOmM NaCl-containing 20ul oligo (dG30mer latex, mixed with beads, temperature 5 to 70 ° C Cycles and hybridizing method [One cycle · Hybridization example: 50 ° C (20 seconds or more) → 55 ° C (20 seconds or more) → 60 ° C (20 seconds or more) → 65 ° C (20 seconds ) → 70 ° C (20 seconds or longer) → 65 ° C (20 seconds or longer) → 60 ° C (20 seconds or longer) → 55 ° C (20 seconds or longer) → 50 ° C (20 seconds or longer)) Then, both were neutralized and then washed 5 times with a washing solution containing lOOmMNaCl warmed to 45 ° C to obtain a complex of BN-CY and oligo (dG) latex beads. To this, a hybridization solution containing 23 ul of 200 mM NaCl was added and placed in a 50 ° C. water bath.

[0050] 2) Mix lul lOOOuM Up-BS and lul lOOOuM Down-BS, 1 to 100, 1 to 10000 mixed pCY and pGBM 2ul, distilled water 5ul, 0.4N NaOH 9ul, about 0 After denaturation (= single-stranded) by treatment at ° C (on ice) for 40 minutes, an equivalent amount (9 ul) of 0.4NHC1 was added for neutralization. The purified closed circular pCY is cleaved with the restriction enzyme Sacl, and the closed circular pCY and the Sacl cleaved pCY are mixed at a ratio of 10: 0, 9.0: 1, 7.5: 2.5, 5.0: 5.0 to obtain linear DNA. This was used as a pCY for pCY: pGBM mixed sample.

[0051] 3) Add the denatured 'neutralized pCY' pGBM and BS mixed solution to the BN-CY and oligo (dG) latus status' bead complex solution prepared in 1). Incubate for 30 minutes, then wash 4 times, 2 times and 1 time with washing solution containing 100 mM, 50 mM and 25 mM NaCl, respectively, warmed to 50 ° C. Washed.

4) 50 ul of 10 mM Tris-ImM EDTA solution was added to the resulting oligo (dG) latex-bead-BN-pCY complex, heated at 70 ° C. for 10 minutes, and ice-cooled. The supernatant was collected by centrifugation.

[0052] <Detection of extracted pCY>

Add 5 ul of the separated and purified pCY solution to 100 ul of competent E. coli, treat on ice for 20 minutes, 42 ° C for 50 seconds, and again on ice for 2 minutes. The cells were cultured for C1 hours, spread on an agar medium containing lOOug / ml ampicillin and j8-gal, and cultured for 16 hours in a 37 ° C incubator. A blue colony having pGBM and a white colony having pCY were counted, and the number and ratio of the colonies were determined. Seven colonies were picked and the 730 base pair DNA sequence inserted into pCY was tested by colony PCR. PCR was performed at 94 ° C. for 3 minutes, 94 ° C. for 30 seconds, 56 ° C. for 30 seconds, and 72 ° C. for 30 seconds for 30 cycles.

[0053] From Table 1, samples containing 90% or more of closed circular double-stranded DNA are very high in the presence of upstream blocking sequence (Up-BS) and downstream blocking sequence (Down-BS)! With this, the target gene (pCY) can be crawled. However, it can be seen that when the ratio of closed circular double-stranded DNA in the sample DNA containing the target gene decreases (the linear or open circular ratio increases), the cloning efficiency of the target gene is greatly decreased. Even in the absence of a blocking sequence, the sample containing 90% or more of the closed circular double-stranded DNA is more efficient in cloning the target gene than the sample with a high linear or open circular rate under the same conditions. Is found to be high. In addition, as long as the sample contains a lot of closed circular double-stranded DNA, high cloning efficiency is observed in the presence of BS as shown in Table 1 even for samples containing RNA (samples pCY_R, pGBM-R). .

[0054] [Table 1] table 1

Effect of linear and open ring DM on pCY extraction using closed ring DM (compared to conventional method) Molecular ratio of pCY / pGBM Number of colonies obtained in the reaction mixture White co π knee-Concentration rate

(weight ratio of pCY / pGBM) BS concentration (UM) White Blue Ratio (times)

(sp_pCY: Sad-cutpCY / pGBM) Up-BS Down- BS (pCY) (pGBM) (%)

1) 1/100 0 0 72 912 7. 3% 7. 3

(10ng: 0ng / 840ng)

2) 1/100 20 20 942 71 93. 0 ¾ 93. 0

(10ng: 0ng / 840ng)

3) 1/100 20 20 737 102 88. 0% 88. 0

(9ng: lng / 840ng)

4) 1/100 20 20 775 443 63. 6% 63. 6

(7.5 ng: 2.5 ng / 840 ng)

5) 1/100 20 20 1072 1764 37. 8 ¾ 37. 8

(5ng: 5ng / 840ng)

0 0 1 152 0. 6% 65. 3

(0. lng: 0ng / 840ng>

7) 1/10000 20 20 101 78 56. 4% 5640. 0

(0. lng: 0ng / 840ng)

8) 1/10000 20 20 121 154 44. 0% 4400. 0

(0.09ng: 0.01ng / 840ng)

20 20 74 376 16.4% 1640. 0

(0.075ng0.025 / 840ng)

10) 1/10000 20 20 38 1067 3. 4% 340. 0

(0. 05 ng: 0. 05ng / 840ng)

11) 1/100 0 0 98 13903 0. 7% 0.7

(Sample 1 pCYlttag / material 2 pGBM840ng>

12) 1/100 20 20 1173 883 57. 0% 57. 0

(Sample 1 pCYlOng / Sample 2 pGBM840 ng)

13) 1/10000 0 0 23 19875 0. 11% 11. 0

(Sample 1 pCYO.lng / Sample 2 pGBM840ng)

14) 1/10000 20 20 82 791 9. 4 ¾ 940. 0

(PCYO.lng of sample 1 / pGBM840ng of sample 2>

15) 1/100 0 0 54 777 6.5% 6.5

(Amount equivalent to pCYlOng of sample pCY-R / Amount equivalent to pGBM840ng of sample GBM-R)

16) 1/100 20 20 826 133 86. 1% 86. 1

(Amount equivalent to pCYlOng of sample CY-R / equivalent to pGBM840ng of sample pGBM-R »

(a): 1), 6), 11), 13) and 15) are the results of the conventional method without BS. 2 to 5), 7 to 10), 12), 14) and 16) are the results of the method of the present invention with BS added. (B): ll) to 14) show the results using SDS'alkali purified sample 1 (pCY) and sample 2 (pGBM) shown in FIG. (C): sp) of l) to 10) indicates sample 3 (closed-ring super-coiled pCY>) in Fig. 1, and Sacl-cut pCY is a sample obtained by cleaving pCY of sample 3 with the restriction enzyme Sacl. Chain pCY is indicated. The pGBM from 1) to 10) was purified in Fig. 1. The pGBM of Sample 4 is shown. (D): In 15) and 16), it was performed using sample pCY-R and sample pGBM-R.

[0056] FIG. 5 shows the results of colony PCR for generating 270 base pair DNA fragments by randomly selecting seven white and blue colonies obtained in the experiment of Table 1. A 270 base pair PCR product of mouse testis-derived cilitestin gene cDNA inserted in pCY was detected only in white colonies.

[Example 2] A sequence having a sequence complementary to a part of the base sequence of the target gene, while being complementary to at least a part of the base sequence of the single-stranded DNA sequence part of the probe DNA binding particle Probes prepared by a method of hybridizing a single-stranded bridge nucleotide for a probe having a cycle of 5 cycles or more within a temperature range of 50 ° C and 70 ° C to the single-stranded DNA sequence of the probe DNA-binding particle. Bridge nucleotide probe Cloning efficiency of target gene using DNA binding particles

[0058] Part of the target gene pCY, which has a complementary sequence to the 30mer base sequence, while complementary to the DNA30mer base sequence of the single-stranded DNA sequence part of the probe DNA-binding particle oligo (dG30mer) latex beads At 60 ° C and 70 ° C under conditions for high-sensitivity single-stranded bridge nucleotides for probes (single-stranded bridge nucleotides for probe for pCY (BN-CY)) to oligo (dG30mer) latex beads Constant temperature hybridization, cycle within the range of 50 ° C to 70 ° C · Hybridization method [One cycle · Hybridization example: (50 ° C (20 seconds or more) → 55 ° C ( 20 seconds or more) → 60 ° C (20 seconds or more) → 65 ° C (20 seconds or more) → 70 ° C (20 seconds or more) → 65 ° C (20 seconds or more) → 60 ° C (20 seconds or more) → The effect of the target gene pCY at 55 ° C (20 seconds or longer → 50 ° C (20 seconds or longer)) on the cloning efficiency was examined.

[0059] The experiment was performed in the same manner as in Example 1, except that the bridge nucleotide for the probe for pCY extraction was hybridized with oligo (dG30mer) latex beads.

As shown in Table 2, 3, 5, 20, 50, 60 in the presence of Up-BS and Down-BS. C, 70. C The ability to obtain a high concentration ratio of the target gene in any case However, the total number of target gene colonies obtained is less than 1/5 in the cycle 'hybridization 5 times or less, 60 ° C constant temperature, 70 ° C constant temperature high pre. Decrease to 1/10. In addition, even with the same 20 cycle cycle 'hybridization', the concentration rate is extremely low at 10 times or less in the conventional method without adding Up-BS or Down-BS. It was recognized that

[0060] [Table 2]

2: Examination of hybridization conditions for pCY extraction probe nucleotide and oligo (dG) for extraction of closed circular DNA pCY (Comparison with conventional method) BS in the eight-ibli pCY / pGBM reaction solution obtained white cone Concentration ratio Concentration of molecular ratio of concentration (uM) Number of percentages (%) (times) Number of times (weight ratio of PCY / PGBM) Up-BS Down-BS White Blue (PCY) (PGBM)

1) 3 times 1/100 20 20 137 34 81.0% 81.0

(10ng / 840ng)

2) 5 times 1/100 20 20 1055 102 91.0% 91.0

(10ng / 840ng)

3) 20 times 1/100 20 20 1042 71 93. 6% 93.6

(10ng / 840ng)

4) 50 times 1/100 20 20 899 65 93.2% 93.2

(10g / 840ng)

5) 60 2 hours 1/100 20 20 196 53 79. 0% 79. 0

(10ng / 840ng)

6) 70 * C2 hours 1/100 20 20 107 32 77.0% 77.0

(10ng 840ng)

7) 20 times 1/100 0 0 68 805 7. 8% 7.8

(10ng / 840ng)

[Example 3] A sequence complementary to a part of the base sequence of the target gene (Tm value A) and a single-stranded probe of the probe DNA binding particle Complementary to at least a part of the base sequence of the DNA binding particle For probes of the target gene using single-stranded bridge nucleotides for probes that have a unique sequence (Tm value B) and the difference between the two Tm values is at least 8 ° C Single-stranded bridge nucleotide probe Recovery to DNA-bound particle solid layer 'cloning'

[0062] The Tm value (Tm value A) of the DNA sequence complementary to a part of the base sequence of the target gene of the single-stranded bridge nucleotide and the DNA sequence complementary to the single-stranded DNA sequence part of the probe DNA binding particle The effect of the difference in Tm value (Tm value B) on the cloning efficiency of the target gene (here pCY) was examined by changing the Tm value B. Use single-stranded bridge nucleotides (BN-CY) for probes for pCY as single-stranded bridge dinucleotides for probes. A bridge nucleotide having various Tm values B was prepared, and cCY cloning was carried out from the pCY / pGBM mixed DNA sample.

pC Y extraction experiment procedure The procedure was the same as in Example 1 except that only samples with a pC Y / pGBM molecular ratio = 1/100 (10 ng / 840 ng) were used. [0063] FIG. 6 shows an example of probe DNA-binding particles and single-stranded bridge nucleotides for probes. Here, BN-1 indicates the sequence (Tm value B) complementary to DNA = oligo (dG30mer) in the single-stranded DNA sequence part of the probe DNA-binding particle, and BN-2 is 1286-13 of the inserted cDNA fragment of pCY. Shows a complementary DNA sequence (Tm value A) to 15 30 bases (inside the rectangle in Table 1).

Table 3 shows the cloning efficiency of pCY when the difference between Tm values (TmB-TmA) is changed to 0.0, 4.1, 8.2, 12.3, 20.5. Until the Tm value difference is 8.2, the cloning efficiency is 85% or more, but below that, even if the Tm value difference is TmB-TmA> 0, the cloning efficiency is considerably lowered. Under these conditions (difference in Tm value TmB-TmA <8.0), effective extraction and cloning of target genes that are present in only 1 in 1000 or 1 in 10000 is difficult.

[0064] [Table 3] Table 3: Effect of bridge nucleotide (BN) Tm value difference (Tm value B—Tm value A) on target gene extraction 'cloning efficiency'

However, the salt concentration was calculated at 150 mM (0.15 M). Up-BS and Down-BS were experimented by adding the same amount as Example 1 in all Examples 1-5.

(From left of column 1; SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 / From column 2, left; SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 / From column 3, left; SEQ ID NO: 12, SEQ ID NO: 13 (SEQ ID NO: 14 / from column 4 left; SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 / from column 5 left; SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20)

[Example 4] Target gene Having a sequence complementary to at least a part of the base sequence of DNA, On the other hand, probe single-stranded bridge nucleotides for probes that have a sequence complementary to at least part of the base sequence of the single-stranded DNA sequence part of the DNA-binding particle DNA binding Produced by enzymatic chemical synthetic covalent binding to the particle The target gene is cloned by the covalently bonded DNA particles.

As shown in Fig. 7, the bridge nucleotide DNA covalently bonded particle is composed of the hybridization DNA of the probe single-stranded bridging nucleotide to the single-stranded DNA sequence part of the probe DNA-binding particle and the Talenow 'fragment' complementary DNA It was prepared by denaturation of double-stranded DNA in an alkaline solution such as NaOH. Dissociation, dissociation, and washing and removal of single-stranded bridged nucleotide DNA.

[0066] Specifically, oligo (dT30mer) latex beads 20 ul (20 mg beads / 2 ul), lOOuM probe for p CY single-stranded bridged nucleotide (BN-CY-2) DNA 10 ul 20 ul Mix with a hybridization solution containing 500 mM NaCl, 2% albumin and add Talenow 'fragment Fragment DNA polymerase (purchased from Takara Bio Inc.) Denatured at 0 ° C to 10 ° C for 40 minutes, and further washed with TE three times. The TE solution was collected and used as a solution for use of bridged nucleotide DNA covalently bonded particles.

[0067] As can be seen from FIG. 7, the bridge nucleotide probe for the probe The probe used for the preparation of the DNA covalent bond particle The single-stranded DNA sequence portion of the DNA-binding particle can be any sequence, and the length of the base sequence Furthermore, the strength of about 3 base pairs that can be hybridized can be up to several hundred bases. Similarly, a single-stranded bridge nucleotide having a sequence that is at least partially complementary to the single-stranded DNA sequence portion of the probe DNA-binding particle and having a partial base sequence of the base sequence of the target gene is also present. As long as the conditions are satisfied, those having an arbitrary base sequence and length can be used, and the single-strand synthetic complementary bridge nucleotide for the probe including the probe DNA sequence synthesized enzymatically based on the single-strand bridge nucleotide itself is used. It is bound to solid-phase beads and has properties that do not degrade even under alkaline denaturation conditions of double-stranded DNA. Because of this property, it is possible to simultaneously denature a closed circular double-stranded DNA sample containing the gene of interest and probe DNA complex particles (which can have a solid layer of any material that is resistant to alkali!). It is possible to make the cloning of the target gene easier, quicker and more accurate, such as being hardly affected by salt concentration and temperature during washing. It is a thing.

[0068] <Bridge nucleotide probe for probe> Extraction of pCY with DNA covalently bonded particles' purification. Cloning>

Next, Table 4 shows the results of extraction, purification, and cloning of pCY from the pCY · pGBM mixed DNA sample force using the bridge nucleotide probe DNA covalently bonded particles for probes prepared in Fig. 7.

The pCY extraction operation is

[0069] 1) Take 20 ul of the bridging nucleotide probe DNA covalent bond particle for pCY probe in a 0.5 ml tube, centrifuge at 12000 rpm for 10 minutes, discard the supernatant, and collect the probe bridging nucleotide probe DNA covalent bond particle. .

2) lul lOOOuM Up-BS and lul lOOOuM Down-BS, mixed 1 to 100 Figure 1 Samples 3 and 4 pCY 1 ul and pGBM lul DNA sample, distilled water 5ul, 0.4N NaOH 9 Add ul together and treat at about 0 ° C (on ice) for 40 min to denature the DNA = single strand, then add an equivalent (9 ul) of 0.4N HC1 and mix on ice. Neutralized.

[0070] 3) Bridge nucleotide probe for denatured 'neutralized probe DNA covalently bonded particles, pCY' pGBM, BS mixed solution containing 23ul of 150mM NaCl and mixed at 50 ° C After incubating for 1 hour and 30 minutes, the plate was washed twice and twice with a washing solution containing 100 mM and 50 mM NaCl each warmed to 55 ° C., and further washed twice with a washing solution containing 25 mM NaCl at room temperature.

4) 50 ul of 10 mM Tris-ImM EDTA solution was added to the obtained bridged nucleotide probe DNA covalent bond particle-pCY complex, heated at 70 ° C. for 10 minutes, and cooled on ice. The supernatant was collected by centrifugation.

Detection of extracted pCY>

The extracted pCY was detected in the same manner as in Example 1. The results are shown in Table 4.

[0071] [Table 4] Table 4: Probe nucleotides for probes Closed circular DNA OC by DNA covalently bonded particles

Extraction of pCY from DNA samples containing PCY

Molecular ratio of pCY / pGBM White colony obtained in reaction solution Concentration rate

(weight ratio of pCY / pGBM) BS concentration (uM)

(sp-pCY / sP-pGBM) Up-BS Down-BS White Blue (pCY) (pGBM)

1) 1/100 0 0 10 210 14.1% 14. 0

(10ng / 840ng)

2) 1/100 20 20 452 11 98. 0% 98. 0

(10ng / 840ng)

20 20 289 20 93.5% 935. 0

(lng / 840ng)

4) 1/10000 20 20 151 21 87. 8% 8780. 0

(0. lng / 840ng)

5) 1/100000 20 20 95 48 66.4% 66400. 0

(0. 01ng 840ng)

[0072] As shown in Table 4, the extraction of the target gene using the bridge nucleotide probe DNA covalent bond particles for probes' cloning method was performed using the bridge nucleotide probe DNA probes for probes shown in Examples 1 to 3. The extraction showed significantly better extraction 'cloning efficiency'.

[Example 5] Single-stranded DNA complementary to the adjacent upstream region of the target gene base sequence corresponding to the probe DNA (upstream blocking sequence, Up-BS) and one complementary to the downstream base sequence (Detection) extraction of target gene by adding strand DNA (downstream blocking sequence, Down-BS)-purification 'cloning'

Table 5 shows 30 mer (30 bases) of base sequence 1 base upstream and 1 base downstream (space base sequence between probe DNA and BS = 1 base) of the base sequence of the target gene complementary to the probe DNA for pCY. The results are obtained using a single-stranded BS of length. Compared with the conventional method (data 1, 3, and 5 in Table 2) without BS, a large enrichment rate can be seen. In addition, in the case of a very small amount of target gene DNA that exists only in parts per million or parts per million, the system of the present invention can be repeated in a short period of time (multi-cycle cloning method). The target gene can be extracted, purified, and cloned easily, quickly and accurately.

[0074] [Table 5] Table 5: Up-BS and Down-BS enrichment effect on pCY extraction (compared with conventional method)

Molecular ratio of CY / pGBM Number of colonies obtained in the reaction solution

(weight ratio of pCY / pGBM) BS concentration (uM) White blue ratio (times)

Up-BS Down-BS (pCY) (pGBM) (%)

1) 1/100 0 0 80 780 .9.3 ¾ 9.3

(10ng / 840ng)

2) 1/100 20 20 3110 180 94.5% 94.5

(10ng / 840ng)

3) 1/1000 0 0 4 537 0.7% 7.4

(lng / 840ng)

4) 1/1000 20 20 1003 147 87.2% 872.0

(lng / 840ng)

5) 1/10000 0 0 1 152 0.6% 65.3

(0. lng / 840ng)

6) 1/10000 20 20 98 73 57.4% 5740.0

(0. lng / 840ng)

7) 1/1000000 50 50 86 2536 3.3% 33000.0

(0.03ng / 25.2ug)

8) lOng 50 50 42 0 100.0% 1000000.0 Extracted once from all colonies in 7) above Mixed DNA sample

9) 1/10000000 50 50 8 3792 0.21 ¾ 21000.0

(0.003ng / 25.2ug)

10) lO ng 50 50 25 12 67.5% 6750000.0 Extracted once from all colonies of 9) above Mixed DNA sample

1), 3) and 5) are the results of the conventional method without BS.

2), 4), 6) 7), 8), 9) and 10) are the results of the method of the present invention with BS added.

The extraction method was the same as in Example 1 for the second round of multi-cycle cloning in 8) and 10).

PCY and pGBM used in the experiment are the DNA samples of Samples 3 and 4 in FIG.

[0075] Table 6 uses single-stranded BSs of various lengths starting from one base upstream and one base downstream of the pCY probe DNA base sequence (space base sequence between probe DNA and BS = 1 base). This is a result of testing the influence of the length of the BS base sequence on the cloning efficiency.

[0076] [Table 6]

Table 6: Up-BS and Down-BS enrichment effects on pCY extraction

(The shadow of the base sequence length of BS)

BS length pCY / pGBM Resulting white in reaction: 3 Roni Concentration rate

(Salt salmon: mer) Molecular ratio BS concentration (uM) Percentage (%) (times)

(weight ratio of pCY / pGBM) Up-BS Down- BS White Blue

(PCY) (pGBM)

1) No BS 1/10000 0 0 1 152 0. 60% 65. 3

(0. lng / 840ng)

2) 10 mer 1/10000 20 20 9 712 1. 20% 120. 0

(0. lng / 840ng)

3) 15 mer 1/10000 20 20 38 727 5. 20% 520. 0

(0. lng 840ng)

4) 20 mer 1/10000 20 20 51 668 7. 63% 763. 0

, (0.lng / 840ng)

5) 25 mer 1/10000 20 20 67 547 11. 00% 1100. 0

(0. lng / 840ng)

1/10000 20 20 490 364 57. 37% 5737. 0

(0. lng / 840ng)

7) 40 mer 1/10000 20 20 645 937 40. 80% 4080. 0

(0. lng 840ng)

8) 50 mer 1/10000 20 20 253 446 36. 20% 3620. 0

(0. lng / 840ng)

9) 100 mer 1/10000 20 20 72 331 17. 86% 1786. 0

(0. lng / 840ng)

10) 30 mer 1/100000 100 100 106 208 33.76% 33700. 0

(0. 01ng / 840ng)

[0077] As shown in Table 6, the length of the base sequence of BS can be varied in various ways, starting from one base upstream and one base downstream of the probe DNA base sequence (space base sequence between probe DNA and BS = 1 base). In the same experiment, an extraction amplification effect was seen at a length of 15 mer or more. In particular, with 30mer, the pCY: pGBM ratio is 1/10000 with a high concentration ratio exceeding 50% (amplification rate exceeds 5000 times), and even with a ratio of 1/100000, BS concentration is 30% with lOOuM (amplification rate 30000 times). A high concentration ratio exceeding

[0078] In experiments using Up-BS and Down-BS, the length of the base sequence of BS was fixed at 30 mer, and the space base distance from the end of the probe DNA base sequence was varied. A high extraction amplification effect was observed.

In addition, the concentration of BS added to the reaction solution is the final concentration (corresponding to 3 of the above extraction operation) luM or more, and the effect of increasing the extraction efficiency can be seen. Table 7 shows the effect of BS base distance from the upstream (5 'upstream) and downstream (3' downstream) base points of the target gene sequence complementary to the probe DNA sequence.

[Table 7] Table 7: Up-BS and Down-BS enrichment effect on pCY extraction (upstream (5 'upstream), downstream (3' downstream) of target gene sequence complementary to BS probe DM sequence Influence of base distance from

Base point of 30merBS pCY / pGBM Obtained white colony in reaction solution Molecular ratio of distance from concentration rate BS concentration (uM) —number ratio (%) (times)

0® base: ier) (weight of PCY / pGBM: ratio) Up- BS Down-BS White Blue

(pCY) (pGBM)

1) No BS 1/100 0 0 7 53 11. 7% 11. 7

(10ng / 840ng)

2) 0 mer 1/100 20 20 301 56 84. 3% 84. 3

(10ng / 840ng)

3) 1 mer 1/100 20 20 409 26 94. 0% 94. 0

(10ng / 840ng)

3) 3 mer 1/100 20 20 338 45 88. 2% 88.2

(10ng / 840ng)

4) 5 mer 1/100 20 20 124 42 75. 6% 75. 6

(10ng / 840ng)

5) 7 mer 1/100 20 20 64 48 57. 1% 57.1

(10ng / 840ng)

6) 10 mer 1/100 20 20 17 33 34. 0% 34. 0

(10ng / 840ng)

7) 30 mer 1/100 20 20 9 39 18. 8% 18. 8

(10ng / 840ng)

8) 60 mer 1/100 20 20 11 67 14. 1% 14. 1

(10ng / 840ng)

[0080] When the base sequence length of the BS is fixed at 30 mer and the space base distance from both ends of the probe DNA base sequence is changed and separated, a high extraction amplification effect exceeding 50% is seen up to 7 mer. Force to be used The effect of SB is lost rapidly when the distance is longer. In addition, up-B S and Down-BS alone have some effect (up-BS alone has a pCY: pGBM ratio = 1/100, 65-45 times enrichment, and Down-BS alone has 35-25 times enrichment) Although it is obtained, it is much lower than the effects of Up-BS and Down-BS coexisting.

[0081] [Example 6] Closed circular double-stranded DNA containing the target gene is denatured at a low temperature (0 ° C to 10 ° C) in an alkali (NaOH, KOH, etc.) solution of 0.1N to 0.4N (normative). To a method of partially single-stranded According to the target gene cloning

Table 8 shows the results of temperature conditions during alkaline denaturation of the closed circular double-stranded DNA containing the target gene (here pCY). Except for the alkali denaturation temperature conditions of the DNA samples, the same experimental procedure as in Example 1 was performed.

Table 8 shows the results. Alkaline denaturation of DNA samples at 0 ° C (on ice) yielded very high cloning efficiencies, while significant reductions in cloning efficiency were seen at 25 ° C (room temperature) and 37 ° C. Table 8 shows that BS is indispensable also in this example. Alkaline denaturation of the DNA sample was performed with 0.2N NaOH (final concentration) for 40 minutes in the same manner as in Example 1. At 10 minutes or 20 minutes denaturation, the yield of the extracted plasmid itself decreased even at a temperature of 1, different.

[Table 8] Table 8: Temperature conditions for DNA sample denaturation for pCY extraction

DNA sample pCY / pGBM BS in reaction solution Number of colonies obtained White colony Concentration rate Density temperature molecular ratio concentration (uM) White blue ratio (%) (times)

(Weight ratio of PCY / PGBM) Up- BS Down-BS (pCY) (pGBM)

1) (TC 1/100 0 0 84 881 8. 7% 8.7

(10ng / 840ng)

2) (TC 1/100 20 20 6 180 180 97. 2% 97.2

(10ng / 840ng)

3) 1/100 at 25 0 0 91 854 9.6% 9. 6

(10ng / 840ng)

4) 25 "C 1/100 20 20 4310 2250 65.7% 65.7

(10ng / 840ng)

5) At 37 1/100 0 0 101 1881 5. 0% 5. 0

(10ng / 840ng)

6) At 37 1/100 20 20 1520 3020 33. 5 33.5

(10ng / 840ng)

1), 3), 5) are the results of the conventional method without BS. 2), 4) and 6) are the results of the method of the present invention with BS added.

[Example 7] Closed circular double-stranded target gene DNA sample and probe DNA binding particles are hybridized under low salt concentration (100 to 200 mM) to target gene to solid particles. Extraction · Recovery · Cloning

Table 9 examines the effect of salt concentration of the solution during hybridization of the denatured sample DNA to the BN-CY oligo (dG) latex bead complex on the efficiency of the extracted 'purified' clawing of pCY. It is a result. [0084] [Table 9] Table 9: Effect of hyperpredation solution salinity on BN-CY-oligo (dG) latex bead complex of denatured DM sample on the efficiency of extraction and purification of pCY Concentration PCY / pGBM BS in reaction solution obtained White: 3D 2 Concentration rate

(nM) Molecular ratio concentration (uM) Number of colonies (times)

(weight ratio of pCY / pGBM) Up-BS Down-BS White Blue (%)

(PCY) (pGBM)

1) 110. 4 1/100 20 20 1095 93 92. 1% 92. 1

(10ng / 840ng)

2) 156. 4 1/100 20 20 781 90 89.7% 89. 7

(10ng / 840ng)

3) 202. 4 1/100 20 20 534 131 81.2% 81.2

(10ng / 840ng)

4) 248. 4 1/100 20 20 173 60 74. 0% 74. 0

(10g / 840ng)

5) 317. 4 1/100 20 20 194 108 64. 2% 64. 2

(10ng / 840ng)

6) 363. 4 1/100 20 20 122 77 61. 3% 61. 3

(10ng / 840ng)

7) 409. 4 1/100 20 20 105 104 50. 2% 50. 2

(10ng / 840ng)

8) 547. 4 1/100 20 20 100 99 50. 2% 50. 2

(10ng / 840ng)

The salt concentration was shown as the sum of the values calculated by multiplying the NaCl concentration by the pH adjustment Tris HC1 concentration of 0.6.

[0085] At a salt concentration from approximately lOOmM to 200mM, a cloning efficiency exceeding 80% can be obtained. At 250mM or more, it has a low cloning efficiency of less than 70%, especially around 500mM used in Northern hybridization. At salt concentration, the cloning efficiency decreases to about 50%. When the cloning of the target gene pCY is carried out at these salt concentrations under the severe conditions of pCY / pGBM 1/1000 and 1/10000, it is further reduced to 5% or less.

[0086] [Example 8] Extraction of actin gene cDNA clone from mouse brain cDNA plasmid library

The mouse brain cDNA plasmid library used for the experiment was purchased from Takara Bio Inc. This is transfected into Escherichia coli, cultured in large quantities, and closed with various ratios of linear DNA and open circular DNA by the SDS-al strength method, CsCl ultracentrifugation method and DNA adsorption membrane method shown in Example 1. A circular cDNA plasmid library was prepared and used for experiments.

Single-stranded probe DNA for extraction of the following actin gene, single-stranded Up-BS (final concentration = 10 Extraction 'purification' cloning was performed in the same manner as in [Example 1] except for 0 uM), single-stranded Down-BS (final concentration = 100 uM) and PCR primers.

[0087] [Chemical 5]

5 '-GGAGAnACTGCTCTGGCTCCTAGCACCATCCCCCCCCCCCCCCCCCCCCCCCCCCCCCC-3' (SEQ ID NO: 21)

[0088] A single-stranded bridge oligonucleotide sequence (BN-actin) composed of 30 bases of 1023-1052 of the cDNA fragment of the actin gene and poly C30 base (BN-actin) was synthesized and probe DNA for extracting the actin gene cDNA plasmid clone Used as.

Oligo (dG) latex 'beads purchased from JSR Corporation were used. The BN-actin probe hybridizes with this latex bead oligo (dG) at the poly C moiety and is immobilized on the bead solid layer.

<Actin probe Blocking sequence upstream and downstream of DNA sequence (

BS)>

Single-stranded Up-BS (upstream blocking sequence)

[0089] [Chemical 6]

5'-TGTACCCAGGCATTGCTGACAGGATGCAGA -3 '(SEQ ID NO: 22)

30 bases of 992-1021 of the cDNA fragment of the actin gene were synthesized and used.

Single-stranded Down-BS (downstream blocking sequence)

[0090] [Chemical 7]

5'-GCGCTCAGaGGAGCAATGATCTTGATCTT -3 '(SEQ ID NO: 23) 30 bases of 1083-1054 (minus strand) of the cDNA fragment of the actin gene were synthesized and used. <Actin primer for PCR>

[0091] [Chemical 8] Prime 1: 5 '-GACGACATGGAGAAGATCTG-3' (316-335)

Primer 2: 5'-TAGGAGCCAGAGCAGTAATC-3 '(1045-1026)

(Top; SEQ ID NO: 24 / Bottom; SEQ ID NO: 25)

[0092] As shown in Table 10, in the conventional method without BS, from mouse mouse cDNA plasmid library lug containing actin gene cDNA clone, 0 in sample 5 (0%), 6 in sample 6 (0.56 %), 24 (4.07%) in sample 7 and 4 (1.21) actin-positive plasmid colonies in sample 8. On the other hand, in the present invention using BS, two samples (0.23%) were obtained in sample 5, 16 samples (3,20%) in sample 6, and

101 (43.72%), 28 samples (22.22%) of actin-positive plasmid colonies were obtained. Even with the conventional method that does not add BS, it can be clonable with a probability of about 4% (4 out of 100) with a sample rich in closed circular DNA, which is the target molecule of the present invention. The actual gene clone can be cloned with a very high probability of about 44% (44 out of 100).

[0093] [Table 10] Table 10: Extraction, purification, and cloning of cDM plasmid clone from a mouse brain cDNA plasmid library according to the present invention (purification without cloning, comparison with conventional methods) Actin used -BS Number of colonies obtained Actin primer PCR positive

DNA sample Up-BS Down- BS (number per total extract) PCR positive

(uM) (uM) Percentage of colonies (%

1) .Sample 5 0 0 腦 0 0 0.00%

2) Sample 5 100 100 887 2 pieces 0.23%

3) Sample 6 0 0 1072 6 pieces 0.55%

4) Sample 6 100 100 497 16 pieces 3. 20%

5) Sample 7 0 0 589 24 24 4. 07%

6) Sample 7 100 100 231 101 101 43. 72%

7) Sample 8 0 0 330 pieces 4 pieces 1. 21%

8) Sample 8 100 100 126 28 22. 22.% Experiments 1) and 3) are results of the conventional method in which 40% or more of open circular DNA is mixed and BS is not included. Experiments 2), 4), 5), 6), 7), and 8) are the results of the “method of the present invention”.

Experiment 10 in Table 10) Among the actin-positive colonies that were also obtained, 2 clones of Escherichia coli were further cultured in liquid, and the plasmid was extracted and purified, and the restriction enzyme EcoR for excising insert DNA was purified. Figure 8 shows the results of PCR using DNA that had been cleaved with 1 and Notl and purified. Clearly, clones containing almost full-length actin cDNA (1.26 k base pairs) were obtained.

[0095] [Example 9] HPRT (hypoxanthine'guanine from mouse brain cDNA plasmid library

'Host Ribon'nore' Tofunsufuefu ¹ ~ ~ zero (Hypoxanthine guanine phosphoribosyl transferase ^) gene cDNA clone of extraction 'refining and claw - ing

The mouse brain cDNA plasmid library used for the experiment was purchased from Takara Bio Inc. This is transferred to Escherichia coli, cultured in large quantities, and subjected to SDS-alkaline and CsCl ultracentrifugation, and a closed circular cDNA plasmid library that contains almost no linear or open circular DNA (from 95% or more closed circular DNA). And used for the experiment.

Except for the following single-stranded probe DNA for HPRT gene extraction, single-stranded Up-BS (final concentration = 100 uM), single-stranded Down-BS (final concentration = 100 uM), and PCR primers Extraction 'purification' cloning was performed in the same manner as in Example 1.

[0096] [Chemical 9]

5 '-GGGTAGGCTGGCCTATAGGCTCATAGTGCACCCCCCCCCCCCCCCCCCCCCCCCCCCCCC-3,

(SEQ ID NO: 26)

A bridge oligonucleotide sequence (BN—HPRT) consisting of 30 bases of 899-870 of the cDNA fragment of HPRT gene and poly C30 base was synthesized and used as probe DNA for extraction of HPRT gene cDNA plasmid clone.

Oligo (dG) latex 'beads purchased from JSR Corporation were used. The BN-HPRT probe hybridizes with the latex 'bead oligo (dG) at the poly C moiety and is immobilized on the bead solid layer.

Single-stranded Up-BS (upstream blocking sequence)

[0097] [Chemical 10] 5 '-TTTCTTACAAGATAAGCGACAATCTACCAG -3,

(SEQ ID NO: 27)

30 bases of HPRT gene cDNA fragment (930-90 minus strand) were synthesized and used. Single-stranded Down-BS (downstream blocking sequence)

[0098] [Chemical 11]

5 '-TGTCAGTTGCTGCGTCCCCAGACTTTTGAT-3'

(SEQ ID NO: 28)

30 bases of 839-868 of HPRT gene cDNA fragment were synthesized and used.

[0099] [Chemical 12] Prime 1: 5 '-GGACTGATTATGGACAGGAC-3' (154-173)

Primer 2: 5'-GAGAGCTTCAGACTCGTCTA-3 '(1097-1078)

(Top; SEQ ID NO: 29 / Bottom; SEQ ID NO: 30)

[0100] As shown in Table 11, in the conventional method without BS, 0 (0%) in sample 5 and 1 in sample 6 (0.59) from the mouse brain cDNA plasmid library lug containing the HPRT gene cDNA clone. %), 6 (1.35%) in sample 7 and 1 (1.02) HPRT positive plasmid colony in sample 8. On the other hand, in the present invention using BS, 1 sample (0.16%) for sample 5, 8 samples (4.06%) for sample 6, 47 (3 7.90%) for sample 7, and 11 (16.41%) for sample 8 Actin-positive plasmid colonies were obtained. Even with the conventional method without adding BS, cloning is possible with a probability of 1.35% (1.35 per 100) in samples rich in closed circular DNA, which is the target molecule of the present invention. The actual gene clone can be cloned with a very high probability of about 37.90% (about 38 out of 100).

[0101] [Table 11] Table 11: HPRT gene cDNA from a mouse brain cDNA plasmid library according to the present invention

Extraction, purification, and cloning of plasmid clones (Comparison with conventional methods that do not contain BS)

Actin-BS used Actin primer obtained PCR positive colony

Percentage of positive PCR by DM sample Up-BS Down-BS α

(uM) (uM) (Number of 抝 colonies per total extract ()

1) Sample 5 0 0 984 0 0 0.00%

2) Sample 5 100 100 601 pieces, 1 piece 0.16 ¾

3) Sample 6 0 0 509 3 pieces 0.59%

4) Sample 6 100 100 197 8 8 4. 06 ¾

5) Sample 7 0 0 444 6 6 1.35%

6) Sample 7 100 100 124 47 37. 90%

7) Sample 8 0 0 98 1 pc 1.02%

8) Sample 8 100 100 67 11 16 16. 41%

Experiments 1) and 3) are the results of a method that contains 40% or more of open circular DNA and does not contain BS. Experiment 2), 4), 5), 6),

7) and 8) are the results of the “method of the present invention”.

[0102] Experiments in Table 11 6) Strength Among the obtained HPRT-positive colonies, 2 clones of E. coli were further cultured in liquid, the plasmid was extracted and purified, and digested with restriction enzymes EcoRl and Notl for excision of insert DNA. Figure 9 shows the results of PCR using purified DNA. A clone was obtained containing almost the full length HPRT cDNA (l.2k base pairs).

[Example 10] Extraction of HPRT gene cDNA clones from mouse brain cDNA plasmid library using bridge nucleotide DNA covalently bonded particles for HPRT gene probe 'Purification' Cloning

In accordance with the method for preparing covalent nucleotides of bridged nucleotide DNA in Example 4, the HPRT gene cDNA fragment consisting of 30 bases of 899-870 and poly C30 base strength is composed of the following sequence for the bridge oligonucleotide (BN— HPRT)

[0104] [Chemical 13]

5 '-GGGTAGGCTGGCCTATAGGCTCATAGTGCACCCCCCCCCCCCCCCCCCCCCCCCCCCCCC-3'

(SEQ ID NO: 31)

After synthesizing and hybridizing latex polybead oligo (dG) at the poly C moiety, single-stranded bridged nucleotide DNA covalent binding particles for HPRT probe were prepared with talen fragment DNA.

Using this single-stranded bridged nucleotide DNA covalent bond particle for HPRT probe, the HPRT gene from the mouse brain cDNA plasmid library by the same extraction procedure as in Example 4 was used. Extraction of the child cDNA clones were performed by 'purification' cloning. The concentration of BS for HPRT and the primer for PCR were the same as in Example 9.

From the mouse brain cDNA plasmid library lug containing the HPRT gene cDNA clone, 64 plasmid colonies were obtained. As a result of colony PCR, as shown in Table 12, it was found that 57 (89%) of them were HPRT positive colonies. On the other hand, all the colonies obtained by the conventional method not containing BS were negative colonies.

[0106] [Table 12] Table 1 2: Extraction, purification and cloning of HPRT gene cDM plasmid clones from mouse brain cDNA plasmid library according to the method of the present invention (comparison with conventional method without BS)

HPRT-BS Number of obtained colonies HPRT primer PCR positive colonies

Percentage of PCR positive by Up-BS Down-BS (number per total extract)

(uM) (uM) Number of colonies (%)

1) 100 100 64 57 89%

2) 0 0 31 0 0 0

Experiment 1) is the result of the method of the present invention, and Experiment 2) is the result of the conventional method not including BS.

[0107] Among the HPRT-positive colonies obtained, 5 clones of E. coli were further cultured in liquid, and the plasmid was extracted and purified, cut with restriction enzymes EcoRl and Notl for excision of the insert DNA, and purified DNA Figure 10 shows the results of PCR using A clone was obtained containing almost the full length HPRT cDNA (1.2 k base pairs).

[Example 11] A closed circular double-stranded DNA sample containing a gene of interest is treated in the presence of a blocking sequence for 10 to 60 seconds under a temperature condition of 94 ° C to 98 ° C, and then immediately 0 Cloning of the target gene by rapid cooling to ° C to 4 ° C, denaturation, and partial single-strand cloning. Closed circular double-stranded DNA containing the target gene (pCY) is cloned in the presence of a blocking sequence. Table 13 shows the results of cloning of the target gene when it was denatured by high-temperature treatment and rapid cooling. High-temperature denaturation of DNA samples · Except for conditions of rapid cooling in ice, the same experimental procedure as in Example 1 was performed. A closed circular double-stranded DNA sample containing the gene of interest in a TE solution (10 mM Tris, ImM EDTA, pH 8.0) and BS are treated at a temperature range of 96 ° C to 98 ° C for 10 to 60 seconds, and immediately The DNA was denatured by quenching in ice water, partially unified, and hybridized with probe DNA complex particles. Although not shown in Table 13, this method also uses B s was essential.

[0109] [Table 13] Table 1 3: Extraction of PCY · Temperature conditions for heat denaturation of DNA samples against Kung Jung

DM sample pCY / pGBM termination-ratio BS concentration in reaction solution (uM) number of mouth knees obtained white colony concentration concentration-denaturation temperature ratio), ['-one-one ^ color ratio (%) ( (Times) ο ο ο ο ο (pCY) (pGB)

1) 4 ^y C 20 20 1599 1201 57. 丄% 57. 1 second

2) 94 ° C 1/100 20 20 4812 1635 74. 6% 74. 6 Paper ^

3) 94 ^y C 20 20 5104 1011 84. 7% 84. 7 seconds

4) 96 ^y C 20 20 1608 989 62.0% 62.0 seconds

5) 96 ° C 1/100 20 20 4905 1107 81. 6% 81. 6 Paper ^

6) 96 ^y C 20 20 6780 427 94. 0% 94.0 seconds

5) 9S ^y C 20 20 2715 706 79.4% 79. 4 seconds

6) 98 ° C 1/100 20 20 3924 215 94. 8% 94. 8

2) 9S ^y C 20 20 3057 160 95. 0% 95.0 seconds

In the presence of BS, the cloning efficiency was 10% or higher for all temperature treatments. Industrial applicability

[0110] By the separation, purification, and cloning method according to the present invention, closed circular double-stranded DNA of any origin, size, sequence, etc. including the target gene can be rapidly (within 24 hours) accurately, efficiently and efficiently. Separation, purification, and cloning are possible. It can be used in all industrial fields that use genes, including the pharmaceutical field.

Claims

The scope of the claims

[1] A method for extracting, purifying, and cloning a target gene using a closed circular double-stranded DNA as a target DNA molecule,

By hybridizing the closed circular double-stranded DNA to the probe DNA complex particle containing a DNA sequence complementary to at least a part of the base sequence of the target gene and a solid particle, the base of the target gene is obtained. The closed circular double-stranded DNA in which the sequence is incorporated is collected in the solid layer particles, eluted with the elution solution, introduced into the host cell, and the target gene is rapidly extracted and purified. And how to.

[2] The probe DNA complex particle is

A probe DNA-binding particle comprising a single-stranded DNA sequence portion comprising a single-stranded bridge nucleotide complementary binding sequence for probes bound to the solid particle,

A DNA sequence complementary to at least a part of the base sequence of the target gene, and a DNA sequence complementary to the probe single-stranded bridge nucleotide complementary binding sequence for the probe DNA-binding particle. For single-stranded bridge nucleotides for probes,

The single-stranded bridge nucleotide complementary binding sequence for probes is formed by complementary binding at a complementation rate of 80% or more.

The extraction, purification, and cloning method according to claim 1, wherein the method is a single-stranded bridge nucleotide probe DNA probe particle for probe.

[3] The probe DNA complex particle is

A probe DNA-binding particle having a single-strand sequence partial force bound to the solid particle has a DNA sequence (Tm value A) complementary to at least a part of the base sequence of the target gene, and the probe DNA-binding particle. A single-stranded bridge nucleotide for a probe comprising a DNA sequence portion complementary to at least a portion of the single-stranded DNA sequence portion (Tm value B), wherein the difference between Tm value A and Tm value B is at least 8 For single-stranded bridge nucleotides for probes with a temperature of ° C or higher (Tm value B—Tm value A> 8 ° C)

2. The extraction, purification and cloning method according to claim 1, wherein the method is a single-stranded bridge nucleotide probe DNA-binding particle for probes formed by complementary binding.

[4] Particle force of the probe DNA complex Single-stranded DNA sequence of the probe DNA-binding particle 2. The single-stranded bridge nucleotide probe for DNA probe, wherein the nucleotide comprises a nucleotide sequence containing a DNA sequence complementary to at least a part of the base sequence of the target gene. Extraction, purification and cloning methods.

[5] From 0 to 7 bases upstream in the upstream region adjacent to the base sequence of the target gene corresponding to a sequence complementary to at least a part of the base sequence of the target gene in the probe DNA complex particle 20 to 50-base single-stranded DNA (upstream blocking sequence, Up-BS) and 20-50 base-sequence single-stranded DNA (downstream blocking sequence, Down-BS) starting from 0-7 bases downstream in the adjacent downstream region: In the presence of a concentration of lOuM or more, and the ratio of the number of BS molecules to the number of target gene molecules is at least 1000 times; a closed circular double-stranded DNA sample containing the target gene is hybridized with the probe DNA complex particle. The extraction, purification, and cloning method according to any one of claims 1 to 4, wherein

[6] Performing denaturation of a closed circular double-stranded DNA sample containing the gene of interest in a 0.1N to 0.4N alkaline solution at a temperature of 0 ° C to 10 ° C for 20 minutes to 1 hour. The extraction, purification, and cloning method according to any one of claims 1 to 5,

[7] A closed circular double-stranded DNA sample containing the gene of interest is treated in the presence of the blocking sequence for 10 to 60 seconds under a temperature condition of 94 ° C to 98 ° C, and then immediately from 0 ° C to 6. The extraction, purification, and cloning method according to any one of claims 1 to 5, wherein the extraction, purification, and cloning are performed by rapid cooling to 4 ° C and denaturation.

[8] The closed circular double-stranded DNA sample containing the target gene is subjected to noble hybridization with the probe DNA complex particles at a salt concentration of 50 to 350 mM, according to any one of claims 1 to 7. Extraction, purification and cloning methods.

[9] The single-stranded bridged nucleotide probe DNA probe-binding DNA according to claim 2.

[10] A single strand for a probe having a sequence complementary to a part of the base sequence of the target gene and a sequence complementary to at least a part of the base sequence of the single-stranded DNA sequence part of the probe DNA binding particle The bridge nucleotide is nominated in the single-stranded DNA sequence portion of the probe DNA-binding particle for 5 cycles or more within a range of 50 ° C. to 70 ° C.,

The single-stranded bridge nucleotide probe probe according to claim 9, which is prepared by:

[11] The single-stranded bridged nucleotide probe probe according to claim 4, wherein the DNA is covalently bonded.

[12] A probe consisting of a single-stranded DNA sequence that binds to a solid particle A sequence that is complementary to at least a part of the single-stranded DNA sequence of the DNA-binding particle and at least a part of the base sequence of the target gene The single-stranded bridge nucleotide having the sequence is hybridized with the single-stranded DNA sequence portion of the probe DNA-binding particle, and the base sequence of the target gene of the single-stranded bridge nucleotide is synthesized using a DNA synthase. Nucleotides containing a sequence complementary to at least a part of the sequence are covalently bound enzymatically and chemically to the single-stranded DNA sequence portion of the probe DNA-binding particle;

The single-stranded bridge nucleotide probe probe according to claim 11, which is prepared by:

[13] The cloning method according to any one of claims 1 to 8, wherein a closed circular double-stranded DNA contained in a recovered product from a host cell containing the target gene after being concentrated by the method according to any one of claims 1 to 8 is used as a target DNA molecule. .