JP2002281981A - METHOD FOR PRODUCING LABEL-INTRODUCED NUCLEIC ACID USING Rec A PROTEIN - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for selectively and conveniently introducing a label into a desired site of a double strand nucleic acid in high site selectivity. SOLUTION: Disclosed is a method for producing a label-introduced nucleic acid comprising a process in which a target nucleic acid is bonded to a first nucleic acid probe through Rec A protein by adding the Rec A protein and a first nucleic acid probe homologous to a terminal region of the target nucleic acid to a sample containing the target nucleic acid into which the label is introduced so that a part of the first nucleic acid probe projects from the target nucleic acid, a process in which the Rec A protein bonded to the target nucleic acid is dissociated from the target nucleic acid, and a process in which the label-introduced nucleic acid is produced by hybridizing a labeled second nucleic acid probe to the projected part of the first nucleic acid probe.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for producing a labeled nucleic acid.

[0002]

2. Description of the Related Art The use of labeled nucleic acids in the analysis of gene expression, mutation and polymorphism, and in the detection and / or isolation of DNA in the diagnosis and treatment of clinical genetic diseases is extremely difficult. It is beneficial. For example, D
It is used as a DNA probe on an NA chip (generally also called a DNA microarray), as a primer in PCR, and as a DNA probe in in situ hybridization.

[0003] Such a labeled nucleic acid is synthesized by the polymerase chain reaction (hereinafter referred to as PCR), and labeled simultaneously or sequentially, or produced by cloning using Escherichia coli or the like and further modified. Manufactured.

In the case of synthesis by PCR, a labeling substance is generally added simultaneously with synthesis by using a primer having a labeling substance or performing a normal PCR process using dNTPs having a labeling substance. I do. Or P
It is possible to obtain a nucleic acid to which a labeling substance has been added by chemically modifying a nucleic acid synthesized by CR or cloning.

[0005] However, in the case of PCR, particularly when the target nucleic acid has a long base sequence, sufficient amplification may not be obtained. In addition, non-specific molecules may be amplified. In such a case, it is necessary to identify and purify the obtained nucleic acid, but such an operation requires much time and labor. Generally, PCR uses a large excess of primers. So, for example,
When used as a probe for a DNA chip, it is necessary to remove the remaining probe to lower the background.

In addition, when a labeling substance is chemically or biochemically added to a synthesized nucleic acid, the following problem arises. For example, when a modification is performed by a nick translation method using an enzyme, modification is performed on all nicks generated in the sequence, and it is difficult to selectively modify a specific site. .

When it is desired to label only one end using polynucleotide kinase or terminal transferase, the labeling substance is added to the end of the double strand by the enzyme, and then the labeling substance at the unnecessary end is added. It must be removed by restriction enzymes. Furthermore, when a label is attached to only one end by linking an adapter to the blunt end by ligation, one end to be treated is made a blunt end prior to the attaching step,
It is necessary to use the other as a cohesive end, or to remove an unnecessary labeled part of the end with an enzyme after giving.
However, since the site selectivity of an enzyme is limited, it is difficult to selectively label a specific site of a nucleic acid by a conventional method using such an enzyme. In the case of a double-stranded nucleic acid, its ends are equivalent,
Only one end is blunt end (or cohesive end)
This requires a complicated operation and a long time in the conventional technology.

[0008]

In view of the above situation, an object of the present invention is to provide a high site selectivity and a simple method.
An object of the present invention is to provide a method for selectively introducing a label to a desired site of a double-stranded nucleic acid.

[0009]

Means for Solving the Problems To solve the above problems, the present invention provides a method for introducing a label into a nucleic acid, wherein a sample containing a target nucleic acid to which the label is to be introduced is provided with a RecA protein, By adding a first nucleic acid probe homologous to the terminal region of the target nucleic acid, the first nucleic acid probe via the RecA protein so that a part of the first nucleic acid probe protrudes from the target nucleic acid. A step of binding a probe to the target nucleic acid; a step of dissociating the RecA protein bound to the target nucleic acid from the target nucleic acid;
Introducing a label into a nucleic acid by hybridizing a labeled second nucleic acid probe.

Here, in order to help understand the following detailed description of the present invention, an outline of the method of the present invention will be described with reference to FIG.

[0011] In the first step of the method of the present invention, the DN is added to the target nucleic acid to be labeled, generally the target DNA 1.
A first probe DNA 2 having a sequence homologous to one terminal region of A and RecA protein 3 are added. Re
The cA protein 3 binds to the first probe DNA 2 to form a probe DNA / RecA protein complex 4. Subsequently, the RecA protein binds the first probe DNA2 to the homologous region by searching for a region homologous to the first probe DNA2 present in the terminal region of the target DNA1. In a later step, the first probe DNA 2 is hybridized with the labeled second probe DNA 6, so that the first probe DNA 2
Is the target DN such that it protrudes from the terminal end of the target DNA1.
It is bound to A1.

In the first step, RecA is added to one end region of the target DNA 1 by the action of the RecA protein.
The first probe DNA 2 binds via the protein 3 to form a triple-stranded DNA structure 5 in the terminal region of the target DNA 1.

In the second step, RecA bound to the first probe DNA 2 is obtained by performing a protein removal reaction.
Protein 3 is removed. When the first probe DNA 2 is bound to the terminal region, RecA protein 3
, The triple-stranded DNA structure 5 is maintained. In the second step, by removing the RecA protein 3 from the triple-stranded DNA structure 5, the first probe DN
The second probe 6 can be easily hybridized to the protruding portion of A2.

In the third step, the first probe DNA 2
The label is introduced into the terminal region of the target nucleic acid by hybridizing the labeled second probe 6 to the protruding portion of the target nucleic acid. In order to bind the label firmly, the second probe 6 is preferably ligated to the terminal region of the target nucleic acid, if necessary. After the second probe 6 is ligated, the first probe DNA 2 becomes unnecessary, and may be dissociated from the target DNA 1.

The above is an outline of the method of the present invention.
Since the specific reactions and structures shown in are only described for the purpose of facilitating understanding,
In practice, those details may not match the drawings. That is, we are not bound by any particular theory.

[0016]

DESCRIPTION OF THE PREFERRED EMBODIMENTS <Introduction> The present invention provides a method for producing a labeled nucleic acid using a RecA protein.

According to the present invention, the RecA protein is synthesized from the three-stranded structure under the specific conditions, that is, when the triple-stranded structure is formed in the terminal region of the target nucleic acid. Has been made based on the discovery of the present inventors that the triple-stranded structure is maintained even after dissociation. It is known that a triple-stranded structure is formed via the RecA protein and the RecA protein. However, in the conventional method, 6
When a probe as short as about 0 mer is used, there is a problem that the probe is repelled from the formed triple strand. Therefore,
The present invention has been achieved by finding means for solving this problem.

Hereinafter, embodiments of the present invention will be described in detail.

<First Step> To carry out the method of the present invention, first, a sample containing a target nucleic acid to which a label is to be introduced is added to the sample.
The ecA protein and a first nucleic acid probe homologous to a terminal region in the target nucleic acid into which the label is to be introduced are added.

In the present specification, the “target nucleic acid” can be a polynucleotide consisting of any simple nucleotide and / or modified nucleotide. The target nucleic acid can be freely selected by the practitioner of the present method. Target nucleic acids are typically DNA, such as cDNA, genomic DNA, and synthetic DNA, and RNA, such as mRNA, total RNA, hnRNA, and synthetic RNA. "Simple nucleotides"
Include adenine, guanine, thymine, cytosine, and uracil. "Modified nucleotide" includes, for example, inosine, acetylcytidine, methyladenosine,
Phosphoric acid esters containing methylguanosine and the like are included.

A “sample containing a target nucleic acid” is an untreated sample collected from an organism, for example, genomic DNA, mRNA,
It can be a biological sample containing a plasmid. The “sample containing a target nucleic acid” may be a sample obtained by performing various operations or treatments on the untreated sample. Such operations or treatments can be nucleic acid extraction operations, amplification operations, treatments with enzymes including restriction enzymes, ligases, polymerases, nucleases, and other treatments well known in the field of genetic engineering, and combinations thereof.

The “nucleic acid extraction operation” can be, but is not limited to, phenol extraction and ethanol precipitation.

"Amplification" typically involves PCR, or a variant thereof, such as reverse transcription PCR, reverse PCR, 5'RA.
CE, 3′RACE.

Alternatively, the “sample containing a target nucleic acid” may be a sample containing an artificially prepared nucleic acid, or a sample prepared by subjecting the sample to each of the above-described treatments.

More specifically, the "sample containing a target nucleic acid" is a sample obtained at each stage of gene cloning, for example, a DNA library, a sample containing a target mRNA, a sample containing a first strand cDNA, an adapter, Can be a sample containing the first-strand cDNA to which the PCR product has been added, or a sample containing the plasmid into which the PCR product has been subcloned.

As used herein, the term "RecA protein" refers to a region present in one strand of a double-stranded nucleic acid, to which a single-stranded nucleic acid homologous to the region is bound to form a three-stranded nucleic acid. A protein capable of forming a chain structure. The RecA protein has homologous recombination, D
It is known to be involved in repair of NA or expression of the SOS gene of Escherichia coli. Among the RecA proteins, the RecA proteins of Escherichia coli and λ phage are the most famous. However, proteins having a structure and function similar to the RecA protein of E. coli are known to be widely distributed in organisms other than E. coli.
These proteins are generally called RecA-like proteins. In the present specification, the “RecA protein” includes not only the RecA protein of Escherichia coli and λ phage, but also a RecA-like protein.
Further, it may be a partial fragment of the RecA protein having RecA activity.

As described above, the RecA protein does not bind a single-stranded nucleic acid to a double-stranded nucleic acid at random, but binds to a homologous region present in one strand of the double-stranded nucleic acid. "Homologous" between two nucleic acids means that the nucleic acids are identical or similar to the extent that they can form a specific triple-stranded structure via the RecA protein. "Similar" means, for example, that two base sequences have at least 60%, preferably 80%,
More preferably 90%, even more preferably 95% or more identity.

Since the RecA protein has a function as described above, when the RecA protein and a homologous first nucleic acid probe are added to the sample, the first nucleic acid probe is added to one strand of the target nucleic acid. Binds to homologous parts present in

When the region into which the label is to be introduced is not located at the terminal region, a part of the target nucleic acid may be excised so that the region is located at the terminal region, and then the method of the present invention may be applied. . Alternatively, once the nucleic acid is circular, the nucleic acid may be cut again so that the specific region comes to the terminal region. When the target nucleic acid is a circular nucleic acid, the circular target nucleic acid is cleaved before or after the formation of the triple-stranded nucleic acid such that the region to be subjected to the desired treatment is located at the terminal region. do it.

As used herein, the term "terminal region" of the target nucleic acid refers to the 400th, more preferably 200th, more preferably 150th, and more preferably, the terminal end of the target nucleic acid and the terminal end of the target nucleic acid. A region located between the 100th base, more preferably the 80th base, still more preferably the 60th base, still more preferably the 50th base, still more preferably the 40th base, still more preferably the 20th base, still more preferably the 10th base (each terminal End).

The shape of the terminal end of the terminal region may be either blunt end or protruding end (generally called cohesive end).

The first nucleic acid probe to be bound to the terminal region of the target nucleic acid has at least 20 bases or more, more preferably 30 bases or more, still more preferably 40 bases or more.
Most preferably it has a length of 50 bases or more. As described in detail in the following experimental examples, if the first nucleic acid probe is too short, a triple-stranded structure is not formed stably, so that the first nucleic acid probe having an appropriate length according to the length of the target nucleic acid is used. Must be used.

In the subsequent step, a labeled second nucleic acid probe is hybridized with the first nucleic acid probe. Therefore, a portion of the first nucleic acid probe must bind to the target nucleic acid such that it preferably projects at least 5 nucleotides, more preferably at least 10 nucleotides, from the target nucleic acid. Part of the first nucleic acid probe is
It may be an intermediate part or a terminal part of the probe. Here, the intermediate part is a part of the probe that does not include the terminal end, and the terminal part is a part of the probe that includes the terminal end.

The base sequence of the protruding portion of the first nucleic acid probe may be any sequence as desired.

As described in detail in the following experimental examples, when the first nucleic acid probe binds to a target nucleic acid via the RecA protein, a triple-stranded structure may be present depending on the orientation of the first nucleic acid probe. May not be formed. Therefore, in order to carry out the method of the present invention, it is necessary to use a nucleic acid probe having an appropriate orientation. Typically, a triple-stranded structure is formed by using a nucleic acid probe having a sequence homologous to a strand having a 5 'end in a terminal region where a triple-stranded structure is to be formed.

In order to obtain a stable triple-stranded structure, the terminal existing outside (ie, the terminal end side of the target nucleic acid) of both ends of the triple-stranded structure is the 100th terminal from the terminal end of the target nucleic acid. It is preferably located outside the base, more preferably the 50th base, further preferably the 20th base, even more preferably the 10th base.

The RecA protein is a protein that originally catalyzes homologous recombination in the presence of ATP. Therefore, if ATP is present in the sample, homologous recombination proceeds, and the triple-stranded structure disappears immediately. Resulting in. Therefore, when performing the method of the present invention, when at least 4.8 mM of ATP is present in the sample when the triple-stranded nucleic acid is formed and after the triple-stranded nucleic acid is formed, Must not be. Preferably, the concentration of ATP is 0.48
Most preferably, it is less than or equal to mM and ATP is not present in the sample.

In order to form a triple-stranded structure via the RecA protein, a substance capable of substituting the function of ATP,
For example, an ATP analog such as ATP-γS must be added.

<Second Step> Following the previous step, a step of dissociating the RecA protein bound to the target nucleic acid is performed. In this step, by dissociating the RecA protein from the target nucleic acid,
Hybridization of the labeled second nucleic acid probe to the first probe is facilitated.

The triple-stranded structure formed in the terminal region of the target nucleic acid is maintained even if the RecA protein dissociates from the region. Therefore, in this step, R
When the ecA protein is dissociated, a triple-stranded structure formed without the recA protein is obtained.

The dissociation of the RecA protein from the target nucleic acid can be performed by removing protein such as adding phenyl / chloroform or phenol alone, adding a surfactant such as SDS, adding a protease, or using SDS +
It can be performed by a simple operation such as addition of a degrading enzyme.

<Third Step> Subsequently, a labeled nucleic acid is obtained by hybridizing a labeled second nucleic acid probe to the protruding portion of the first nucleic acid probe.

The label in the second nucleic acid probe may be any commonly used detectable label and any label that can be specifically separated. For example, the detectable label may be any detectable label,
For example, luminescent substances such as fluorescent substances and chemiluminescent substances, radioisotopes, enzymes, haptens, antigens, antibodies and the like can be used. Further, the “label that can be specifically separated” may be any one of a binding pair having high affinity for each other and specifically binding, for example, one of avidin or streptavidin and biotin , One of an antigen and an antibody. The label may be present at any position in the second nucleic acid probe, for example, it can be labeled at the end of the probe or inside the probe.

The second nucleic acid probe may be optionally
A plurality of labels, that is, two or more labels may be provided in one probe.

[0045] The second nucleic acid probe typically has a base sequence complementary to the protruding portion of the first nucleic acid probe. However, the base sequence may not be completely complementary to the protruding portion of the first nucleic acid probe as long as it can hybridize with the protruding portion. Further, the second nucleic acid probe may be shorter or longer than the protruding portion.

The above is the basic operation of the method of the present invention. As described below, an operation in which each of these operations is modified may be performed. It may be added.

As a new operation, following the last step, an operation of ligating the second nucleic acid probe to the target nucleic acid can be mentioned. If the second nucleic acid probe is ligated to the terminal end of one strand of the target nucleic acid, a nucleic acid into which the label has been firmly introduced can be obtained. Ligation can typically be accomplished with enzymes such as ligase.

After performing such an operation, an operation of dissociating the first nucleic acid probe bound to the target nucleic acid from the target nucleic acid to eliminate the triple-stranded structure may be performed. In order to eliminate the triple-stranded structure, for example, heat treatment, alkali treatment, enzyme treatment (eg, DNA polymerase treatment, etc.) can be used. Alternatively, it is also useful to introduce a mutation into the first nucleic acid probe in advance so that the triple-stranded structure can be easily dissolved. The nucleic acid probe into which the mutation has been introduced has a weak binding property to the target nucleic acid, and thus can easily eliminate the triple-stranded structure.

Further, after the basic operation of the present invention is completed, an operation of converting the target nucleic acid into a single strand may be performed. In order to make the target nucleic acid single-stranded, for example, a known method such as alkali treatment or heat treatment can be used.

Furthermore, the method of the present invention can be repeated a plurality of times for one nucleic acid to introduce a plurality of labels into one nucleic acid. In the case of performing the treatment multiple times, preferably, the triple-stranded structure in the nucleic acid obtained after the first operation is eliminated, and then the second operation is performed. This makes it possible to introduce a desired label into a plurality of desired sites. Any nucleic acid obtained by each of these operations belongs to the scope of the present invention. The nucleic acid obtained in the embodiment of the present invention can be used, for example, as a probe for a Southern hybridization method and a probe for a DNA chip. Further, the present nucleic acid may be used for direct cloning of a gene.

The obtained nucleic acid may be used as a double-stranded DNA molecule partially having a triple-stranded structure. Or a double-stranded DNA molecule or a double-stranded D molecule partially having a triple-stranded structure
Used as NA molecule, and in the middle of use process, normal DN
A A single-stranded DNA molecule may be obtained by denaturing under denaturing conditions. Alternatively, it may be used as a labeled single-stranded DNA molecule, an unlabeled single-stranded DNA molecule, or a mixture thereof by denaturation in the same manner. It can be used freely as desired by the user. If treated as a double-stranded DNA molecule or a double-stranded DNA molecule partially having a triple-stranded structure, since the labeled strand is covered, unnecessary hybridization with other nucleic acids, Unnecessary binding due to the complementary sequence in the strand DNA can be prevented and it is easy to handle.

For example, a double-stranded D partially having a triple-stranded structure
When a DNA chip is manufactured using a labeling substance of NA molecules or double-stranded DNA molecules, it is possible to easily manufacture DNA having a DNA probe at a very high density as compared with conventional ones. . Thereby, the efficiency of hybridization is improved. Further, a DNA chip obtained by immobilizing a probe composed of the above molecule on a support using a labeling substance and then removing an unlabeled strand is free from steric hindrance by a partner strand,
Hybridization can be performed efficiently.

Further, according to the embodiment of the present invention, it is possible to put a label on DNA of an arbitrary length, and the label is inserted only near the end of one side chain of DNA or in a non-terminal region. Can be. Therefore, it is also possible to directly detect the presence or absence of a specific DNA molecule using the label as an index. Such a labeled probe is also included in the scope of the present invention.

According to a further aspect of the present invention, there are also provided a labeled nucleic acid produced by the above-described method, and a carrier on which the labeled nucleic acid is immobilized.

Conventional general methods for producing a DNA chip include a method of directly synthesizing DNA on a solid support and a method of electrostatically immobilizing DNA on a solid support by spotting. However, the direct synthesis method described above can only immobilize DNA of at most 10 bp. Further, it is difficult to increase the density of the DNA to be immobilized by the method of immobilizing DNA on a solid carrier by static electricity. Furthermore, the DNA chip thus obtained is
The binding strength of DNA to a solid carrier is weak, and it is difficult to use it repeatedly. Furthermore, stable hybridization is difficult to achieve due to the structure of DNA that can be taken on a DNA chip.

The carrier on which the nucleic acid produced using the labeled nucleic acid provided by the present invention is immobilized solves the problems caused by the conventional methods as described above. That is, a nucleic acid having a long base sequence can be immobilized, the density of immobilized DNA can be increased, and stable hybridization can be achieved.

Examples of the method of immobilizing the labeled nucleic acid on the carrier include a method of forming an amino group on the surface of the carrier, and a method of arranging a substance specifically binding to the label introduced into the nucleic acid on the surface of the carrier. .

The method of forming an amino group on a solid surface is a method known per se, for example, forming an amino group by silane treatment, applying a mask to a necessary portion, and immobilizing a labeled nucleic acid by a photoreaction. It is possible to

In the method of arranging a substance which specifically binds on the surface of a carrier, for example, biotin is used as a label contained in a labeled nucleic acid, and avidin or streptavidin is arranged on the surface of the carrier, and these are specifically bound. Can be achieved. By using the labeled nucleic acid of the present invention, it is possible to immobilize the labeled nucleic acid at a high integration rate. Such an effect is obtained because the probe is immobilized on the carrier only by the label portion introduced to the end of the nucleic acid. Therefore, by using the carrier of the present invention, it is possible to stably hybridize the target nucleic acid in the detection sample with the probe.
In the above, an example using biotin and avidin has been described, but the present invention is not limited to this. As used herein, the term "binding pair" refers to a member capable of forming a bond with each other, as long as the probe of the present invention can be immobilized on a solid support, a substance that binds to each other with high affinity. Or a functional group, a part of a molecule or a residue. Generally, a binding pair is a moiety or residue of a group or molecule capable of forming a biological specific bond with one another, or a functional group or moiety of a molecule capable of forming a covalent bond chemically. Alternatively, it may be a residue. Such a covalent bond may be formed, for example, via a spacer derived from a bifunctional organic compound. As a binding pair that forms a biological specific bond,
Although not limited thereto, for example, combinations of biotins and avidins, antigens (or antigenic determinants) and antibodies, oligosaccharides and lectins, and the like can be used. When a DNA-immobilized solid support produced according to an embodiment of the present invention is used as a DNA chip, it is preferable to select biotins and avidins. That is, when biotins and avidins are used, they can be used repeatedly with high precision in a hybridization operation, and the immobilized DNA can be easily peeled off and recovered. Therefore, it is possible to reuse the solid support. As biotins, for example, biotin, biocytin, desthiobiotin, oxybiotin, and derivatives thereof that can form a stable complex with avidin may be used. On the other hand, as avidins,
For example, it may be used by selecting from avidin, streptavidin, and their variants capable of forming a stable complex with biotin. Here, “capable of forming a stable complex” means that the dissociation constant of the biotin-avidin complex (10
^-15 M) means that a complex having a dissociation constant close to that of M can be formed. The “variant” means a modified or fragment of naturally occurring avidin or streptavidin, or a recombinant thereof. Also, either of the binding pairs may be used as a label,
Therefore, either may be arranged on the carrier surface. A functional group or a part or residue of a molecule which can form a chemical covalent bond can also be used for immobilizing a protein or nucleic acid to a solid phase via a covalent bond. In the present invention, usable functional groups capable of forming a covalent bond chemically or a part or residue of a molecule may be those generally used for the immobilization. For example, there are an amino group, a hydroxyl group, a sulfhydryl group, a carboxyl group, an isocyanate group, a thioisocyanate group, and the like, and an atomic group containing these groups. When providing a DNA carrying an amino group, for example, as a substrate in a substitution reaction, N
⁶ - (6-aminohexyl) dATP can also be used. Of the above binding pairs, those that are preferred for use as labels in nucleic acid probes are those that do not adversely affect homologous recombination. For example, biotins, a member of a binding pair, for example, an isocyanate or thioisocyanate group or an atomic group having these as a functional group (for example, an alkylene chain which may be interrupted by a C1-16 oxygen atom) can be exemplified. . In addition, an isocyanate or thioisocyanate group or an atomic group having these as a functional group makes it possible to easily obtain a solid support having an amino group on the surface. The carrier on which the labeled nucleic acid is to be immobilized is not particularly limited, for example, a silicon substrate, a filter made of nitrocellulose or nylon,
Examples include, but are not limited to, microtiter plates made of polypropylene or polyethylene, and glass plates such as slide glass. Therefore, the carrier on which the labeled nucleic acid is immobilized includes, but is not limited to, a nucleic acid-immobilized carrier called a so-called DNA chip or microarray. In addition, any material may be used regardless of a natural substance or a synthetic resin, and any shape may be used as long as it does not adversely affect the mutual bond formation between the bond pairs. The shape that the solid support can take is not limited to the above example, and may be, for example, a flat plate, a microwell, a bead, a stick, or the like.

The surface properties of the solid support are generally preferably non-porous. Further, for convenience of operation, the solid support may be processed into a form of a magnetic material or an electrode.

Hereinafter, the present invention will be described in more detail with reference to Experimental Examples and Examples, but it is not intended to limit the scope of the present invention in any way.

Example 1 Dependence of Each Reaction Component on Triple-Strand Formation M13mp18RF DNA as a target DNA linearized with a restriction enzyme SnaBI, and a 60-mer oligonucleotide having the sequence of the terminal site of the target DNA 1 was prepared. The oligonucleotide is T
4 Polynucleotide kinase and [γ
The 5 'end was labeled with ³² P using [ ³² P] ATP. The triple-strand formation reaction between the target DNA and the labeled oligonucleotide 1 was 1 pmol of the labeled oligonucleotide 1, 3.0 μg of the RecA protein, 4.8 m
MATP-γS, 200 ng of target DNA,
20 mM magnesium acetate, 30 mM Tris acetate (pH
In 7.2), the mixture was kept at 37 ° C. for 30 minutes. After the reaction,
0.5% (W / Vol) SDS and 0.7 mg / ml proteinase K were added, and the mixture was kept at 37 ° C. for 30 minutes to remove proteins. 1% for half the amount
Agarose gel electrophoresis was performed. After electrophoresis, the gel was stained with ethidium bromide and a photograph of the DNA was recorded. The result is shown in FIG. After the gel was placed on a filter paper and dried with a gel dryer, an autoradiogram of the gel was taken, and the signal from the labeled probe was recorded on an X-ray film. The result is shown in lane 1 of FIG.

Lane M is a DNA size marker whose size is shown at the left end of the drawing. This size marker is
λ DNA is cut with a restriction enzyme HindIII, and T4Pol
nucleide kinase and [γ-
^This was labeled at the 5 'end with ³² P using [ ³² P] ATP. Lane 2 is the same as lane 1 except that the reaction was performed without adding RecA. Lane 3 is AT
The same as lane 1 except that the reaction was performed without adding P-γS. Lane 4 is the same as lane 1 except that the reaction was performed without adding RecA and ATP-γS. Lane 5 is the same as lane 1 except that the reaction was performed using ³² P-labeled oligonucleotide 2. Lane 6
^This is the same as Lane 1 except that the reaction was performed using ³² P-labeled oligonucleotide 3. Lane 7, as the target ^DNA, limiting the pBR 32 2 DNA enzyme ScaI
The same reaction as in lane 1 was performed, except that the one cleaved in step 1 was used and the ³² P-labeled oligonucleotide 3 having the sequence of the terminal site was used.

Sequence of oligonucleotide 1 5'-agaggctttg aggactaaag actttttcat gaggaagttt
ccattaaacg ggtaaaatac-3 'Sequence of oligonucleotide 2 5'-gtattttacc cgtttaatgg aaacttcctc atgaaaaagt ctt
tagtcct caaagcctct-3 'Sequence of oligonucleotide 3 5'-cactgcataa ttctcttact gtcatgccat ccgtaagatg ctt
ttctgtg actggtgagt-3 '.

It can be said from these results that, as shown in lane 1, all the reaction components must be added to the reaction in order to form a triple strand. In addition, it is necessary to use an oligonucleotide having a sequence having a sequence direction such as oligonucleotide 1 as the oligonucleotide used at that time.

Example 2 Sequence Directionality of Oligonucleotides Used in Triple-Strand Formation Reaction Lane 1 in FIG. 3 (A) was obtained by performing the same reaction as lane 1 in FIG. 2 (A). Lane 2 shows the result of the same reaction as lane 1 except that ³² P-labeled oligonucleotide 2 was used. Lane 3 shows the result of the same reaction as lane 1 except that labeled oligonucleotide 4 was used. Lane 4 shows the result of the same reaction as lane 1 except that labeled oligonucleotide 5 was used.
(B) shows a photograph of the same agarose gel as in (A) stained with DNA.

It can be said from these results that a triple strand can be formed at both terminal sites of the linear target DNA, and the oligonucleotide used at that time is a sequence having one direction of both terminal sequences of the target. There must be.

Sequence of oligonucleotide 4 5'-tgttttagtg tattctttcg cctctttcgt tttaggttgg tgc
cttcgta gtggcattac-3 'Sequence of oligonucleotide 5 5'-gtaatgccac tacgaaggca ccaacctaaa acgaaagagg cga
aagaata cactaaaaca-3 '.

Example 3 Length of Oligonucleotide Required for Triplex Formation Reaction Lane 1 in FIG. 4 (A) is the same as lane 1 in FIG. 2 (A) except that oligonucleotide 6 was used. The result of having performed the same reaction as shown in FIG. Lane 2 shows the result of performing the same reaction as lane 1 in FIG. 2 (A). Lane 3 is a reaction performed in the same manner as in lane 1, except that labeled oligonucleotide 7 in which the 5 'end of the oligonucleotide used in lane 1 has been trimmed by 30 mer was used. Lane 4 shows the position of the 5 'end of the oligonucleotide used in lane 1 of 40 m.
Except for using the labeled oligonucleotide 8 which was cut off,
The same reaction as in lane 1 was performed. Lane 5
The same reaction as in lane 1 was performed, except that the labeled oligonucleotide 9 in which the 5 'end of the oligonucleotide used in lane 1 was trimmed by 50 mer was used. Lane 6 is a reaction performed in the same manner as in lane 1, except that the labeled oligonucleotide 10 in which the 5 'end portion of the oligonucleotide used in lane 1 has been trimmed by 60 mer was used. Lane 7 is a reaction performed in the same manner as in lane 1, except that the labeled oligonucleotide 11 in which the 5'-terminal portion of the oligonucleotide used in lane 1 was reduced by 70 mer was used. FIG. 4 (B) shows the results obtained by staining the gel with ethidium bromide after electrophoresis and recording a photograph of the DNA.

Sequence of oligonucleotide 6 5'-acgagggtag caacggctac agaggctttg aggactaaag ac
tttttcat gaggaagttt ccattaaacg ggtaaaatac -3 'Sequence of oligonucleotide 7 5'-aggactaaag actttttcat gaggaagttt ccattaaacg gg
taaaatac -3 'Sequence of oligonucleotide 8 5'-actttttcat gaggaagttt ccattaaacg ggtaaaatac-
3 ′ Sequence of oligonucleotide 9 5′-gaggaagttt ccattaaacg ggtaaaatac -3 ′ Sequence of oligonucleotide 10 5′-ccattaaacg ggtaaaatac -3 ′ Sequence of oligonucleotide 11 5′-ggtaaaatac -3 ′.

It can be said from these results that the length of the oligonucleotide required for the triplex formation is preferably 40 me
It turns out that r or more is necessary.

Example 4 Positional Relationship of Oligonucleotide Sequences Required for Triplex Formation Reaction Lane 1 in FIG. 5 (A) was obtained by performing the same reaction as lane 1 in FIG. 2 (A). . Lane 2 is the target DN
The same reaction as in lane 1 was performed, except that oligonucleotide 12 having the sequence of the terminal site except the terminal 10 bases of A was used. Lane 3 shows the result of the same reaction as lane 1 except that oligonucleotide 13 having the sequence of the terminal site leaving the terminal 20 bases of the target DNA was used. Lane 4 shows the result of the same reaction as lane 1 except that oligonucleotide 14 having the sequence of the terminal site remaining at the terminal 30 bases of the target DNA was used. FIG. 5 (B) shows the results obtained by staining the gel with ethidium bromide after electrophoresis and recording a photograph of the DNA.

Sequence of oligonucleotide 12 5'-caacggctac agaggctttg aggactaaag actttttcat g
aggaagttt ccattaaacg -3 'Sequence of oligonucleotide 13 5'-acgagggtag caacggctac agaggctttg aggactaaag ac
tttttcat gaggaagttt -3 'Sequence of oligonucleotide 14 5'-cagcatcgga acgagggtag caacggctac agaggctttg agg
actaaag actttttcat -3 '.

It can be said from these results that the oligonucleotide sequence necessary for the formation of the triplets has a target DNA sequence starting from a sequence which enters the interior of the DNA strand from the end of the target DNA to 20 bases, if desired. It turns out to be desirable.

Example 5 Dependence of Each Reaction Component on Labeling of Target DNA pBluescriptIISK was used as target DNA.
+ DNA was linearized with a restriction enzyme NotI, and oligonucleotide 15 having the sequence of the terminal site of the target DNA was prepared. The triple-strand formation reaction between the target DNA and oligonucleotide 15 is 50 pmo.
1 oligonucleotide 15, 5.0 μg RecA protein, 4.8 mM ATP-γS, 200 ng of target DNA, 20 mM magnesium acetate, 30 mM
Incubation was carried out at 37 ° C. for 30 minutes in M tris acetate (pH 7.2). After the reaction, 0.5% (W / Vol) SDS,
Add 0.7 mg / ml proteinase K and add 3 mg at 37 ° C.
The protein was removed by keeping it warm for 0 minutes. Then, 40 μl of TE buffer (10 mM Tris-HC)
1, 1 mM EDTA) to make up to 60 μl, perform phenol / chloroform extraction once, and then use S-40.
The operation of the 0 spin column (manufactured by Amersham Pharmacia Biotech) was performed once, and unreacted oligonucleotide 1
5 was removed. After performing ethanol precipitation and dissolving the concentrated DNA in 10 μl of distilled water, 1 pmol of
³² P-labeled oligonucleotide 16, 20 mM Tri
s-HCl (pH 8.3), 25 mM KCl, 10 m
M MgCl2, 0.5 mM NAD, 0.01% Tr
itonX-100, 5 unit Ampligase
Ligation was performed by adding DNA ligase and reacting at 50 ° C. for 60 minutes. After the ligation reaction, extract phenol / chloroform for 1 hour.
After performing chloroform extraction once, ethanol precipitation was performed. After dissolving the dried DNA pellet in 8 μl of distilled water, 100 mM NaCl, 10 mM
Tris-HCl, 10 mM MgCl2, 1 mM d
ithiothreitol (pH 7.4), 10un
It Scal was added and the mixture was kept at 37 ° C for 120 minutes. Half of the mixture was subjected to 1% agarose gel electrophoresis, and the other half was subjected to 0.7% alkaline agarose gel electrophoresis according to a conventional method. For the gel after agarose gel electrophoresis, the gel was stained with ethidium bromide after electrophoresis, and a photograph of the DNA was recorded. After the gel was placed on a filter paper and dried with a gel dryer, an autoradiogram of the gel was taken and the signal from the labeled probe was converted to X.
Recorded on line film. The result is shown in lane 1 of FIG. Lane 2 is without ATP-γS
The result of performing the same reaction as in Lane 1 is shown. Lane 3
The result of performing the same reaction as in Lane 1 without adding Ligase is shown. Lane 4 shows the result of performing the same reaction as lane 1 without adding oligonucleotide 15. Lane 5 shows the result of the same reaction as lane 1 except that oligonucleotide 17 was added instead of oligonucleotide 15. Lane 6 shows the results of the same reaction as in lane 1, except that ³² P-labeled oligonucleotide 16 was added during the triple-strand formation reaction and the ligation reaction was performed at 60 ° C. for 60 minutes. FIG. 6 (B) shows the results obtained by staining the gel with ethidium bromide after electrophoresis and recording a photograph of the DNA.

It can be said from these results that the highest target DNA labeling efficiency can be obtained only when all the reaction components are contained in the reaction solution. Also, the labeling method is
Two methods are conceivable.

Sequence of oligonucleotide 15 5'-GCCAAGCGCG CAATTAACCC TCACTAAAGG GAACAAAAGC TGG
AGCTCCA CCGCGGTGGCGGCCgc ggc cgc gg -3 'Sequence of oligonucleotide 16 5'- c cgc ggc cgc-3' Sequence of oligonucleotide 17 5'-GCCAAGCGCG CAATTAACCC TCACTAAAGG GAACAAAAGC TGG
AGCTCCA CCGCGGTGGCGGCC-3 '.

Example 6 Confirmation of Ligation in Labeling of Target DNA FIG. 7 (A) shows the results of subjecting the same sample as in FIG. 6 to 0.7% alkaline agarose gel electrophoresis. FIG. 7 (B)
Shows that the restriction enzyme treatment after the ligation reaction in Example 5 was carried out using 50 mM potassiu instead of ScaI.
m acetate, 20 mM tris-aceta
te, 10 mM Magnesium umucatat
e, 1 mM dithiothreitol (pH7.
9), 100 μg / ml BSA, 10 unit Nl
The sample reacted at 37 ° C. for 120 minutes with aIV was 4.5
The results of% denaturing gel electrophoresis are shown.

It can be said from these results that the highest target DNA labeling efficiency is obtained only when all the reaction components are contained in the reaction solution, and that the labeled probe is covalently bound to the target DNA. .

Example 7 Labeling of Target DNA by Triple-Strand Formation Lane 1 in FIG. 8 (A) shows the result of performing the same reaction as lane 1 in FIG. Lane M is a DNA size marker, the size of which is shown at the left end of the drawing. This size marker cuts λ DNA with the restriction enzyme HindIII,
Polynucleotide kinase and [γ-
^This was labeled at the 5 'end with ³² P using [ ³² P] ATP. Lane 2 is the same as lane 1 except that the reaction was performed without adding RecA. Lane 3 is AT
The same as lane 1 except that the reaction was performed without adding P-γS. Lane 4 is the same as lane 1 except that the reaction was performed without adding RecA and ATP-γS. FIG. 8C shows the result of subjecting the same sample as in FIG. 8A to 0.7% alkaline agarose gel electrophoresis.

Example 8 Lane 1 in FIG. 9 (A) shows the result of performing the same reaction as lane 1 in FIG. Lane 2 shows oligonucleotide 1
The result of performing the same reaction as in Lane 1 without adding 5 is shown. Lane 3 shows that instead of oligonucleotide 15,
The result of performing the same reaction as in Lane 1 by adding oligonucleotide 17 is shown. Lane ^4, except for using ³² P-labeled oligonucleotide 3 having the terminal sequence of restriction enzyme ScaI fragment ^pBR 3 2 2 DNA, it having been subjected to the same reaction as in Lane 1. FIG. 9 (B) shows the results obtained by staining the gel with ethidium bromide after electrophoresis and recording a photograph of the DNA. FIG. 9 (C) shows the results of subjecting the same sample as in FIG. 9 (A) to 0.7% alkaline agarose gel electrophoresis.

Example 9 Lane 1 in FIG. 10 (A) shows the result of performing the same reaction as lane 1 in FIG. Lane 2 shows the result of the following reaction. PBluescript as target DNA
ptIISK + DNA was linearized with a restriction enzyme NotI, oligonucleotide 15 having the sequence of the terminal portion of the target DNA, and oligonucleotide 16 having a sequence complementary to the 10-mer sequence from the end of oligonucleotide 15 were prepared. . Oligonucleotide 16
Is T4Polynucleotide kinase
And [γ- ³² P] ATP were used to label the 5 ′ end with ³² P. Target DNA and oligonucleotide 15
The triplex formation reaction between 5 pmol oligonucleotide 15, 3.0 μg RecA protein, 4.8
mM ATP-γS, 200 ng of target DNA
Was incubated in 20 mM magnesium acetate, 30 mM Tris acetate (pH 7.2) at 37 ° C. for 30 minutes. Furthermore, 20 mM Tris-HCl (pH 8.3), 25 mM
mM KCl 10 mM MgCl2 0.5 mM N
AD, 0.01% Triton X-100, 5uni
ts Ampligase DNA Ligase, 1
pmol oligonucleotide 16 and 60 ° C at 60 ° C.
Incubated for a minute. After the reaction, 0.5% (W / Vol) SD
S, 0.7 mg / ml proteinase K was added, and 37 ° C.
For 30 minutes to remove proteins.
After performing a 1% agarose gel electrophoresis on the half of the gel, placing the gel on a filter paper and drying it with a gel dryer, taking an autoradiogram of the gel, and taking a signal from the labeled probe on an X-ray film. Recorded. FIG.
(B) shows the gel stained with ethidium bromide after electrophoresis,
The result of having recorded the photograph of DNA is shown. FIG. 101 (C)
Shows the result of electrophoresis of the same sample as in FIG. 10A with 0.7% alkaline agarose gel.

Example 10 Confirmation of Ligation in Labeling of Target DNA FIG. 11 shows that the restriction enzyme treatment after ligation reaction in Example 9 was performed by using 50 mM potass instead of ScaI.
ium acetate, 20 mM tris-ace
state, 10mM magnesium ace
state, 1 mM dithiothreitol (p
H7.9), 100 μg / ml BSA, 10 units
A sample reacted with NlaIV at 37 ° C. for 120 minutes
The results of 4.5% denaturing gel electrophoresis are shown.

[0084]

According to the method of the present invention, it is possible to introduce a label selectively to a desired site of a double-stranded nucleic acid easily and in a short time with a high site selection system. Specifically, even if the ends of the nucleic acids are equivalent to each other, it is possible to introduce a label only at the desired end.

By using the method of the present invention, it is not necessary to perform further steps essential for the conventional PCR method, for example, steps such as identification of the obtained nucleic acid and purification of the target nucleic acid.

Further, the carrier obtained by the present invention is DN
The A probe is provided immobilized at high density.

[0087]

[Sequence List] SEQUENCE LISTING <110> Aisin Cosmos R & D Co., LTD. <120> Method of a preparation of a nucleic acid introduced a label with RecA protein <130> A000007765 <140> <141> <160> 17 <170 > PatentIn Ver. 2.0 <210> 1 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: <400> 1 agaggctttg aggactaaag actttttcat gaggaagttt ccattaaacg ggtaaaatac 60 <210> 2 <211 > 60 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: <400> 2 gtattttacc cgtttaatgg aaacttcctc atgaaaaagt ctttagtcct caaagcctct 60 <210> 3 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: <400> 3 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt 60 <210> 4 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: <400> 4 tgttttagtg tattctttcg cctctttcgt tttaggttgg tgccttcgta gtggcattac 60 <210> 5 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> Descripti on of Artificial Sequence: <400> 5 gtaatgccac tacgaaggca ccaacctaaa acgaaagagg cgaaagaata cactaaaaca 60 <210> 6 <211> 80 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: <400> 6 acgagggtag caacggctac agaggctttg aggactaaag actttttcat gaggaagttt 60 ccattaaacg ggtaaaatac 80 <210> 7 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: <400> 7 aggactaaag actttttcat gaggaagttt ccattaaacg ggtaaac 50g <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: <400> 8 actttttcat gaggaagttt ccattaaacg ggtaaaatac 40 <210> 9 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: <400> 9 gaggaagttt ccattaaacg ggtaaaatac 30 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: <400> 10 ccattaaacg ggtaaaatac 20 <210> 11 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> Description of Artific ial Sequence: <400> 11 ggtaaaatac 10 <210> 12 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: <400> 12 caacggctac agaggctttg aggactaaag actttttcat gaggaagttt ccattaaacg 60 <210 > 13 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: <400> 13 acgagggtag caacggctac agaggctttg aggactaaag actttttcat gaggaagttt 60 <210> 14 <211> 74 <212> DNA < 213> Artificial Sequence <220> <223> Description of Artificial Sequence: <400> 14 cagcatcgga acgagggtag caacggctac agaggctttg aggactaaag actttttcat 60 <210> 15 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: <400> 15 gccaagcgcg caattaaccc tcactaaagg gaacaaaagc tggagctcca ccgcggtggc 60 ggccgcggcc gcgg 74 <210> 16 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: <400> 16 ccgcggccgc 10 <210> 17 <211> 64 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequenc e: <400> 17 gccaagcgcg caattaaccc tcactaaagg gaacaaaagc tggagctcca ccgcggtggc 60 ggcc 64

[Brief description of the drawings]

FIG. 1 is a scheme showing reactions that are assumed to proceed in embodiments of the present invention.

FIG. 2 is a photograph of electrophoresis showing the results of Experimental Example 1.

FIG. 3 is a photograph of electrophoresis showing the results of Experimental Example 2.

FIG. 4 is a photograph of electrophoresis showing the results of Example 3.

FIG. 5 is a photograph of electrophoresis showing the results of Experimental Example 4.

FIG. 6 is a photograph of electrophoresis showing the results of Experimental Example 5.

FIG. 7 is a photograph of electrophoresis showing the results of Experimental Example 6.

FIG. 8 is a photograph of electrophoresis showing the results of Experimental Example 7.

FIG. 9 is a photograph of electrophoresis showing the results of Experimental Example 8.

FIG. 10 is a photograph of electrophoresis showing the results of Experimental Example 9.

FIG. 11 is a photograph of electrophoresis showing the results of Experimental Example 10.

[Explanation of symbols]

1. 1. Target DNA First probe DNA
3. RecA protein4. Probe DNA / RecA protein complex
5. 5. Triple-stranded DNA structure Second probe DNA

 ──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Michio Oishi 1532-3 Yana, Kisarazu-shi, Chiba Inside the Kazusa DeNA Institute (72) Inventor Osamu Ohara 1532-3 Yana, Kisarazu-shi, Chiba Foundation F-term in Kazusa DeNA Research Laboratories (reference) 4B024 AA20 CA01 HA08 HA12 HA19 4B029 AA23 BB20 CC03 4B063 QA01 QA05 QA11 QA17 QA18 QA19 QQ42 QR32 QR82 QS14 QS25 QS34 QX02

Claims (4)

[Claims] 1. A method for producing a nucleic acid into which a label has been introduced, the method comprising:
By adding a protein and a first nucleic acid probe homologous to the terminal region of the target nucleic acid, a part of the first nucleic acid probe projects from the target nucleic acid, and A step of binding a first nucleic acid probe to the target nucleic acid; a step of dissociating the RecA protein bound to the target nucleic acid from the target nucleic acid; and a second label attached to a protruding portion of the first nucleic acid probe. Producing a nucleic acid into which a label has been introduced by hybridizing the nucleic acid probe. 2. A method for producing a nucleic acid into which a label has been introduced, wherein each step according to claim 1 further comprises a step of ligating the second nucleic acid probe to the target nucleic acid. 3. A method for producing a nucleic acid into which a label has been introduced, wherein in each of the steps according to claim 1 and 2, the first nucleic acid probe bound to the target nucleic acid is used as the target nucleic acid. A method further comprising the step of dissociating from 4. A carrier on which a nucleic acid produced by the method according to claim 1 is immobilized.