JP2003159058A - Method for hybridizing double-stranded nucleic acid containing target sequence with capturing probe nucleic acid and method for analyzing base sequence in sample containing specimen double-stranded nucleic acid utilizing the same hybridization - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for hybridizing a target double-stranded nucleic acid with a capturing probe nucleic acid with a high efficiency. <P>SOLUTION: This method is to hybridize the double-stranded nucleic acid containing a target sequence with the capturing probe nucleic acid. The capturing probe nucleic acid comprises a sequence complementary to the target sequence. The method comprises the following steps of (1) denaturing the double- stranded nucleic acid and providing a single-stranded nucleic acid and (2) hybridizing the single-stranded nucleic acid with the capturing probe nucleic acid in the presence of a recombination inhibiting nucleic acid containing a sequence complementary to a part of the single-stranded nucleic acid obtained in (1) and different from the target sequence and the complementary sequence thereto. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for hybridizing a double-stranded nucleic acid with a capture probe nucleic acid for capturing the double-stranded nucleic acid.

[0002]

2. Description of the Related Art In recent years, a genetic testing technique using a nucleic acid chain fixed array (generally called a DNA array) as a nucleic acid detection sensor has attracted attention (Beattie).
et al. 1993, Fodor et al.
1991, Khrapko et al. 1989,
Southern et al. 1994).

DNA arrays have 10 different sequences.
It is an array made of glass or silicon substrate of several cm square on which ¹ to 10 ⁵ kinds of DNA nucleic acid chains are immobilized. When analyzing a test solution, first, a test solution gene labeled with a fluorescent dye or a radioisotope (RI) is reacted on an array, or a mixture of an unlabeled test solution gene and a labeled oligonucleotide. Are reacted on the array under conditions allowing sandwich hybridization. If the test solution has a sequence complementary to the DNA nucleic acid chain on the array, a signal (eg, fluorescence intensity or RI intensity) derived from the label can be obtained at a specific site. Therefore, if the sequence and position of the immobilized DNA nucleic acid chain are known, the base sequence present in the test solution gene can be easily examined. In addition, since a large amount of information on a nucleotide sequence can be obtained from a small amount of sample, a DNA array is highly expected as a sequencing technique as well as a gene detection technique (Pease et al. 1994, Par.
inov et al. 1996).

On the other hand, most naturally occurring nucleic acids exist as double strands. When actually analyzing a test nucleic acid as described above using a DNA array, first, the test nucleic acid is often amplified by a PCR reaction or the like prior to such analysis. The PCR product obtained by such amplification is generally double-stranded. Therefore, in the analysis by the DNA array, it is first necessary to denature the double strand to be analyzed into a single strand.

When a double-stranded nucleic acid is analyzed by a conventional method using a DNA array, the target double-stranded nucleic acid is heated to be single-stranded to obtain a single-stranded nucleic acid sample, To D
After mixing with a solution containing NA probe, it is generally cooled to allow hybridization. Such a conventional method has the following problems.

[0006] For example, when the length of the probe nucleic acid and the length of the single-stranded nucleic acid of the sample are equal, the binding probabilities of these are equal.
Therefore, in addition to the above conditions, the number of probe nucleic acids and
If the number of single-stranded nucleic acids is the same, the number of single-stranded nucleic acids in the sample is 5
It will be recombined with a probability of 0%.

If the length of the probe nucleic acid and the length of the sample single-stranded nucleic acid are the same, if the number of probe nucleic acids is made larger than the number of the sample single-stranded nucleic acids, the recombination rate of the sample single-stranded nucleic acids is reduced. It is possible to However, in such a case, a large number of probe nucleic acids are required, so that immobilizing them on a DNA chip would result in an excessively high density of probe nucleic acids.

Further, for example, when the length of the probe nucleic acid is made shorter than the length of the sample single-stranded nucleic acid, the binding rate of the probe nucleic acid and the sample single-stranded nucleic acid becomes higher than that of the sample single-stranded nucleic acids ( This is due to the short regions of complementarity). However, when electrochemical detection is performed using an intercalating agent that selectively binds only to the hybridized nucleic acid region, the hybridized region is shortened, so that the binding rate of the intercalating agent decreases, and The amount of detected electric charge becomes small.

Further, when only a DNA probe having a complementarity to the single-stranded nucleic acid on one side obtained by single-stranded formation is used, 50% of the single-stranded nucleic acid on the specimen present in the reaction system is wasted. Become.

[0010]

In view of the above situation, it is an object of the present invention to provide a method for hybridizing a target double-stranded nucleic acid with a capture probe nucleic acid with high efficiency.

[0011]

The present invention is
Focusing on the fact that a short base sequence has a faster binding rate than a long base sequence, it was achieved by a unique idea based on this and earnest research for solving the above problems.
That is, (I) a method of hybridizing a double-stranded nucleic acid containing a target sequence and a capture probe nucleic acid, wherein the capture probe nucleic acid contains a sequence complementary to the target sequence, and the following steps: 1) denaturing the double-stranded nucleic acid into a single strand, and (2) complementing a part of the single strand obtained in (1) and the target sequence and its complementary sequence; Hybridizing the single strand with the capture probe nucleic acid in the presence of a recombination-inhibiting nucleic acid containing a different sequence. Soybean method; and (II) a method for analyzing a base sequence in a sample containing a sample double-stranded nucleic acid, which comprises the following steps: (1) denaturing the sample double-stranded nucleic acid to form a single strand And (2) obtained in (1) above. The single strand obtained in the above (1) in the presence of a recombination-inhibiting nucleic acid which is complementary to a part of the single strand and which contains a sequence different from the target sequence and its complementary sequence, and the target. Detecting the target sequence in the sample double-stranded nucleic acid by hybridizing with a capture probe nucleic acid containing a sequence complementary to the sequence, and (3) detecting the hybridization generated in (2) above. The method for analyzing a base sequence in a sample containing a sample double-stranded nucleic acid, comprising:

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION According to the present invention, a method for hybridizing a target double-stranded nucleic acid with a capture probe nucleic acid with high efficiency is provided.

Specifically, according to the present invention, there is provided a method of hybridizing a double-stranded nucleic acid containing a target sequence and a capture probe nucleic acid, wherein the capture probe nucleic acid is a sequence complementary to the target sequence. And (1) denaturing the double-stranded nucleic acid into a single strand, and (2) complementary to a part of the single strand obtained in (1) above, and A double-stranded nucleic acid containing a target sequence, which comprises hybridizing the single strand with the capture probe nucleic acid in the presence of a recombination-inhibiting nucleic acid containing a sequence different from the target sequence and its complementary sequence. There is provided a method for hybridizing a probe nucleic acid for capture with a nucleic acid.

The hybridization method according to the present invention comprises the detection and analysis of a sample double-stranded nucleic acid and / or a target sequence contained in a sample, for example, gene expression analysis such as appearance / disappearance of gene expression, single nucleotide polymorphism in the genome. Type (Single Nucleotide Polymorphism, hereafter referred to as SNP, and when there are multiple SNPs), analysis of polymorphisms such as microsatellite sequences, diagnosis of disease by detection of disease-related genes, prediction of incidence, infection It is useful for detection of virus, analysis of virus type, and toxicity test. It can also be used in clinical diagnosis and prediction and various basic researches. For example, it can be used when a genetic test is required, such as food inspection, quarantine, pharmaceutical inspection, forensic medicine, agriculture, livestock, fisheries and forestry.

Conventionally, when a sample double-stranded nucleic acid is analyzed using a base sequence detection chip on which a probe nucleic acid is immobilized, first, the sample double-stranded nucleic acid is denatured to 1
A single strand is formed, and the obtained single strand is hybridized with the immobilized probe nucleic acid. In the case where there is a problem here, for example, the sample that has been made single-stranded by denaturation by heating or the like does not hybridize with the probe nucleic acid to be bound and is reproduced between the original nucleic acid chains (hereinafter, also referred to as rear annealing). ) If you do. Alternatively, there is a case where the original nucleic acid chains hybridize to each other in the other portion even though the single-stranded nucleic acid is bound to the probe nucleic acid to be bound.

According to the present invention, the problems in the conventional method can be solved by a simple and effective means.

The term "double-stranded nucleic acid containing a target sequence" as used herein refers to a double-stranded nucleic acid containing a target sequence which is the sequence to be detected and / or analyzed. The double-stranded nucleic acid that can be detected and / or analyzed in the method according to the present invention may be any naturally occurring nucleic acid or any artificially synthesized nucleic acid. For example, examples of natural double-stranded nucleic acids include cells and tissues derived from humans and non-human animals, such as cells, tissues, blood, body fluids, various excretions, cells and tissues derived from plants, and bacteria, fungi,
It may be a double-stranded nucleic acid prepared as desired by a method known per se from any material derived from organisms and microorganisms such as viruses, amoeba, protozoa and cultured cells. The artificially synthesized nucleic acid may be a double-stranded nucleic acid obtained by an operation such as synthesis or gene recombination by a method known per se.

As used herein, the term "nucleic acid" refers to various naturally occurring DNAs and RNAs, as well as artificially synthesized nucleic acids such as peptide nucleic acids, morpholino nucleic acids, methylphosphonate nucleic acids and S-oligo nucleic acids. Nucleic acid analogs and the like are used as a general term. The term "double-stranded nucleic acid" used herein refers to a state in which two nucleic acids, which are any of the above-mentioned nucleic acids, are hybridized in the same kind or different kinds.

The term "base sequence detection chip" as used herein refers to a chip for the purpose of detecting a base sequence, and generally means what is called a DNA microarray or a DNA chip. . For example, the base sequence detection chip that can be used in accordance with the present invention may be one in which a probe nucleic acid is immobilized on a general substrate, and such a base sequence detection chip is a general method known per se. Can be manufactured by

As used herein, the terms "complementary", "complementary" and "complementarity" may be complementary in the range 50% to 100%, preferably 100% complementary. Say. Also, as used herein, "homologous", "homologous"
The terms "homology" and "homology" need only be homologous in the range of 50% to 100%, and preferably 100% are homologous.

Basically, the method of hybridizing the double-stranded nucleic acid containing the target sequence with the probe nucleic acid for capture according to the present invention is hybridization utilizing the complementarity of the base sequences, and therefore the base in the sample is used. It may be used as a means to analyze a sequence. By such an analysis, for example, it is possible to detect whether or not the target sequence is present in the sample double-stranded nucleic acid. In addition, it is used in place of the conventional hybridization method, for example, the conventional method that is carried out when analyzing the base sequence of a double-stranded nucleic acid sample by hybridization using a nucleic acid probe immobilized on a base sequence detection chip. May be.

The method for analyzing the nucleotide sequence in the sample according to the present invention can be carried out by utilizing the method of hybridizing the double-stranded nucleic acid containing the target sequence according to the present invention and the capture probe nucleic acid. is there. For example, one example of such a method is as follows. That is, a method for analyzing a base sequence in a sample containing a sample double-stranded nucleic acid, which comprises the following steps; (1) denaturing the sample double-stranded nucleic acid into a single strand, and ( 2) Obtained in (1) above in the presence of a recombination-inhibiting nucleic acid that is complementary to a part of the single strand obtained in (1) above and contains a sequence different from the target sequence and its complementary sequence. Sample double-stranded by hybridizing the single-stranded DNA with a probe nucleic acid for capture containing a sequence complementary to the target sequence, and (3) detecting the hybridization generated in (2) above. A method for analyzing a base sequence in a sample containing a sample double-stranded nucleic acid, comprising detecting the target sequence in the nucleic acid.

Examples of the sample double-stranded nucleic acid to be analyzed by the method according to the present invention are derived from plants such as cells, tissues, blood, body fluids and various excretions derived from humans and non-human animals. Naturally occurring cells and tissues, etc., and derived from organisms and microorganisms such as bacteria, fungi, viruses, amoeba, protozoa and cultured cells 2
It may be a double-stranded nucleic acid, a double-stranded nucleic acid obtained by artificial synthesis, or a double-stranded nucleic acid obtained by an operation such as gene recombination. The term "target sequence" as used herein refers to the sequence that one wishes to detect.

According to the present invention, the capture probe nucleic acid may be immobilized on the base sequence detection chip.

The method for hybridizing a double-stranded nucleic acid containing a target sequence and a probe nucleic acid for capturing according to the present invention, and the method for detecting whether or not a target sequence is present in a sample double-stranded nucleic acid are the present invention. Various changes can be made without departing from the range.

3. Examples Hereinafter, embodiments of the present invention will be described with reference to the drawings. Figure 1,
2, 4 and 5 schematically show the existing state of the nucleic acid contained in the reaction system. All reactions are carried out in a known reaction vessel or a chip for detecting a nucleotide sequence, which contains a solution under conditions suitable for a nucleic acid hybridization reaction known per se. Further, in the following examples, the "double-stranded nucleic acid" corresponds to the above-mentioned "double-stranded nucleic acid containing the target sequence" or "sample double-stranded nucleic acid".

Example 1 An example of the hybridization method according to the present invention will be described with reference to FIG.

First, the double-stranded nucleic acid 1 that is in a double-stranded state at room temperature is denatured by heating to obtain a first single-stranded nucleic acid 2 and a second single-stranded nucleic acid 3. Here, SNPs of double-stranded nucleic acid
The existence area 4 includes SNPs to be detected. The sequence of SNPs existing region 4 is the target nucleic acid in this example.

The recombination-inhibiting nucleic acid 5 is added while being heated, and then cooled, and the recombination-inhibiting nucleic acid 5 is hybridized with a part of the first single-stranded nucleic acid 2. The partially double-stranded first single-stranded nucleic acid 2 and second single-stranded nucleic acid 3 thus obtained are added to the base detection chip 6. Since the capture probe nucleic acids 7a and 7b are fixed in advance on the base detection chip 6, the capture probe nucleic acid 7a is
The single-stranded nucleic acid 2 hybridizes with the second single-stranded nucleic acid 3 hybridizes with the capture probe nucleic acid 7b.

The length of the double-stranded nucleic acid 1 is 5 bases to 10 bases.
It may be 10,000 bases. Here, an example is shown in which the SNP site is included as the target sequence to be detected, but the target sequence may include any sequence as desired. Also, multiple target sequences may be present.

The recombination inhibiting nucleic acid 5 is complementary to a part of the base sequence of the first single-stranded nucleic acid 2 contained in the double-stranded nucleic acid 1. The length of the base sequence of the recombination-inhibiting nucleic acid 5 can be changed as desired, but it is preferably about 3 to about 99,000 bases. First one of recombination-inhibiting nucleic acids 5
The site for the double-stranded nucleic acid 2 may be any site as long as it is a site different from the target sequence.

In the above example, since the SNPs existing region 4 of the double-stranded nucleic acid 1 is the target sequence, the capture probe nucleic acid 7
a and 7b have a base sequence complementary to the SNPs existing region 4. As described above, the target sequence is not limited to the SNPs existing region and may be arbitrarily selected by the practitioner. Therefore, the capture probe nucleic acids 7a and 7b are composed of a base sequence complementary to the selected target sequence, for example, the sample 2
It is sufficient that the double-stranded nucleic acid has a sequence complementary to the sequence containing the sequence or base to be detected.

The capture probe nucleic acid 7a is a probe for capturing the first single-stranded nucleic acid 2 by hybridization. The capture probe nucleic acid 7b is a probe for capturing the second single-stranded nucleic acid 3.

The length of the capture probe nucleic acids 7a and 7b may be from 1 base to 99,999 bases, preferably from 3 bases to 99,000 bases, and is longer than the lengths of the first and second single-stranded nucleic acids. short.

The site of each single-stranded nucleic acid to which each of the complementary probe nucleic acids 7a and 7b binds is preferably near the 3'or 5'end of each single-stranded nucleic acid. The capture probe nucleic acids 7a and 7b may or may not be complementary to each other. Therefore, the capture probe nucleic acids 7a and 7b are both 3'side or 5'of the single-stranded nucleic acid.
Even if it is complementary to the sequence of, one side is 5'and the other side is 3 '
It may be complementary to the side.

Here, prior to addition to the base sequence detection chip 6, the denaturation of the double-stranded nucleic acid 1 and hybridization with the recombination-inhibiting nucleic acid 5 are carried out in an arbitrary container. However, the order of performing each step is not limited to this.

For example, the double-stranded nucleic acid 1 and the recombination inhibiting nucleic acid 5 are added to the base sequence detection chip 6 at room temperature, and then,
The same reaction may be performed by sequentially performing heat treatment and cooling treatment. Alternatively, the double-stranded nucleic acid 1 is heated and denatured to 1
After forming the single strand, the single strand obtained may be added to the base sequence detection chip 6 before or after the addition of the recombination-inhibiting nucleic acid 5, and then cooled. Alternatively, denaturation may be performed after the recombination-inhibiting nucleic acid 5 is added to the double-stranded nucleic acid. Alternatively, the recombination-inhibiting nucleic acid 5 can be prepared by a process such as heating, cooling and various additions at a timing other than the above.
The hybridization of the single-stranded nucleic acid derived from the sample double-stranded nucleic acid 1 and the capture probe nucleic acid 7 may be carried out in the presence of. Hybridization methods achieved by such variations are also within the scope of the invention.

As the reaction conditions such as temperature and reaction time used here, it is possible to use general conditions known per se, but the selection is made according to the type of base sequence to be detected, the type used. It may be arbitrarily selected by the practitioner according to conditions such as the type of probe nucleic acid to be used.

Here, the room temperature is 10 ° C. to 30 ° C. Here, the heating temperature may be a temperature necessary for denaturing the double-stranded nucleic acid, and may be, for example, 30 ° C to 100 ° C.
The cooling temperature may be a temperature necessary for achieving hybridization, and is, for example, -200 ° C to 10 ° C.
Good. The heating and cooling rates may be heating and cooling rates known per se that are suitable for denaturing and hybridizing nucleic acids.

Base sequence detection chip 5 used here
The substrate may be a substrate such as a glass substrate or a silicon substrate on which a capture probe nucleic acid having a sequence complementary to the target base sequence or a sequence capable of capturing the target base sequence is immobilized. For example, a current detection type base sequence detection chip as described in Japanese Patent No. 2573443 or a fluorescence detection base sequence detection chip may be used. The present invention is also a method for increasing the efficiency of hybridization with a capture probe nucleic acid used for detecting a double-stranded nucleic acid. Therefore, the form of the substrate provided in the base sequence detection chip is not limited to the plate-shaped substrate, and may be spherical, rod-shaped, or column-shaped. Alternatively, the sample double-stranded nucleic acid and the capture probe nucleic acid not immobilized on the substrate may be hybridized in a liquid.

Example 2 An example of the hybridization method according to the present invention will be described with reference to FIG.

First, the double-stranded nucleic acid 1 which is in a double-stranded state at room temperature is denatured by heating to obtain a first single-stranded nucleic acid 2 and a second single-stranded nucleic acid 3. Here, SNP of double-stranded nucleic acid 1
The s existing region 4 includes SNPs to be detected. As in Example 1, the sequence of SNPs existing region 4 is used as the target sequence.

After the recombination-inhibiting nucleic acid 5 is added while being heated, it is cooled and the recombination-inhibiting nucleic acid 5a is hybridized with a part of the first single-stranded nucleic acid 2. Then, the recombination-inhibiting nucleic acid 5b is added to hybridize the second single-stranded nucleic acid 3 with the recombination-inhibiting nucleic acid 5b. Part 2 obtained by this
The first single-stranded nucleic acid and the second single-stranded nucleic acid 3 that have become single-stranded are added to the base sequence detection chip 5 and hybridize to the capture probe nucleic acids 7a and 7b.

Basic conditions and applicable ranges of various reactions and various nucleic acids used may be as described in Example 1. Similarly, the recombination-inhibiting nucleic acid 5a may be as described for the recombination-inhibiting nucleic acid 5 in Example 1. The recombination-inhibiting nucleic acid 5b may be as described for the recombination-inhibiting nucleic acid 5 of Example 1, except that its sequence has complementarity with a partial sequence of the second single-stranded nucleic acid 3.

In the above method, the recombination-inhibiting nucleic acid 5
Although a and 5b were added sequentially, they may be added simultaneously. In that case, the nucleic acid sequences of the recombination-inhibiting nucleic acids 5a and 5b preferably have low complementarity.

Similar to Example 1, also in Example 2, recombination-inhibiting nucleic acids 5a and 5b are prepared by a process of heating, cooling and various additions at timings other than the above.
The hybridization of the single-stranded nucleic acid derived from the double-stranded nucleic acid 1 and the capture probe nucleic acids 7a and 7b may be carried out in the presence of. By doing so, the number of types of recombination-inhibiting nucleic acids is increased, so that variations in the process can be increased. Hybridization methods achieved by such variations are also within the scope of the invention.

Example 3 FIGS. 3 (a) to 3 (f) show arrangement patterns of detection probes 7a and 7b immobilized on a base sequence detection chip 5 which can be used in Examples 1 and 2 according to the present invention. It is a schematic diagram which shows an example. The black squares indicate the area on which the detection probe 7a is immobilized, and the open squares indicate the area on which the capture probe nucleic acid 7b is immobilized. In each area, a plurality of detection probes 7 are immobilized independently of each other detection probe. These patterns are provided for illustrative purposes and are not intended to limit the invention. Therefore, it is not always necessary to immobilize the same number of both probes. Furthermore, if desired, other types of detection probes may be used for capturing probe nucleic acid 7a.
It may be immobilized together with and b.

The detection probes 7a and 7b fixed to the base sequence detection chip 5 used in the present invention are
Both probes may be mixed and immobilized on the base sequence detection chip 5. However, in order to prevent the rear annealing of the first single-stranded nucleic acid 2 and the second single-stranded nucleic acid 3, they are separated from each other by a distance such that they do not intersect with each other or do not mix, that is, respectively. It is preferable that each type is fixed in an independent area. Such an interval may be, for example, 10 μm to 100 μm. Immobilization of the detection probe on the base sequence detection chip 5 may be achieved by an immobilizing means known per se, for example, by immobilizing it by means using general spotting or photoreaction. Good.

A base sequence detection chip having a capture probe nucleic acid in such a pattern is also within the scope of the present invention.

Further, the above-mentioned base sequence detection chip is
It can be manufactured by a technique known per se.
Furthermore, the basic conditions and applicable range for the nucleotide sequence detection chip 5 used here may be as described in Example 1.

Example 4 (Reference Example) A further example of the hybridization method according to the present invention will be described with reference to FIG.

First, the double-stranded nucleic acid 1 which is in a double-stranded state at room temperature is denatured by heating to obtain a first single-stranded nucleic acid 2 and a second single-stranded nucleic acid 3. Here, SNPs of double-stranded nucleic acid
The existence area 4 includes SNPs to be detected. As in Examples 1 and 2, the sequence of SNPs existing region 4 is also used as the target sequence here.

Next, the mediating probe nucleic acid 8 is kept heated.
Add a and 8b. The intermediary probe nucleic acid 8b has a sequence complementary to a part of the sequence of the first single-stranded nucleic acid 2 and a sequence 9 complementary to the capture probe nucleic acid 7c immobilized on the base sequence detection chip 5. Including. Similarly, the intermediary probe nucleic acid 8a is complementary to the sequence complementary to a part of the sequence of the second single-stranded nucleic acid 2 and the capture probe nucleic acid 7d immobilized on the base sequence detection chip 5. Includes Sequence 9. Therefore, by cooling the obtained mixture, the mediating probe nucleic acids 8b and 8a are respectively added to the first single-stranded nucleic acid and the second single-stranded nucleic acid.
Hybridize to the single-stranded nucleic acid of.

Next, the obtained reaction solution is added to the base sequence detection chip 5. Thereby, the complex of the first single-stranded nucleic acid 2 and the intermediary probe nucleic acid 8b hybridizes to the capture probe 7c, and the complex of the second single-stranded nucleic acid 2 and the intermediary probe nucleic acid 8a becomes the capture probe 7d. Hybridize.

The intermediary probe nucleic acid 8 used here is
It has a function as a recombination-inhibiting nucleic acid, and also has a role of mediating the hybridization between the single-stranded nucleic acid 2 derived from the double-stranded nucleic acid 1 and the capture probe nucleic acid 7. By using such an intermediary probe nucleic acid 8, in addition to achieving highly efficient hybridization between the double-stranded nucleic acid 1 and the capture probe nucleic acid 8, the hybridization on the base sequence detection chip 5 is stabilized. Can be carried out. In addition, the capture probe nucleic acid 8
If the sequences of 8a and 8b are designed to be different, it is also possible to use the base sequence detection chip 5 on which the capture probe nucleic acid 8 is immobilized in the pattern as described in Example 3.

For the sequences complementary to the first and second single-stranded nucleic acids 2 and 3 contained in the intermediary probe nucleic acids 8a and 8b, a sequence near the 3'or 5'end of each single-stranded nucleic acid is selected. Preferably.

The sequence 9 complementary to the capturing nucleic acid contained in the intermediary probe nucleic acids 8a and 8b, that is, the arbitrary base sequence 9 may be a base sequence arbitrarily selected by the practitioner. For example, it may be designed considering stable reactivity,
The sequences of other bases that may exist in the same reaction system may be taken into consideration and designed so as to have low complementarity with those sequences.
Moreover, the intermediary probe nucleic acids 8a and 8b may include a further sequence in addition to the sequence complementary to the single-stranded nucleic acid 2 and the arbitrary base sequence 9. Further, if desired, one arbitrary base sequence 9 and the other arbitrary base sequence 9 may have the same base sequence or different base sequences.

The length of the intermediary probe nucleic acids 8a and 8b may be 1 to 99,999 bases, preferably 3 to 99,000 bases, and the first and second 1
It is shorter than the length of the double-stranded nucleic acid.

Further, capture probe nucleic acids 7a and 7b
May further include a sequence other than the sequence complementary to the arbitrary base sequence 9.

In the above method, the mediating probe nucleic acid 8
Although two kinds of the intermediary probe nucleic acids 8 of a and the intermediary probe nucleic acid 8b are used, only one of them may be used.

In the same manner as in Examples 1 and 2, the reaction is carried out in the presence of the intermediary probe nucleic acids 8a and / or 8b by a process such as heating, cooling and various additions at a timing other than the above. The single-stranded nucleic acid derived from the double-stranded nucleic acid 1 may be hybridized with the capture probe nucleic acid 7.

The basic conditions and applicable range of various reactions and various nucleic acids used may be the same as those described in Examples 1 to 3.

Example 5 A further example of the hybridization method according to the present invention will be described with reference to FIG. First, the double-stranded nucleic acid 1 that is in a double-stranded state at room temperature is denatured by heating to obtain the first single-stranded nucleic acid 2 and the second single-stranded nucleic acid 3. Here, in the SNPs existing region 4 of the double-stranded nucleic acid, the SNPs to be detected
It is included. Similar to the above example, here, the sequence of the SNPs existing region 4 contained in one of the single chains is used as the target sequence.

Next, the nucleic acid 5a that inhibits recombination while being heated
And the intermediary probe nucleic acids 8a and 8b are added. Then, when cooled, the first single-stranded nucleic acid 2, the recombination-inhibiting nucleic acid 5a, and the mediating probe nucleic acid 8b hybridize. At the same time, the intermediary probe nucleic acid 8a hybridizes to the second single-stranded nucleic acid 3.

Next, the recombination inhibiting nucleic acid 5b is added. The mixture of the first single-stranded nucleic acid complex and the second single-stranded nucleic acid complex having a double-stranded portion in a part thus obtained is added to the base sequence detection chip 5. As a result, the first single-stranded nucleic acid complex hybridizes with the capture probe nucleic acid 7c, and the second single-stranded nucleic acid complex hybridizes with the capture probe nucleic acid 7c.
hybridizes to d.

In the above example, two types of recombination-inhibiting nucleic acid 5
a and 5b and two kinds of mediating probe nucleic acids 8a and 8
Although the method using b has been shown, the types of recombination-inhibiting nucleic acid and mediating probe nucleic acid are not limited to these, and can be further increased.

In the same manner as in Examples 1, 2 and 4, two nucleic acid molecules in the presence of the recombination-inhibiting nucleic acid 5 and the intermediary probe nucleic acid are subjected to a process of heating, cooling and various additions at a timing other than the above. Hybridization between the single-stranded nucleic acid derived from the strand nucleic acid 1 and the capture probe nucleic acid 7 may be performed. Hybridization methods achieved by such variations are also within the scope of the invention.

Furthermore, the basic conditions and applicable range of various reactions and various nucleic acids used are described in Examples 1 to 3.
The conditions and ranges according to the description may be used.

Example 6 In the present invention, the above-described hybridization method according to the present invention is utilized, and following those processes, the capture probe nucleic acid 7 on the chip for detecting a base sequence and the single-stranded nucleic acid or the intermediary probe nucleic acid are used. It is also possible to detect the target sequence in the double-stranded nucleic acid of the sample by detecting the hybridization that occurs between the nucleic acid and the nucleic acid. In one aspect of the present invention, a method for analyzing the base sequence in such a sample is also provided.

The method for detecting the hybridization between the capture probe nucleic acid 7 and the single-stranded nucleic acid 2 or the intermediary probe nucleic acid 8 may be any means known per se, for example, fluorescence detection. Method, RI intensity detection method, electrochemical detection method and the like can be used.

Further, specifically, the method for analyzing the base sequence in the sample described above can be carried out using the examples shown in Examples 1 to 5 described above. Such methods are also included in the present invention.

Furthermore, the recombination-inhibiting nucleic acid or the intermediary probe nucleic acid can be prepared by any means known per se, for example,
Labeling substances such as fluorescence, dyes, luminescence, RI and enzymes may be labeled. By detecting those labeled substances,
A method for confirming that no rear annealing due to the sample nucleic acid has occurred is also included in the scope of the present invention.

[0073]

INDUSTRIAL APPLICABILITY According to the present invention, there is provided a method for hybridizing a target double-stranded nucleic acid with a capture probe nucleic acid with high efficiency.

[Brief description of drawings]

FIG. 1 is one of the hybridization methods according to the present invention.
The flowchart which shows an example.

FIG. 2 is a flowchart showing another example of the hybridization method according to the present invention.

FIG. 3 is a plan view showing an example of a nucleotide sequence detection chip used in the hybridization method according to the present invention.

FIG. 4 is a flowchart showing a further example of the hybridization method according to the present invention.

FIG. 5 is a flowchart showing a further example of the hybridization method according to the present invention.

[Explanation of symbols]

1. Sample double-stranded nucleic acid 2. First single-stranded nucleic acid 3.
Second single-stranded nucleic acid 4. SNPs existing area 5. Recombination-inhibiting nucleic acid 6. Substrate 7. Detection probe
8. Mediating probe 9. Arbitrary base sequence

Claims (11)

[Claims] 1. A method for hybridizing a double-stranded nucleic acid containing a target sequence and a capture probe nucleic acid, wherein the capture probe nucleic acid contains a sequence complementary to the target sequence, wherein the following steps: 1) denaturing the double-stranded nucleic acid into a single strand, and (2) complementing a part of the single strand obtained in (1) and the target sequence and its complementary sequence; Hybridizing the single strand with the capture probe nucleic acid in the presence of a recombination-inhibiting nucleic acid containing a different sequence. How to soybean. 2. At least two kinds of the recombination-inhibiting nucleic acids are used, and the first recombination-inhibiting nucleic acid is complementary to a part of the first single strand contained in the double-stranded nucleic acid, and has a target. A second recombination-inhibiting nucleic acid comprising a sequence different from the sequence and its complementary sequence, is complementary to a part of the second single strand contained in the double-stranded nucleic acid, and the target sequence and its complementary sequence. A method for hybridizing a double-stranded nucleic acid containing a target sequence according to claim 1 with a probe nucleic acid for capture, which comprises a sequence different from 3. The capture probe nucleic acid is at least 2
The first capture probe nucleic acid contains a sequence complementary to a part of the first single strand contained in the double-stranded nucleic acid, and the second capture probe nucleic acid contains the double-stranded nucleic acid. A target sequence according to claim 1 or 2, which comprises a sequence complementary to a part of the second single strand contained in the nucleic acid.
A method of hybridizing a single-stranded nucleic acid and a capture probe nucleic acid. 4. A method for hybridizing a double-stranded nucleic acid containing a target sequence and a capture probe nucleic acid, which comprises the steps of: (1) denaturing the double-stranded nucleic acid into a single strand. And (2) an intermediary probe nucleic acid containing the single strand obtained in (1) above, a sequence complementary to the target sequence and an arbitrary sequence, and a probe nucleic acid for capture containing the sequence complementary to the arbitrary sequence In the presence of a recombination-inhibiting nucleic acid having a sequence complementary to a part of the single strand but different from the sequence contained in the intermediary probe nucleic acid and the capture probe nucleic acid and its complementary sequence. The method for hybridizing a double-stranded nucleic acid containing a target sequence comprising: 5. At least two kinds of the recombination-inhibiting nucleic acids are used, and the first recombination-inhibiting nucleic acid is complementary to a part of the first single strand contained in the double-stranded nucleic acid, The second recombination-inhibiting nucleic acid comprises a sequence different from the sequence contained in the intermediary probe nucleic acid, and is complementary to a part of the second single strand contained in the double-stranded nucleic acid, The method for hybridizing a double-stranded nucleic acid containing a target sequence according to claim 4, which contains a sequence different from the contained sequence, and a probe nucleic acid for capture. 6. At least two kinds of the intermediary probe nucleic acids are used, the first intermediary probe nucleic acid comprises a sequence complementary to the target sequence and a first arbitrary sequence, and the second intermediary probe nucleic acid is the above-mentioned 6. A double-stranded nucleic acid containing a target sequence according to any one of claims 4 and 5, which contains a sequence homologous to the target sequence and a second arbitrary sequence, and a probe nucleic acid for capture. How to soybean. 7. The capture probe nucleic acid is at least 2
And the first capture probe nucleic acid is the first
7. The double-stranded chain containing a target sequence according to claim 6, characterized in that the second capture probe nucleic acid contains a sequence complementary to the arbitrary sequence described in (1) above, and the second capture probe nucleic acid contains a sequence complementary to the second arbitrary sequence. A method of hybridizing a nucleic acid and a capture probe nucleic acid. 8. The base sequence detection chip is provided with the capture probe nucleic acid, and the capture probe nucleic acid is included in the base sequence detection chip.
A method for hybridizing a double-stranded nucleic acid containing the target sequence according to any one of 1 to 4 and a probe nucleic acid for capture. 9. The first capture probe nucleic acid and the second capture probe nucleic acid are independently immobilized for each type in different regions of the base sequence detection chip. Or a method of hybridizing a double-stranded nucleic acid containing the target sequence according to any one of 8 and a capture probe nucleic acid. 10. A method for analyzing a base sequence in a sample containing a sample double-stranded nucleic acid, comprising the steps of: (1) denaturing the sample double-stranded nucleic acid into a single strand. And (2) in the presence of a recombination-inhibiting nucleic acid that is complementary to a part of the single strand obtained in (1) above and contains a sequence different from the target sequence and its complementary sequence, (1) above A sample is obtained by hybridizing the single strand obtained in 1 above with a capture probe nucleic acid containing a sequence complementary to the target sequence, and (3) detecting the hybridization generated in (2) above. A method for analyzing a base sequence in a sample containing an analyte double-stranded nucleic acid, which comprises detecting the target sequence in the double-stranded nucleic acid. 11. A method for analyzing a base sequence in a sample containing a sample double-stranded nucleic acid, comprising the steps of: (1) denaturing the sample double-stranded nucleic acid into a single strand. And (2) in the presence of a recombination-inhibiting nucleic acid that is complementary to a part of the single strand obtained in (1) above and contains a sequence different from the target sequence and its complementary sequence, (1) above Hybridizing the single strand obtained in (1), a mediating probe nucleic acid containing a sequence complementary to the target sequence and an arbitrary sequence, and a capture probe nucleic acid containing a sequence complementary to the arbitrary sequence, and ( 3) Specimen 2 comprising detecting the target sequence in the specimen double-stranded nucleic acid by detecting the hybridization generated in (2) above
A method for analyzing a base sequence in a sample containing a double-stranded nucleic acid.