CROSS REFERENCE TO RELATED APPLICATIONS

This application is a national stage filing under section 371 of International Application No. PCT/JP2013/069694, filed on Jul. 19, 2013, and published on Jan. 23, 2014 as WO 2014/014106, which claims priority to Japanese Application No. 2012-161834, filed on Jul. 20, 2012, and to Japanese Application No. 2012-270614, filed on Dec. 11, 2012. The entire contents of each of said applications are hereby incorporated herein by reference.

SEQUENCE LISTING

This application contains a Sequence Listing, created on Dec. 1, 2014; the file, in ASCII format, is designated H0178302.txt and is 8.34KB in size. The file is hereby incorporated by reference in its entirety into the present application.

TECHNICAL FIELD

The present invention relates to a method for photocoupling with a probe comprising photo-responsive nucleotides.

BACKGROUND ART

In recent years, in order to establish personalized medicine, which is a of made-to-order treatment for individual patients, research has been proposed to reveal the relationship between specific gene polymorphisms of patients and drug sensitivity or drug response.

In particular, with advances in genome science, pharmacokinetics and polymorphisms of enzymes, proteins, and the like involved in drug response are being rapidly elucidated. In human genome analysis, single nucleotide polymorphisms (SNPs) are attracting attention as the most frequent polymorphism markers. The SNPs are already known to be useful in elucidating the relationship between various diseases and drug response. In addition, it is also known that a haplotype analysis using a plurality of SNPs is useful in analyzing disease susceptibility.

Especially in the medical field, a use in the diagnosis of disease prevalence using SNPs detection results, a use in the selection of an effective administration drug, a use in the prediction of side effects, and the like, are considered, and it is expected to lead to the improvement of QOL of patients as well as an increase in therapeutic effect. Many techniques have been reported for determining the bases of such SNPs sites, so-called typing. As a technique for typing single nucleotide polymorphism of DNA, a TaqMan PCR method, an Invader method, and the like, are known as techniques having high sequence selectivity enough to identify a single base substitution.

However, in the detection of acquired mutations as malignancies, since the wild-type nucleic acid molecule derived from normal cells, which are major components of a sample to be analyzed, is the background, it is often impossible to detect mutations such as a single base substitution with high sensitivity in the analysis methods as described above.

Therefore, as a particularly effective analysis technique as an alternative to the above methods, a use of photo-responsive nucleotides is disclosed. A photo-responsive nucleotide and a target nucleotide which are hybridized with each other can form photocoupling by carrying out photo-irradiation at a specific wavelength. This photocoupling occurs through the formation of an intermolecular covalent bond between the photo-responsive nucleotide molecule and the target nucleotide molecule, caused by a photoreaction of an artificial base moiety. The molecules photocoupled with each other in this way do not merely assemble with only a thermal stability, and thus, even if they are placed under conditions where a complementary duplex dissociates, the molecules do not dissociate and remain bound (Patent literature 1).

An analysis technique of detecting mutated genes using photo-responsive nucleotides is disclosed, based on the properties of the photo-responsive nucleotides, i.e., the findings that the photocoupling reaction proceeds very rapidly in about 1 second, and that the photocoupling does not occur if the hybridization is not complete (Patent literature 2).

Patent literatures 1 and 2 disclose the identification of SNPs using the photo-responsive nucleotides. However, they only disclose examples in which the use of the photo-responsive nucleotides is useful in selective amplification of mutated genes, based on the findings that photocoupling occurs when a probe containing the photo-responsive nucleotide is hybridized with a target site having a sequence complementary to the probe.

Therefore, in view of such conventional technical common knowledge, it has been considered that the photo-responsive nucleotide binds only with the target site having a complementary sequence, or a target nucleotide sequence in the vicinity of the target site, because it is necessary for the photocoupling that the probe containing the photo-responsive nucleotide should be hybridized with the target site having its complementary sequence, and the photocoupling is formed by the binding of the photo-responsive nucleotide with the target site, or the target nucleotide sequence in the vicinity of the target site.

CITATION LIST

Patent Literature

[Patent literature 1] JP 2009-254279 A

[Patent literature 2] WO 2012/033190

SUMMARY OF INVENTION

Technical Problem

The present inventors found that, in the reaction where a probe containing a photo-responsive nucleotide (i.e., a photocoupling probe) was photocoupled with a target nucleotide, the photocoupling proceeded only up to a certain percentage regardless of the amount of the target nucleotide, that is to say, the photocoupling stagnated. The present inventors conducted intensive studies based on the findings, and as a result, found that, as factors that stagnated the photocoupling, when the probe containing the photo-responsive nucleotide was photo-irradiated for the photocoupling with the target nucleotide, photocoupling in the self-sequence of the probe occurred. That is to say, it was presumed that since the photo-responsive nucleotide in the probe was assembled and photocoupled with a base capable of photocoupling with the photo-responsive nucleotide, a probe that could not hybridize with the target site and/or could not photocouple with the target nucleotide was accumulated depending on the photo-irradiation time (energy), and as a result, the photocoupling against the target nucleotide stagnated.

Additionally, in view of conventional, technical common knowledge, in the case where the amplification of a wild-type gene is selectively suppressed (amplification-suppressed), and a mutated gene is selectively amplified, the maximum advantage of the photo-coupling method using photo-responsive nucleotides, as disclosed in Patent literature 2, is the fact that the target nucleotide which has been once photocoupled by photo is not cleaved in the heat denaturing step of a PCR reaction step, that is to say, that the target nucleotide which has been once photocoupled by photo can be brought into a non-equilibrium system.

However, the present inventors found that even when it is photo-irradiated at a wavelength capable of photocoupling (365 nm), not only the probe was photocoupled with the target nucleotide, but also part of the probe which had been photocoupled with the target nucleotide was released through a photo-cleavage reaction, which does not naturally occur. That is to say, it was considered that the light equilibrium state in which the photocoupling reaction and the photocoupling-cleaving reaction concurrently occurred was a factor that stagnated the photocoupling against the target nucleotide.

An object of the present invention is to solve the problem that the photocoupling with a target nucleotide using a probe containing photo-responsive nucleotides stagnates, and to provide a method of improving photocoupling efficiency, and a kit thereof.

Solution to Problem

The present inventors intensively studied the means for solving the problem, and as a result, found that in order to avoid the stagnation of the photocoupling efficiency, the effective concentration of the probe containing the photo-responsive nucleotide was maintained in a reaction solution by suppressing photocoupling in the self-sequence of the probe, and that hybridization could be promoted and the photocoupling efficiency could be improved by increasing the substantial concentration of the probe containing the photo-responsive nucleotide, locally around the target site.

The present invention relates to:

[1] a photocoupling method, characterized by hybridizing a target site present in a nucleic acid sample with a first probe having a sequence complementary to the target site and containing a photo-responsive nucleotide, in a reaction solution, and carrying out photocoupling by photo-irradiation, wherein self-assembly caused by the photo-responsive nucleotide contained in the first probe is suppressed,
[2] the photocoupling method of [1], characterized in that the self-assembly caused by the photo-responsive nucleotide contained in the first probe is suppressed by co-existing with a second probe being highly complementary to the first probe,
[3] the photocoupling method of [1] or [2], wherein being highly complementary means a state in which the first probe and the second probe are complementary to one another, and a base to be photocoupled with the photo-responsive nucleotide itself in the first probe under predetermined photocoupling conditions hybridizes with the second probe,
[4] the photocoupling method of any one of [1] to [3], wherein a target nucleotide contained in the target site present in the nucleic acid sample is photocoupled with the photo-responsive nucleotide contained in the first probe,
[5] the photocoupling method of any one of [1] to [4], wherein the second probe contains a photo-responsive nucleotide,
[6] the photocoupling method of any one of [1] to [5], wherein the first probe and the second probe contain photo-responsive nucleotides, and a third probe having a sequence complementary to the first probe and/or the second probe is used so that the photo-responsive nucleotide or the photo-responsive nucleotides present in the first probe and/or the second probe cannot be photocoupled itself in a non-complementary region between the first probe and the second probe,
[7] the photocoupling method of [1], characterized by hybridizing a target site present in a nucleic acid sample, a first probe having a sequence complementary to the target site and containing a photo-responsive nucleotide, and a fourth probe having a sequence complementary to the target site and containing a target nucleotide, so that they are placed adjacent in a reaction solution, and carrying out photocoupling by photo-irradiation between the target nucleotide contained in the fourth probe and the photo-responsive nucleotide contained in the first probe, wherein photocoupling of the first probe itself is suppressed by co-existing with a second probe being highly complementary to the first probe,
[8] the photocoupling method of any one of [1] to [7], characterized by using the first probe in which a nucleotide that self-assembles with the photo-responsive nucleotide in the first probe is substituted with a nucleotide not capable of photocoupling with the photo-responsive nucleotide, and characterized in that photocoupling of the first probe itself is suppressed,
[9] the photocoupling method of [8], wherein the nucleotide not capable of photocoupling with the photo-responsive nucleotide is a purine base,
[10] the photocoupling method of [8], wherein the nucleotide not capable of photocoupling with the photo-responsive nucleotide is a synthetic base obtained by artificially converting a pyrimidine ring,
[11] the photocoupling method of any one of [1] to [10], wherein an anionic substance is contained in the reaction solution,
[12] the photocoupling method of any one of [1] to [11], characterized in that at least one photocoupling probe is contained at a concentration of 0.1 μmol/L or more in the reaction solution,
[13] A method for gene analysis, using the photocoupling method of any one of [1] to [12],
[14] the method for gene analysis of [13], which is a method for gene detection or a method for nucleic acid amplification,
[15] a method for mutated nucleic acid detection, characterized in that the method for nucleic acid amplification described in claim14is a method for detecting the presence or absence of the mutated nucleic acid, by selectively amplifying a nucleotide sequence for amplification containing a target site of the mutated nucleic acid,
[16] a photocoupling kit, characterized by comprising a first probe having a sequence complementary to a target site present in a nucleic acid sample, and containing a photo-responsive nucleotide, and a second probe being highly complementary to the first probe,
[17] the photocoupling kit of [16], wherein the first probe is capable of photocoupling with a target nucleotide contained in the target site present in the nucleic acid sample,
[18] the photocoupling kit of [16], further comprising a fourth probe containing a target nucleotide capable of photocoupling with the photo-responsive nucleotide contained in the first probe,
[19] the photocoupling kit of any one of [16] to [18], wherein the second probe contains a photo-responsive nucleotide, and
[20] the photocoupling kit of any one of [16] to [19], wherein the first probe is a probe in which a nucleotide that self-assembles with the photo-responsive nucleotide in the first probe is substituted with a nucleotide not capable of photocoupling with the photo-responsive nucleotide.

Advantageous Effects of Invention

According to the present invention, in a gene analysis method using photo-responsive nucleotides, the photocoupling efficiency between the target site and a probe containing the photo-responsive nucleotide can be improved. That is to say, the present invention is effective in a gene analysis method that requires high sensitivity and high specificity, because photocoupling against the target nucleotide can be specifically and effectively carried out.

