JP2005204558A - Probe for detecting nucleic acid and method for detecting nucleic acid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a probe for detecting nucleic acid, excellent in detection accuracy and having two or more probe domains, and to provide a detection method using such a probe. <P>SOLUTION: The probe for detecting nucleic acid comprises n nucleic acid-natured probe domains( wherein, n≥2 ) and (n-1) non-nucleic acid-natured spacer domains, wherein the probe domains are connected with each other via the non-nucleic acid-natured spacer domains. This probe is usable for detecting e.g. human leukocyte antigen(HLA), T-cell receptor gene, erythrocyte-type determining gene and Rh antigen gene. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

Field of Invention

  The present invention relates to a probe for detecting a mutation or polymorphism of a target nucleic acid. Specifically, the present invention relates to a nucleic acid detection probe capable of simultaneously identifying genetic information contained in two or more discontinuous regions existing in the same nucleic acid, and a nucleic acid detection method.

  After the human genome has been deciphered, the entire sequence of the human chromosome has been clarified, and it is expected that research on the information of individual genes will be pursued energetically in the future. Until now, the relationship between many diseases and gene mutations and polymorphisms has been studied, and the relationship between single gene diseases and multifactorial diseases and causative genes has been clarified one after another. Polymorphisms have been reported for many genes, and these are useful markers for individual identification and pathogenesis gene search. It has also been reported that red blood cell ABO polymorphisms and polymorphisms found in the HLA gene are useful for determining suitability for blood transfusion or bone marrow transplantation.

Under such circumstances, a technique for simply and accurately detecting a target gene or a gene mutation or gene polymorphism present in a region containing the gene is required.
Many methods have been reported as methods for detecting gene mutations or polymorphisms. For example, a method for detecting a mutation or polymorphism of a gene based on the sequence information of the entire target fragment can be mentioned. As such a method, specifically, for example, PCR-SSCP method (see, for example, Proc. Natl. Acad. Sci. USA 86, 2766 (1989) (Non-patent Document 1)), PCR-PHFA method (See, for example, Nucl. Acids Res. 22, 1541- (1994) (non-patent document 2)), PCR-DGGE method (for example, Proc. Natl. Acad. Sci. USA 86, 232 (1989) (non-patent document) Reference 3)), PCR-RSCA method, Sequencing method and the like. In these methods, theoretically, even if a mutation or polymorphism exists in any region within the fragment, the difference in the structure of the single strand, the difference in the annealing method, the difference in the denaturation method, the heteroduplex Mutations or polymorphisms can be detected by changing the structure of or the decoded sequence itself and detecting the difference.

  Examples of the method for detecting a gene mutation or polymorphism include a method for detecting a gene mutation or polymorphism in a very limited region. Examples of such methods include PCR-SSP method, Invader method, PCR-RFLP method and the like. In these methods, mutations or polymorphisms existing in a very limited region affect the extension reaction from the primer end, cleavage reaction by Cleavase, and cleavage reaction by restriction enzyme. Polymorphism can be detected.

  Other methods include, for example, PCR-SSO method, Taqman method, Light Cycler method and the like. These methods utilize the difference in affinity between a DNA molecule having a mutation or polymorphism in a target region and a synthetic oligonucleotide having a sequence corresponding to the target region. That is, in these methods, when a mutation or polymorphism is present in the binding region of a synthetic oligonucleotide, the affinity with the synthetic oligonucleotide changes, thereby causing binding of the oligonucleotide probe, cleavage by polymerase, or fluorescence energy. A change is made in the presence or absence of metastasis and detected. Thereby, mutation or polymorphism can be detected.

HLA (Human Leukocyte Antigen, human leukocyte antigen) plays an important role in recognition of self and non-self, and there are many polymorphisms as its gene. Major genes include HLA-A, -B, -C, -DR, -DP, and -DQ. When bone marrow transplantation or the like is performed, the type of these genes is examined in advance between the bone marrow supplier and the recipient, and by adapting them, the therapeutic effect by bone marrow transplantation or the like can be greatly improved.
So far, several tens to hundreds of polymorphic types have been reported for each gene. However, it is not easy to determine the allele of these genes. In particular, it is very difficult to find a person with a suitable genotype in the case of an unrelated person. For this reason, in general, a suitable person is searched among many donors registered in a public bone marrow bank.

As a method for determining these gene polymorphisms, for example, see PCR-SSP method, PCR-SSO method, Direct sequencing method and the like (for example, today's transplantation, vol.7 SUPPL (1994) (Non-patent Document 4). )
Of these, the PCR-SSO method is suitable for multi-sample processing and is most widely used. In this method, oligonucleotide probes corresponding to a large number of regions exhibiting polymorphism in each HLA gene are set, and the genotype is determined from the reaction mode. However, since human genes consist of two sets, one derived from the father and one derived from the mother, and the HLA gene is very polymorphic, the genotype cannot often be determined by the PCR-SSO method using conventional probes. There is. For example, when the sequences of bases at two adjacent positions # 1 and # 2 of one allele are A and T, respectively, and the sequences of bases at the corresponding positions of the other allele are T and G, the usual method Then, A and T are detected from position # 1, and T and G bases are detected from position # 2. As a result, the same base is detected when one allele has A and G at each position and the other allele has T and T. That is, the physically connected state and the state that is not so cannot be distinguished by a normal PCR-SSO method. Since it is common for a continuous protein to be transcribed and translated from a continuous gene, it is important to know the sequence of the gene for each allyl unit. As described above, when the sequence cannot be specified for each allyl unit, an accurate polymorphism cannot be determined.

  Therefore, Saito et al. Have proposed a probe for detecting a nucleic acid having two specific sequence regions separated from each other by a nucleobase in one probe molecule (International Publication WO 03/027309 ( (See Patent Document 1)). The two specific sequence regions present in the probe each form a complementary strand with the target nucleic acid sequence on the sample nucleic acid under hybridization conditions. If a target region complementary to two specific regions on the probe is present in the analyte nucleic acid, which is a separate strand, the two specific regions are physically located on the two strands. The complementary strands that are formed will not be double strands of sufficient strength. However, when target regions complementary to two specific regions are present in the same strand, the formed complementary strand becomes a double strand having sufficient strength. Therefore, by using the probe, a target region complementary to two specific sequence regions exists physically separated on two strands, and on a single strand, It can be distinguished from the case of being physically close based on the strength of the double strands formed.

However, in the detection probe, since the spacer that separates the two specific sequence regions is a normal nucleic acid, even if the sequence is carefully selected, there is some difference between the portion other than the target nucleic acid sequence in the sample nucleic acid. May show interactions and as a result, unexpected duplexes may be formed. In addition, the specific sequence region in the detection probe and the spacer are hydrogen-bonded, and the probe itself forms a higher-order structure within the molecule or between the molecules, so that sufficient hydrogen bonding with the sample nucleic acid is not possible. In some cases. In such a case, even if the nucleic acid is not complementary to the detection probe, a non-specific double strand is formed between them, or a complementary strand is originally formed having a complementary sequence. The sequence may not be able to form a sufficiently strong duplex. As a result, nucleic acid detection accuracy may be reduced.
Therefore, there has been a demand for a method for determining a gene polymorphism that is simple and further improves detection accuracy.

The literature cited in the above description is listed below.
International Publication No. WO 03/027309 Proc. Natl. Acad. Sci. USA 86, 2766 (1989) Nucl. Acids Res. 22, 1541- (1994) Proc. Natl. Acad. Sci. USA 86, 232 (1989) Today's transplant, vol.7 SUPPL (1994)

Summary of the Invention

  The present inventors have recently formed non-specific double strands by using non-nucleic acid structures such as alkyl chains as spacers between the probe regions in a detection probe having two or more probe regions. For this reason, it has been found that the problem of reduction in detection accuracy that can occur when the spacer has a nucleic acid structure can be remarkably suppressed. The present invention is based on such knowledge.

  Therefore, an object of the present invention is to provide a nucleic acid detection probe having two or more probe regions with excellent detection accuracy, and a detection method using such a probe.

  The nucleic acid detection probe according to the present invention comprises n (where n is 2 or more) nucleic acid probe regions and (n-1) non-nucleic acid spacer regions. A probe, wherein the probe region and the probe region are connected via a non-nucleic acid spacer region.

A method for detecting whether a target nucleic acid sequence is present in a sample nucleic acid according to the present invention comprises:
(A) a step of bringing a sample nucleic acid possibly containing a target nucleic acid sequence into contact with a nucleic acid detection probe according to the present invention under hybridization conditions;
Here, each probe region in the nucleic acid detection probe is complementary to n target regions present in the target nucleic acid sequence, and
The nucleic acid detection probe is configured to form a specific hybrid with the target nucleic acid when both of the target regions are present in the same strand, and (b) the nucleic acid detection probe and Determining the presence of the target nucleic acid sequence in the sample nucleic acid based on the presence or absence of a specific hybrid formed with the target nucleic acid.

  The nucleic acid detection probe set according to the present invention comprises at least two types of nucleic acid detection probes according to the present invention.

A kit for detecting the presence of a target nucleic acid sequence in a sample nucleic acid according to the present invention comprises:
(I) a probe for detecting a nucleic acid according to the present invention, and (ii) a primer used for amplifying a sample nucleic acid.

  According to the nucleic acid detection probe of the present invention, when detecting a target nucleic acid having two or more target regions in the same strand, a non-specific reaction between the probe and the target nucleic acid can be effectively suppressed. Therefore, the detection accuracy of the target nucleic acid can be further improved as compared with the conventional case. According to the present invention, genetic information contained in two or more discontinuous regions existing in the same strand of a sample nucleic acid can be simultaneously identified. Therefore, the probe according to the present invention can be used for gene diagnostic techniques, and can also be applied to medical products or food testing.

DETAILED DESCRIPTION OF THE INVENTION

As described above, the nucleic acid detection probe according to the present invention comprises n nucleic acid probe regions and (n-1) non-nucleic acid spacer regions, The probe region and the probe region are connected via a non-nucleic acid spacer region. That is, the nucleic acid detection probe according to the present invention comprises n probe regions and a non-nucleic acid spacer region separating each probe region in a single molecule.

Here, n is 2 or more, preferably 2 to 10, more preferably 2 to 5, and particularly preferably 2.
Therefore, for example, when n is 2, the nucleic acid detection probe according to the present invention includes two nucleic acid probe regions and one non-nucleic acid spacer region, and the first probe region and the first probe region Two probe regions are connected via a non-nucleic acid spacer region (FIG. 1).

  The nucleic acid detection probe according to the present invention is used for detection of a target nucleic acid that may be present in a specimen or identification of a nucleic acid to be examined. The target nucleic acid to be detected by the probe according to the present invention may be any target nucleic acid as long as it has a plurality of target regions in the same strand, preferably a human-derived gene, more preferably Is a human leukocyte antigen (HLA) gene, T cell receptor gene, erythroid typing gene, or Rh antigen gene.

  Here, the “probe region” refers to a region that is complementary to the sequence of the target region in the target nucleic acid to be detected in the nucleic acid detection probe according to the present invention. In the present invention, by utilizing the complementarity between the probe region and the target region, the target nucleic acid is detected by utilizing the fact that both can bind specifically. Therefore, if there are n target regions in the target nucleic acid sequence and they need to be detected simultaneously, a probe region that is complementary to each of those target regions is required. Those skilled in the art can appropriately determine the sequence of the probe region according to the nucleic acid to be detected. A plurality of probe regions may be the same or different from each other.

In the present invention, the structure constituting the “probe region” is a nucleic acid structure. Here, the term “nucleic acid” means that the sequence is constituted by oligonucleotides such as DNA or RNA, PNA (Peptide Nucleic Acid), LNA (Locked Nucleic Acid), and the like.
According to a preferred embodiment of the present invention, the probe region is composed of an oligonucleotide, PNA (Peptide Nucleic Acid), or LNA (Locked Nucleic Acid). All of the probe regions included in the probe according to the present invention may be composed of the same one, or may be composed of different ones.
Those skilled in the art can appropriately synthesize the probe region sequence by applying a conventional method based on the target sequence information.

  The length of the probe region is not particularly limited, but is, for example, 5 to 100 bases, preferably 8 to 30 bases.

  In the present invention, the “spacer region” means a region interposed between two probe regions present in the probe according to the present invention. That is, in the probe according to the present invention, each probe region is separated by the spacer region, and the probe according to the present invention constitutes a non-circular single molecule. Therefore, in the present invention, when n probe regions are present, (n-1) spacer regions are present. At this time, the plurality of spacer regions may be the same as or different from each other.

In the present invention, the “spacer region” is a non-nucleic acid structure. Here, the non-nucleic acid is not a nucleic acid or a modified nucleic acid, and may be any as long as it does not form a base pair with another nucleobase. That is, the spacer region does not form a base pair with the base in the intervening region between the target region and the target region present in the target nucleic acid. The “spacer region” is constituted by a spacer. At this time, one spacer region may be composed of two or more spacers.
The spacer preferably does not significantly change the water solubility of the detection probe according to the present invention, and more preferably can be a phosphoramidite derivative that can be used in an ordinary nucleic acid synthesizer. Examples of the nucleic acid that can be introduced at the stage of nucleic acid synthesis include an alkyl chain, an ether chain, a peptide chain, or a sugar chain. These chains may be modified as necessary.

  Therefore, according to a preferred embodiment of the present invention, the spacer constituting the spacer region is one or more selected from the group consisting of an alkyl chain, an ether chain, a peptide chain, and a sugar chain.

According to a more preferred embodiment of the present invention, the spacer is — (O—CH ₂ —CH ₂ ) p [where p is 1 or more, preferably 3 to 10], — (CH 2) q [where q Is 1 or more, preferably 3 to 30], or has a structure of deoxyribose chain (preferably 3 to 10 chain). In the present invention, specific examples of the spacer include those having the following structure:

［上記式中、
ＤＭＴは、ジメトキシトリチル基を示し、
ｉ−Ｐｒは、イソプロピル基を示し、
ＣＮＥｔは、シアノエチル基を示す］。
なお、上記スペーサーの具体例において、ＤＭＴは５’末端の水酸基の保護基であり、Ｐ(Ｏ-ＣＮＥｔ)Ｎ(i-Ｐｒ)_２は、リン酸基の保護基である。

[In the above formula,
DMT represents a dimethoxytrityl group,
i-Pr represents an isopropyl group;
CNEt represents a cyanoethyl group].
In the specific example of the spacer, DMT is a protecting group for a hydroxyl group at the 5 ′ end, and P (O—CNEt) N (i-Pr) ₂ is a protecting group for a phosphate group.

  The length of the spacer region can be appropriately set according to the distance between the target regions in the target nucleic acid sequence. The length of the spacer region preferably gives a distance at which 3 or more carbon atoms are bonded or a degree of freedom equivalent thereto, and more preferably a distance at which 6 to 30 carbon atoms are bonded or It gives the same degree of freedom.

  In the present invention, the “target nucleic acid” refers to a nucleic acid to be used for detection, and this target nucleic acid has a target region that can be used for detection in the sequence in the same strand. It has a plurality. In the target nucleic acid sequence, which sequence is targeted can be appropriately determined by those skilled in the art based on available genetic information on gene mutation or polymorphism.

  According to a preferred aspect of the present invention, in the probe for detecting a nucleic acid, each probe region contained therein is complementary to each of n target regions present in the target nucleic acid sequence, and each of the target regions is When present in the same strand, they are configured to form a specific hybrid with the target nucleic acid. Therefore, when a plurality of target regions are not on the same strand but are present on different strands, the probe of the present invention can be used between the target nucleic acid and the target nucleic acid even if all of the probe regions in the probe form complementary strands. Then, a stable hybrid is not formed. As described above, since the probe of the present invention is configured to form a specific hybrid, the length of the probe region in the probe, the length of the spacer region, the type of spacer, etc. are appropriately selected. can do.

  In the present invention, that the probe region and the target region are “complementary” means that all the corresponding bases in the probe region and the target region are in a state capable of forming a base pair. In the range where no problem occurs in detection, one or several mismatches may be included therein. The number of mismatches that may exist is limited depending on the required detection accuracy and the length of the probe region. In the detection, by appropriately changing the hybridization conditions between the probe and the target nucleic acid, it is possible to eliminate the case where there is a mismatch or to adjust the allowable number of mismatches.

  The probe according to the present invention may further have a region having a labeling substance that can be used in the detection operation, if necessary, and may have any sequence or range within a range that does not affect the detection ability as a probe. It may further contain a modifying substance.

Nucleic acid detection method As described above, the method for detecting whether or not a target nucleic acid sequence is present in a sample nucleic acid according to the present invention,
(A) a step of bringing a sample nucleic acid possibly containing a target nucleic acid sequence into contact with a nucleic acid detection probe according to the present invention under hybridization conditions; and (b) a nucleic acid detection probe; The method includes the step of determining the presence of the target nucleic acid sequence in the sample nucleic acid based on the presence or absence of a specific hybrid formed with the target nucleic acid.

  Here, “hybridization conditions” may vary depending on the probe and target nucleic acid used, but general conditions can be easily set by those skilled in the art. A specific example of the hybridization condition is (1-5) × SSC (0.75 M NaCl, 75 mM sodium citrate), and the temperature at this time is preferably 25 to 80 ° C.

As a method for detecting a nucleic acid according to the present invention, a conventional method used in a normal gene detection method can be applied. Examples of such a method include the following methods (1) to (5):
(1) The detection probe according to the present invention is immobilized on a solid phase in advance, and a sample nucleic acid having a detectable label or a sample nucleic acid to which a label can be easily introduced, and the label has been introduced in advance. A method in which a sample nucleic acid and an immobilized probe are subjected to a base pairing reaction under hybridization conditions, and then a label remaining on a solid phase is detected;
(2) A sample nucleic acid is immobilized on a solid phase in advance, and a probe according to the present invention into which a detectable label is introduced in advance and the immobilized sample nucleic acid are subjected to base pairing under hybridization conditions. A method of detecting the label remaining on the solid phase after the formation reaction;
(3) By binding the probe according to the present invention and the sample nucleic acid to separate suspendable solid phases and bringing them into contact with each other under hybridization conditions, specific double strand formation A method of causing an aggregation reaction and confirming the aggregation;
(4) Fluorescence generated when the probe according to the present invention and a sample nucleic acid are subjected to a base pairing reaction in a liquid phase under a hybridization condition and a specific double strand is formed. A method by detecting energy transfer; and (5) a method of arbitrarily combining (1) to (4) above.

  In the present invention, the method (1) is preferred. In the method (1), the target nucleic acid is easily captured by the immobilized probe according to the present invention, and the other nucleic acids in the sample are not captured. Therefore, the label level on the solid phase is measured. Thus, the target nucleic acid can be easily detected.

  Examples of the solid phase carrier used when the detection probe or the sample nucleic acid according to the present invention is immobilized on a solid phase can be non-specifically adsorbed by the detection probe or the sample nucleic acid according to the present invention, or As long as a functional group can be introduced and a covalent bond can be formed between the functional group and the nucleic acid, any material or any shape can be used. Specific examples include so-called polymer microplates, tubes, and beads. In the present invention, it is preferable to use a microplate or bead shape because of its ease of mechanization.

  Examples of the immobilization method include a chemical bonding method [Nucleic Acids Res., 15, 5373-5390 (1987)]. As a specific example, there may be mentioned a method in which a carrier into which a carboxyl group is introduced and a nucleic acid into which an aminohexyl group is introduced at the 5'-end are bound to each other using a cross-linking agent such as a water-soluble carbodiimide such as EDC.

  Alternatively, the nucleic acid can be directly immobilized on the carrier by non-specific binding such as adsorption. When the carrier is made of polystyrene, the adsorption efficiency can be increased by ultraviolet irradiation or addition of magnesium chloride (Japanese Patent Laid-Open No. 61-219400). Furthermore, a method of immobilizing nucleic acid and protein by chemical bonding or non-specific adsorption by an appropriate method and utilizing non-specific adsorption between the protein and carrier is also effective.

Examples of the method for labeling the probe or analyte nucleic acid according to the present invention include:
(A) a method of directly introducing a label into a nucleic acid to be labeled;
(B) a method of synthesizing a nucleic acid to be labeled or a nucleic acid complementary to the nucleic acid to be labeled using a labeled oligonucleotide primer, and (C) an oligonucleotide primer in the presence of a labeled unit nucleic acid And a method of synthesizing a nucleic acid to be labeled or a nucleic acid complementary to the nucleic acid to be labeled.

  As the method of (A), a method of detecting a biotin derivative by introducing a biotin derivative into a nucleic acid to be labeled with a streptavidin bound with an enzyme [Nucleic Acids Res., 13, 745 (1985)], And the like, and a method of detecting using an enzyme-labeled anti-sulfonated antibody [Proc. Natl. Acad. Sci. USA, 81, 3466-3470 (1984)].

  As the methods (B) and (C), a method of amplifying a specific nucleic acid sequence [BIO / TECHNOLOGY, 8, 291 (1990)] can be used. For example, in the PCR method [Science, 230, 1350-1354 (1985)], by using a labeled primer or by using a labeled mononucleotide triphosphate, a labeled extension product or An amplification product can be obtained. In addition, in the amplification method using Qβ replicase [BIO / TECHNOLOGY, 6, 1197 (1988)], a labeled extension product or amplification product is obtained by using a similarly labeled mononucleotide triphosphate. Can be obtained. Also in nucleic acid amplification methods other than those described above, the extension product or amplification product can be labeled by labeling the mononucleotide triphosphate or oligonucleotide incorporated by the extension reaction or amplification reaction.

  The labeling substance used here may be radioactive or non-radioactive as long as the substance can be detected after the hybridization operation. Non-radioactive labeling substances are preferable from the viewpoints of easy handling, storage, disposal, and the like.

  Non-radioactive labeling substances include, for example, haptens such as biotin, 2,4-dinitrophenyl group, digoxigenin, fluorescein and derivatives thereof (eg fluorescein isothiocyanate (FITC) etc.), rhodamine and derivatives thereof (eg tetramethyl Rhodamine isothiocyanate (TRITC), Texas Red, etc.], fluorescent materials such as 4-fluoro-7-nitrobenzofuran (NBDF) and dansyl, and chemiluminescent materials such as acridine. When the target nucleic acid or the probe according to the present invention is labeled with these, labeling can be performed by any known means (see JP-A-59-93098 and JP-A-59-93099).

  In the present invention, the operation for detecting the presence or absence of a specific hybrid formed between the probe according to the present invention and the target nucleic acid may be appropriately selected and determined according to the type of label present.

  Here, when the label is directly detectable, that is, when the label is, for example, a radioisotope, a fluorescent substance, or a dye, the detection operation is performed with the labeled nucleic acid bound to the solid phase, or the label After the product is bound to the nucleic acid or released from the solution in a state where the labeled product is cleaved from the nucleic acid, the detection operation can be performed by a method corresponding to the label. In addition, when the label is indirectly detectable, that is, when the label is a ligand for a specific binding reaction such as biotin or hapten, a direct signal is used as is generally used for detecting them. The detection operation can be performed using a receptor (for example, avidin or an antibody) bound with an enzyme that catalyzes a reaction that generates a label or a signal.

  According to one preferred embodiment of the present invention, the detection method according to the present invention further comprises a step of amplifying the sample nucleic acid in the sample before step (a). That is, in the detection method according to the present invention, prior to detection, the sample nucleic acid is preferably amplified by a conventional gene amplification method using the sample nucleic acid as a template. Examples of such an amplification method include a method requiring a temperature change such as a PCR method (Polymerease Chain reaction) and a method performed isothermally such as a LAMP method (Loop mediated amplification).

According to another aspect of the present invention, as described above, a kit for detecting the presence of a target nucleic acid sequence in a sample nucleic acid,
There is provided a kit comprising (i) a nucleic acid detection probe according to the present invention, and (ii) a primer used for amplifying a sample nucleic acid.

The kit of the present invention further comprises (iii) a reagent necessary for performing a gene amplification reaction such as PCR using a sample nucleic acid as a template,
(Iv) Reagents necessary for performing a hybridization reaction between the gene amplification product and the probe according to the present invention, and / or (v) Whether a specific hybrid is formed by the gene amplification product and the probe according to the present invention. It may further contain a reagent or the like necessary for determining whether or not. As these reagents, conventional ones can be used as appropriate. For example, examples of the reagent for determining whether or not a hybrid is formed include intercalators that react specifically with double strands.

  Hereinafter, the present invention will be described by way of examples, but these examples do not limit the present invention.

Example 1: Discrimination between HLA-DRB1 * 15011 and DRB1 * 04011 and DRB1 * 0404 Using the nucleic acid detection probe of the present invention, the HLA-DR gene (HLA-DRB1 * 15011, DRB1 * 04011, and DRB1 * 0404) ) Was identified.
Probes 70-2D and 70-2Dt were prepared as nucleic acid detection probes. These are probes that recognize a region corresponding to amino acid numbers 53 to 57 of the HLA-DRB1 * 15011 gene (first probe region) and a region corresponding to numbers 68 to 72 (second probe region). It had a sequence. Here, the probe 70-2D is “Spacer 18” in which the spacer region between the two probe regions is a non-nucleic acid structure, whereas the probe 70-2Dt (SEQ ID NO: 6) corresponds to the spacer region. The probes were different in that the intervening region was thymidylate (TTTT) with four nucleic acid structures.
The probe 70-2D corresponds to a probe according to the present invention, and the probe 70-2Dt corresponds to a comparative example.

  As the HLA-DR gene used, the genes HLA-DRB1 * 15011, DRB1 * 04011, and DRB1 * 0404 were used (SEQ ID NOs: 1, 2, and 3 respectively). Among these, DRB1 * 04011 has a mismatch of 2 bases between the target region and the first probe region of the nucleic acid detection probe, and 2 bases between the second probe region of the nucleic acid detection probe. It was a thing. Similarly, DRB1 * 0404 has a mismatch of 2 bases only between the target region and the first probe region of the nucleic acid detection probe, and DRB1 * 15011 includes the target region and the nucleic acid detection probe. There was no mismatch between the first and second probe regions. The sequence relationship between the probe used and the HLA-DR gene is shown in FIG.

The two types of probes were immobilized on a solid phase according to a conventional method. Specifically, carboxylated polystyrene beads were used as a solid phase carrier, and 5′-aminated oligonucleotides were coupled thereto using EDC to immobilize the probes.
On the other hand, for the three types of genes DRB1 * 04011, DRB1 * 0404, and DRB1 * 15011, a PCR reaction (in this case, the reaction is 94 ° C./30 seconds) using a plasmid incorporating each gene as a template and a biotin identification primer. , 55 cycles at 55 ° C./30 seconds and 72 ° C./30 seconds were performed for 30 cycles) to obtain an amplification product.
The biotin-labeled amplification product obtained for each gene was hybridized with the immobilized probe under hybridization conditions. Streptavidin-phycoerythrin (fluorescent substance) was allowed to act on this, and after washing, the fluorescence intensity remaining on the beads was measured, and the relative values are shown in the results table.

  The results were as shown in Table 1.

The probe 70-2Dt (comparative example) gave strong fluorescence intensity when there was no mismatch in both probe regions, but also strong fluorescence when there was a mismatch in only one region (in the case of DRB1 * 0404) Strength was given. It was expected that the fluorescence intensity showed such a tendency due to non-specific reaction between the nucleic acid spacer and the target nucleic acid region.
On the other hand, the probe 70-2D (the present invention) showed strong fluorescence intensity only for DRB1 * 15011 in which there is no mismatch in both the first region and the second region. As a result, only DRB1 * 15011 could be easily identified.

Example 2: Discrimination between HLA-DRB1 * 0404 and DRB1 * 15011 and DRB1 * 15021 Using the nucleic acid detection probe of the present invention, the HLA-DR gene (DRB1 * 0404, DRB1 * 15011, and DRB1 * 15021) Identification was performed.
Probes 69-2D and 69-2Dt were prepared as nucleic acid detection probes. These are a region corresponding to amino acid numbers 56 to 60 of the HLA-DRB1 * 0404 gene and HLA-DRB1 * 15011 gene (first probe region), and a region corresponding to numbers 68 to 71 (second It had a probe sequence that recognizes the probe region). Here, the probe 69-2D is “Spacer 18” in which the spacer region between the two probe regions is a non-nucleic acid structure, whereas the probe 69-2Dt (SEQ ID NO: 16) corresponds to the spacer region. The probes were different in that the intervening region was thymidylate (TTTT) with four nucleic acid structures.
The probe 69-2D corresponds to a probe according to the present invention, and the probe 69-2Dt corresponds to a comparative example.

  As the HLA-DR gene used, each gene of DRB1 * 0404, DRB1 * 15011, and DRB1 * 15021 (SEQ ID NO: 7) was used. Among these, DRB1 * 15021 had a mismatch of 2 bases only between the target region and the second probe region of the nucleic acid detection probe. On the other hand, DRB1 * 0404 and DRB1 * 15011 completely matched between the target region and the first and second probe regions of the nucleic acid detection probe. The sequence relationship between the probe used and the HLA-DR gene is shown in FIG.

The two types of probes were immobilized on a solid phase according to a conventional method. Specifically, the probe was immobilized in the same manner as in Example 1.
For the three genes DRB1 * 0404, DRB1 * 15011, and DRB1 * 15021, PCR was performed in the same manner as in Example 1, and PCR amplification products were obtained for each of the genes. In the same manner as in Example 1, each gene was fluorescently labeled.
The labeled amplification product obtained for each gene was hybridized in the same manner as in Example 1 under hybridization conditions with the immobilized probe.
The fluorescence intensity of the amplified product captured by the immobilized probe was measured in the same manner as in Example 1.

  The results were as shown in Table 2.

Probe 69-2Dt (comparative example) gave strong fluorescence intensity in the case of DRB1 * 15011 gene with no mismatch in both probe regions. However, although it is similar to the DRB1 * 15011 gene, the fluorescence intensity was low in the case of the DRB1 * 0404 gene with no mismatch in both probe regions. On the other hand, in the case of DRB1 * 15021 gene having a mismatch in the second region, strong fluorescence intensity was shown. Therefore, when the probe 69-2Dt was used, the difference in the base sequence of the targeted region in this example could not be identified.
On the other hand, the probe 69-2D (invention) showed strong fluorescence intensity for DRB1 * 0404 and DRB1 * 15011 genes in which there was no mismatch in both the first region and the second region. .

Example 3: Discrimination between HLA-DRB1 * 15021 and DRB1 * 04011, DRB1 * 0404, and DRB1 * 15011 Using the nucleic acid detection probe of the present invention, the HLA-DR gene (DRB1 * 15021, DRB1 * 04011, DRB1 * 0404 and DRB1 * 15011) were identified.
Probes 70-1D and 70-1Dt were prepared as nucleic acid detection probes. These are probes that recognize a region corresponding to amino acid numbers 56 to 60 (first probe region) and a region corresponding to numbers 68 to 71 (second probe region) of the HLA-DRB1 * 15021 gene. It had a sequence.
Here, the probe 70-1D is “Spacer 18” in which the spacer region between the two probe regions is a non-nucleic acid structure, whereas the probe 70-1Dt (SEQ ID NO: 25) corresponds to the spacer region. The probes were different in that the intervening region was thymidylate (TTTT) with four nucleic acid structures.
The probe 70-1D corresponds to a probe according to the present invention, and the probe 70-1Dt corresponds to a comparative example.

  As the HLA-DR gene used, DRB1 * 04011, DRB1 * 0404, DRB1 * 15011, and DRB1 * 15021 genes were used. Among these, DRB1 * 04011 had a mismatch of 2 bases only between the target region and the first probe region of the nucleic acid detection probe. DRB1 * 0404 has a mismatch of 2 bases between the target region and the first probe region of the nucleic acid detection probe and 2 bases between the second probe region of the nucleic acid detection probe. It was. DRB1 * 15011 had a mismatch of 2 bases only between the target region and the second probe region of the nucleic acid detection probe. DRB1 * 15021 had no mismatch between the target region and the first and second probe regions of the nucleic acid detection probe. The sequence relationship between the probe used and the HLA-DR gene is shown in FIG.

  Preparation of amplification product by PCR, gene labeling, hybridization, and measurement of fluorescence intensity were carried out in the same manner as in Example 1.

  The results were as shown in Table 3.

The probe 70-1Dt (comparative example) is more effective against the DRB1 * 15011 gene having a mismatch in the second region than the fluorescence intensity for the DRB1 * 15021 gene in which there is no mismatch in both the first region and the second region. The fluorescence intensity was higher. Therefore, in the case of the probe 70-1Dt, the DRB1 * 15021 gene could not be accurately detected. This is because the spacer region in the nucleic acid detection probe and the target nucleic acid have some kind of non-specific interaction, or the nucleic acid detection probe itself forms some higher order structure. Estimated.
In contrast, probe 70-1D (invention) showed the strongest fluorescence intensity when applied to the DRB1 * 15021 gene.

It is the figure which showed typically the probe for nucleic acid detection (in the case of n = 2) by this invention. It is a figure which shows the relationship between the HLA-DR gene used in Example 1, and the probe for nucleic acid detection. It is a figure which shows the relationship between the HLA-DR gene used in Example 2, and the probe for nucleic acid detection. It is a figure which shows the relationship between the HLA-DR gene used in Example 3, and the probe for nucleic acid detection.

Claims (12)

a nucleic acid detection probe comprising n (where n is 2 or more) nucleic acid probe regions and (n-1) non-nucleic acid spacer regions,
A probe for nucleic acid detection, wherein the probe region and the probe region are connected via a non-nucleic acid spacer region.   The probe for nucleic acid detection according to claim 1, wherein the probe region is composed of oligonucleotide, PNA (Peptide Nucleic Acid), or LNA (Locked Nucleic Acid).   The nucleic acid detection probe according to claim 1 or 2, wherein the spacer constituting the spacer region is at least one selected from the group consisting of an alkyl chain, an ether chain, a peptide chain, and a sugar chain.   The human leukocyte antigen (HLA) gene, T cell receptor gene, erythrocyte typing gene, and Rh antigen gene are used for detection of a gene selected from the group according to any one of claims 1 to 3. Nucleic acid detection probe. Each probe region is complementary to n target regions present in the target nucleic acid sequence, and
The nucleic acid detection according to any one of claims 1 to 4, which is configured to form a specific hybrid with a target nucleic acid when all of the target regions are present in the same strand. Probe.   The probe for nucleic acid detection according to any one of claims 1 to 5, wherein the number (n) of probe regions is 2. A method for detecting whether a target nucleic acid sequence is present in a sample nucleic acid,
(A) a step of contacting a sample nucleic acid possibly containing a target nucleic acid sequence with the nucleic acid detection probe according to any one of claims 1 to 6 under hybridization conditions;
Here, each probe region in the nucleic acid detection probe is complementary to n target regions present in the target nucleic acid sequence, and
The nucleic acid detection probe is configured to form a specific hybrid with the target nucleic acid when both of the target regions are present in the same strand, and (b) the nucleic acid detection probe and Determining the presence of the target nucleic acid sequence in the sample nucleic acid by the presence or absence of a specific hybrid formed with the target nucleic acid.   The method according to claim 7, wherein the target nucleic acid is a gene selected from the group consisting of a human leukocyte antigen (HLA) gene, a T cell receptor gene, an erythrocyte typing gene, and an Rh antigen gene.   The method according to claim 7 or 8, further comprising a step of amplifying the sample nucleic acid in the sample before step (a).   A probe set for nucleic acid detection, comprising at least two types of probes for nucleic acid detection according to any one of claims 1 to 6. A kit for detecting the presence of a target nucleic acid sequence in a sample nucleic acid,
(I) A kit comprising the nucleic acid detection probe according to any one of claims 1 to 6, and (ii) a primer used for amplifying a sample nucleic acid.   The kit according to claim 11, which is used for detection of a gene selected from the group consisting of a human leukocyte antigen (HLA) gene, a T cell receptor gene, an erythrocyte typing gene, and an Rh antigen gene.

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