JP2014180278A - Nucleic acid analyzing method and assay kit used thereby - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a simple and inexpensive nucleic acid analyzing method which analyzes a mutations of several bases with high accuracy.SOLUTION: A method comprises the following steps of: preparing a primer set to amplify a template sequence comprising a mutation site which consists of a first or a second base; subjecting a sample to an amplification reaction with a primer set in one reaction vessel to obtain an amplification product; reacting a nucleic acid probe complementary to a target sequence and the amplification product; detecting an amount of caused hybridization and analyzing the nucleic acid comprised in the sample. The first primer comprises a sequence complementary to the mutation site which consists of a first base on 3' side, and comprises a sequence complementary to a part of template nucleic acids and a tag sequence. The second primer comprises a sequence complementary to the mutation site which consists of a second base on 3' side, and comprises a sequence complementary to a part of template nucleic acids and a tag sequence.

Description

  Embodiments described herein relate generally to a nucleic acid analysis method and an assay kit used therein.

  Even in one species population, there are diversity in genome base sequences. This diversity is due to, for example, a mutation of several bases. When a single base is mutated in the genome base sequence, if the mutation is found at a frequency of 1% or more in the population, this is called a single nucleotide polymorphism (SNP). In contrast, if the mutation is less than 1% within the population, i.e. if the allele frequency is less than 1%, this is called a mutation.

  The genetic background of the target organism can be examined from several base mutations such as SNP. For genetic diseases for which the causative gene is known, future risk factors for the occurrence of the genetic disease can be diagnosed. It is also expected to identify disease-related genes by linkage analysis using SNPs. For example, it is known that genetic factors such as strength against alcohol mainly depend on the SNP of the aldehyde dehydrogenase gene (ALDH2).

  In recent years, many SNPs of genes expected to be applied clinically have been found. For example, it is greatly influenced by the strength of the enzyme cytochrome P450 (CYP) family involved in drug metabolism, N-acetyltransferase 2 (NAT-2) involved in the metabolism of the antituberculosis drug isoniazid, and the oral anticoagulant warfarin. Affected CYP2C9 and VKORC1. These genes include SNPs that reduce enzyme activity, and if the blood concentration of the drug is kept high for a long time, the effect of the drug is more strongly expressed than necessary in patients who have been administered the drug. Toxic intermediate metabolites may accumulate. On the other hand, if there is a SNP that inhibits the activation of the drug, a treatment such as an increase in dosage is required. By examining such SNPs and determining the type of gene prior to drug administration, an appropriate drug dosage can be determined. Thereby, side effects can be reduced or avoided. In addition, a therapeutic effect can be obtained efficiently.

  Medical treatment performed according to the individual constitution of the patient by using genetic information is called “tailor-made medicine”, “order-made medicine”, or “personalized medicine”. Such medical care improves inappropriate medication and reduces side effects. This can be expected to reduce medical costs.

  In bacteria and viruses, there are cases where specific drugs do not work due to the presence of single nucleotide polymorphisms or single nucleotide mutations. In such a case, it is possible to select an effective treatment method by discriminating the genotypes of bacteria and viruses.

  In the industrial field, SNP analysis is effective for species discrimination of livestock, pets, and even rice. By using genetic analysis to determine the variety, it helps to protect the intellectual property rights of growers and to prevent the production area from being camouflaged.

Japanese Patent No. 3937136 Japanese Patent No. 3926627 Japanese Patent No. 4721803 JP 2003-159100 A

  An object is to provide a simple and inexpensive nucleic acid analysis method for analyzing a mutation of several bases with high accuracy.

  The nucleic acid analysis method according to the embodiment includes a step of preparing a specimen, a step of preparing a primer set that amplifies a template sequence including a mutation site consisting of the first or second base, and a primer set in one reaction vessel. A step of performing an amplification reaction on a specimen to obtain an amplification product, a step of reacting a nucleic acid probe complementary to the target sequence and the amplification product, and a step of detecting the amount of hybridization generated and analyzing the nucleic acid contained in the specimen Including. The primer set includes a first primer, a second primer, and a third primer paired therewith. The first primer includes a sequence complementary to the mutation site consisting of the first base on the 3 'side, a sequence complementary to a part of the template nucleic acid, and a tag sequence. The second primer includes a sequence complementary to the mutation site consisting of the second base on the 3 'side, a sequence complementary to a part of the template nucleic acid, and a tag sequence. The tag sequence loops out when the first and second primers bind to the complementary strand.

The figure which shows the conceptual diagram of embodiment. The figure which shows the conceptual diagram of embodiment. The figure which shows the conceptual diagram of embodiment. The figure which shows one example of a DNA chip. The figure which shows one example of a DNA chip. The figure which shows the example of an amplification reagent integrated DNA chip. The figure which shows the result of carrying out the restriction enzyme process and the electrophoresis of the product made to compete Allele specific reaction. The figure which shows the result detected by the chip | tip.

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

[Definition]
“Nucleic acid” is a term that collectively indicates substances that can represent a part of the structure by a base sequence, such as DNA, RNA, PNA, LNA, S-oligo, and methylphosphonate oligo. For example, the nucleic acid may be a partially or completely artificially synthesized and / or designed artificial nucleic acid, such as naturally occurring DNA and RNA, and mixtures thereof.

  The “specimen” is a target to be subjected to the analysis method according to the present embodiment, and may be any sample that may contain a nucleic acid. The specimen is preferably in a state that does not interfere with the amplification reaction and / or the hybridization reaction. For example, in order to use a material obtained from a living body or the like as a specimen according to the present embodiment, pretreatment may be performed by any means known per se. For example, the specimen may be a liquid.

  The nucleic acid contained in the sample is referred to as “sample nucleic acid”. Of the sample nucleic acid, the sequence to be amplified by the primer is referred to as “template sequence”. A nucleic acid containing a template sequence is referred to as a “template nucleic acid” or “template”.

  The “mutation site” refers to a position where a mutation exists in the nucleic acid to be analyzed. Variations of several bases related to diversity are referred to as “polymorphism” or “mutation” depending on the frequency difference in the population, but for convenience, “polymorphism” and “mutation” are collectively referred to as “ Called "mutation". Embodiments specify several bases constituting one mutation site existing in a template sequence, for example, which base is one base, or what kind of sequence is 2 bases or 3 bases. The

  The “target nucleic acid” refers to an amplification product obtained by amplifying a template nucleic acid or a template sequence using a primer set, for example, a forward primer and a reverse primer. The target nucleic acid includes a target sequence in a part thereof. The target sequence consists of a tag sequence and a partial sequence of the template sequence. The target sequence also includes a base or sequence corresponding to the mutation site. Preferably, the target sequence also includes a region derived from the template sequence. The target sequence is used to detect a target nucleic acid using a nucleic acid probe.

  The region derived from the template sequence means a region in which the template sequence is reflected in a region other than the region to which the primer binds among regions amplified by the primer. Here, the region reflecting the sequence of the template may be the same sequence as the partial sequence included in the template or a sequence complementary to the partial sequence included in the template. The sequence of “region derived from template sequence” is referred to as “sequence derived from template sequence” or “sequence derived from template sequence”.

  Two sequences are “identical” means that two sequences are equal to each other over 90%, 95%, 98% or 100% over their entire length. Two sequences are “complementary” refers to sequences that are complementary to each other by 90%, 95%, 98%, or 100% over the entire length of the two sequences.

  A “nucleic acid probe” is a nucleic acid comprising a sequence that is complementary to a target sequence. The nucleic acid probe may be immobilized on a known solid phase. A preferred nucleic acid probe is used by being immobilized on the surface of a substrate. An apparatus including such a substrate and a nucleic acid probe immobilized on the substrate may be generally performed by a DNA chip.

  A “DNA chip” is an apparatus that analyzes a nucleic acid using a hybridization reaction between a nucleic acid probe having a sequence complementary to the nucleic acid to be detected and the nucleic acid to be detected. The term DNA chip is synonymous with commonly used terms such as “nucleic acid chip”, “microarray” and “DNA array”, and is used interchangeably.

1. Nucleic Acid Analysis Method The nucleic acid analysis method according to the embodiment is a method for clarifying the type and sequence of several base mutations, for example, one base, two consecutive bases or three consecutive bases. It is difficult to detect a mutation of several bases with high accuracy by the conventional method. The embodiment makes it possible to measure such a mutation of several bases accurately and simply.

  For example, the nucleic acid analysis method can determine whether a base or sequence of a mutation site of a certain nucleic acid is a wild type or a mutant type.

  The template sequence amplified by the primer set includes a mutation site in a part thereof. There are mutations consisting of one base, two consecutive bases, or three bases belonging to a salt at the mutation site. The base or sequence of the mutation site determines whether the template sequence is a wild type or a mutant type. That is, according to this method, it is possible to specify the type or sequence of a base at a certain mutation site.

  For example, when determining whether the sample nucleic acid is a wild type or a mutant type, the following primer set is prepared. A first primer for amplifying the wild type; a second primer for amplifying the mutant type; and a third primer for amplifying the template sequence together with the first primer or the second primer. A primer set is used. For example, when the PCR method is used, the first primer and the second primer may be forward primers, and the third primer may be a reverse primer. Alternatively, the first primer and the second primer may be reverse primers, and the third primer may be a forward primer.

  The first primer and the second primer include a base or sequence complementary to the mutation site near the 3 'end so that amplification efficiency depending on the type and sequence of the mutation site base can be achieved. Furthermore, a tag sequence is included in order to more accurately achieve amplification efficiency depending on the type and sequence of the base at the mutation site. When the sequence of the tag sequence included in the first primer and the sequence of the tag sequence included in the second primer are different from each other, it is more advantageous to enable accurate identification of the mutation site type at the time of detection using hybridization described later It is.

  When the tag sequence is introduced into the forward primer, the forward primer is the first primer and the second primer. Reverse primer is the third primer. When the tag sequence is introduced into the reverse primer, the reverse primer is the first primer and the second primer. The forward primer is the third primer.

  For the first primer, the first primer is designed to include a base at the mutation site of the wild-type template sequence or a base complementary to the sequence in the vicinity of the 3 ′ end in the direction in which the first primer extends. The Further, the first primer includes a tag sequence on the 5 'side of the base or sequence corresponding to this mutation site. The tag sequence has a sequence different from the complementary sequence so as not to bind to the template sequence. Thereby, when the first primer binds to the template sequence, the tag sequence loops out from the double-stranded nucleic acid generated by the binding. In other words, the first primer includes three parts, that is, a first sequence, a second sequence, and a tag sequence existing between the first sequence and the second sequence. By the first sequence and the second sequence, the first primer binds to the template sequence, and the tag sequence loops out without binding to the template sequence.

  In the design of the second primer, the base at the mutation site of the mutant template sequence or a base complementary to the sequence is arranged in the vicinity of the 3 'end in the direction in which the second primer extends. Except for this configuration, the first primer is designed in the same manner.

  The third primer is a primer having a sequence that amplifies the template sequence together with the first primer or the second primer. That is, the third primer is present on the 3 ′ side of the complementary strand of the template sequence with respect to the region corresponding to the sequence to which the first primer binds.

  After preparing the primer set, an amplification reaction is performed on the specimen in one reaction tank using the first primer, the second primer, and the third primer. Thereby, in the reaction vessel, the first template and the third primer are used to amplify the wild type template sequence, and the second primer and the third primer are used as the mutant template sequence. Competitively proceeds with the second amplification reaction in which is amplified.

  Thereafter, the amplification product obtained by the amplification reaction is detected using a nucleic acid probe set. The nucleic acid probe set includes a first nucleic acid probe for detecting an amplification product derived from a wild-type template sequence and a second nucleic acid probe for detecting an amplification product derived from a mutant template sequence. . The first nucleic acid probe is a sequence complementary to a target sequence including a sequence corresponding to a wild type mutation site. The second nucleic acid probe is a sequence complementary to the target sequence including a sequence corresponding to the mutation site of the mutant type.

  For example, the target sequence for the first nucleic acid probe includes a mutation site, a region derived from the template sequence, and a tag sequence derived from the first primer. The second nucleic acid probe includes a mutation site, a region derived from the template sequence, and a tag sequence derived from the second primer.

  By detecting the amount of hybridization between the amplification product and the first and second nucleic acid probes, it is determined whether the nucleic acid contained in the specimen is a wild type, a mutant type, or a heterogeneous one.

  In the nucleic acid analysis method of the embodiment, in addition to determining the wild type and / or the mutant type of the nucleic acid such as the gene as described above, the type of the base or the type of the sequence is determined for a specific mutation site. Is possible. Such a method includes the following steps (a) to (e).

(A) Step of preparing a sample containing a template nucleic acid Preparation of a sample containing a template nucleic acid is performed as follows. First, it may be a liquid and / or solid collected from an individual. When the individual is an animal, for example, blood, serum, leukocytes, lymph, cerebrospinal fluid, peritoneal fluid, saliva, semen, tissue, biopsy, oral mucosa, cultured cells, body fluids such as sputum and milk, urine and feces Collect excrement as a sample. Thereafter, the sample is subjected to treatments such as centrifugation, precipitation, extraction and / or separation to make it suitable for nucleic acid amplification and hybridization. When the collected sample is suitable for nucleic acid amplification and hybridization as it is, the collected sample may be used as a specimen. The specimen may be collected not only from samples derived from animals but also from soil, rivers, lakes, seas, and the atmosphere. The sample only needs to contain a template nucleic acid, and as a result of performing the nucleic acid analysis method of the embodiment, it may be determined that the sample does not contain a template nucleic acid.

  The template nucleic acid may be, for example, an individual's genomic DNA, genomic RNA, mRNA or the like. Individuals may be, but are not limited to, humans, non-human animals, plants, and microorganisms such as viruses, bacteria, bacteria, yeasts and mycoplasmas. Extracted directly from microorganisms. Nucleic acid extraction can be performed using, but not limited to, commercially available nucleic acid extraction kits such as QIAamp (manufactured by QIAGEN), Sumitest (manufactured by Sumitomo Metals), and the like.

(B) Preparation of primer set The structure of a template sequence and a primer set is demonstrated referring FIG. The template nucleic acid 1 includes a template sequence 2 to be amplified by the primer set. Template sequence 2 includes mutation site 3. The first primer 4 includes a sequence complementary to a part of the template sequence 2. The first primer 4 includes a site 5 complementary to the mutation site 3 in the vicinity of the 3 ′ end thereof.

  The first primer 4 includes a first sequence 6, a second sequence 7, and a tag sequence 8 included between the first sequence 6 and the second sequence 7. The first sequence 6 is present on the 3 'side of the first primer 4, and the second sequence 7 is included on the 5' side. The first sequence 6 and the second sequence 7 have a sequence complementary to the template sequence 2. Tag sequence 8 is neither identical nor complementary to template sequence 2. With such a configuration, the first primer 4 binds to the template sequence 2 via the first sequence 6 and the second sequence 7. When the first primer binds to the template nucleic acid, the tag sequence loops out from the double strand generated by the binding.

  The third primer 9 is used together with the first primer 4 to amplify the template sequence 2. The third primer 9 binds to the complementary strand 10 of the template nucleic acid 1. The binding site of the third primer 9 to the complementary strand 10 is a region 12 on the template nucleic acid 1 on the 5 ′ side of the region 11 to which the first primer 4 on the template nucleic acid 1 binds with respect to the template nucleic acid 1. Is the region 13 of the complementary strand 10 corresponding to. Therefore, the third primer is complementary to the sequence of the complementary strand 10 and has the same sequence as the region 12 on the template nucleic acid 1.

  Thus, the template nucleic acid 1 has the template sequence 2 and the region 11 to which the first primer 4 binds, the region 12 having the same sequence as the second primer, and between the region 11 and the region 12. And a region 14 derived from an existing template sequence.

  The tag sequence may be composed of a sequence consisting of consecutive bases consisting of A, T, C and / or G, may be composed of a modified nucleic acid known per se, A, T, C, G And / or may be composed of modified bases. The type of nucleic acid constituting the tag sequence is not particularly limited as long as it is a substance capable of representing a partial structure by a base sequence, such as DNA, RNA, PNA, LNA, S-oligo, and methylphosphonate oligo.

  The number of bases in the tag sequence may be 2 bases or more, 3 bases or more, 4 bases or more, for example, 2 bases to 10 bases, preferably 3 bases to 7 bases. The types of bases contained in the tag sequence may be one, one or more, for example, two, two or more, for example, three, four, five, seven, and the like.

For example, A, T, when configuring a tag sequence 4 consecutive bases of C and G, the sequence of 4 ⁵ next 1024 are candidates of the tag sequence. However, in order to avoid the risk of mismatch hybridization, it is preferable to use a sequence composed of a combination of two or more consecutive bases, more preferably three consecutive bases having different lengths, as a tag sequence. The tag sequence is designed to loop out without binding to the template nucleic acid when the first or second primer containing the tag sequence binds to the complementary template nucleic acid. For example, the tag sequence includes a sequence different from the sequence of the template sequence and its complementary strand.

  The length of the tag sequence may be within a range that allows loop-out and that includes a primer that can amplify the template nucleic acid. The tag sequence is preferably 3 to 7 bases continuous.

  The length of the first and second primers may be 13 to 40 bases, for example 15 to 30 bases, excluding the tag sequence.

  The insertion position of the tag sequence into the first and second primers is an important point for specific amplification. Amplification becomes unstable if it is too close to the 3 'end, and specificity is low if it is too far from the 3' end. It may be the second base to the tenth base from the 3 ′ end of the first primer and the second primer, preferably the fourth base to the eighth base from the 3 ′ end, more preferably the fifth base or 6th base. Similarly, the position of the base or sequence corresponding to the mutation site contained in the first primer and the second primer in the primer is also an important point for specific amplification. Preferably, they are the first to fifth bases from the 3 ′ end side of the first primer and the second primer, more preferably the second base or the third base.

  When the PCR method is used as the amplification method, the tag-introducing wild-type amplification primer and the tag-introducing mutant amplification primer may be either a forward primer (F primer) or a reverse primer (R primer).

  Amplification methods include, for example, Polymer chain reaction method (PCR method), Loop mediated isometric amplification method (LAMP method), Isothermal and chimeric priming method (IC method). ), Strand displacement amplification (SDA method), Ligase chain reaction (LCR method), Rolling Circle Amplification method (RCA method), and the like. The obtained amplification product is fragmented or single-stranded as necessary. Examples of means for forming a single strand include heat denaturation, a method using beads, an enzyme, and the like, and a method of performing a transcription reaction using T7 RNA polymerase. Furthermore, a loop forming primer that forms a single strand may be used.

  When the nucleic acid contained in the sample is RNA, it can be converted into complementary strand DNA by, for example, reverse transcriptase before amplification. Both reverse transcriptase and DNA polymerase may be added in the same tube, and reverse transcription and DNA amplification may be performed simultaneously.

  When amplified by the LAMP method or ICAN method and a single-stranded region is present in the product and this single-stranded region is used as a target sequence, it can be directly subjected to the hybridization step.

  Since this single-stranded step is not necessary, the target nucleic acid obtained by amplification preferably has a stem-loop structure. The amplification product having a stem-loop structure can conveniently use the sequence of the single-stranded loop portion for the reaction with the nucleic acid probe.

  For amplification of the target nucleic acid, the LAMP method (for example, see Japanese Patent No. 3313358) is preferably used. The LAMP method is a rapid and simple gene amplification method, and the amplification product has a stem-loop structure. In the LAMP method, six types of primers that recognize a maximum of eight regions of a template nucleic acid and a strand displacement type DNA synthase are used. The template nucleic acid is amplified under isothermal (60-70 ° C.) conditions.

  The eight regions of the template nucleic acid will be described with reference to FIG. The eight regions are defined as an F3 region, an F2 region, an LF region, an F1 region, and a B3c region, a B2c region, an LBc region, and a B1c region in this order from the 5 'end side of the template nucleic acid. The F1c, F2c, F3c, B1, B2, and B3 regions represent regions in the complementary strand of the F1, F2, F3, B1c, B2c, and B3c regions, respectively. The eight kinds of primers shown in FIG. 2 are FIP inner primers having a sequence F1c complementary to the F1 region on the 5 ′ end side and the same sequence F2 as the F2 region on the 3 ′ end side, and the same as B1c on the 5 ′ end side. BIP inner primer having sequence B1c and having sequence B2 complementary to B2c on the 3 ′ end side, F3 primer having the same sequence F3 as F3 region, B3 primer having sequence B3 complementary to B3c region, complementary to LF region An LFc primer having a typical sequence LFc, and an LBc primer having the same sequence LBc as the LBc region. Indispensable for the amplification reaction are the FIP inner primer (FIP primer) and the BIP inner primer (BIP primer), and the F3 primer, B3 primer, LF primer and LB primer are added to increase the amplification efficiency. A loop structure is formed in the amplified product by the LAMP method, and the F2 region and the B2 region become single-stranded regions.

  The first primer and the second primer including the tag sequence may be FIP primers or BIP primers. The tag sequence is included in the vicinity of the 3 'end that is the extension start portion of the FIP primer or BIP primer, that is, in the sequence F2 or sequence B2.

  In the template nucleic acid 1 having the above 8 regions, the sequence F2 on the 3 'side of the FIP primer as the first primer binds to the F2c region of the template nucleic acid 1. This binding is achieved by the first sequence 6 and the second sequence 7 included in the sequence F2. That is, the first sequence 6 and the second sequence 7 are complementary to the sequence of the F2c region of the template nucleic acid 1. The first primer includes a tag sequence 8 between the first sequence 6 and the second sequence 7 of the sequence F2. Tag sequence 8 is neither identical to nor complementary to template sequence 2. With such a configuration, the first primer 4 binds to the template sequence 2 via the first sequence 6 and the second sequence 7. When the first primer binds to the template nucleic acid, the tag sequence loops out from the double strand generated by the binding.

  The third primer 9 is used together with the first primer 4 to amplify the template sequence. When the LAMP method is used, the third primer is a BIP primer. The third primer 9 binds to the complementary strand 10 of the template nucleic acid 1. The binding of the third primer 9 to the complementary strand 10 is performed via the BIP primer sequence B2. That is, the sequence B2 of the third primer 9 is complementary to the B2c region of the complementary strand 10 of the template nucleic acid 1.

  Alternatively, the first and second primers may be BIP primers. In that case, the third primer is an FIP primer. In this case, the primer may be designed in the same manner as the FIP primer.

  When the LF primer and / or LB primer and the target sequence overlap, it is preferable not to add the LF primer and / or LB primer.

  An example of the primer set is a primer set including an FIP primer and a BIP primer as follows.

  Both the first primer and the second primer are FIP primers. The third primer is a primer set that is a BIP primer. The FIP primer has a sequence F1c complementary to the sequence of the F1 region on the 5 'end side, and the same sequence F2 as the sequence of the F2 region on the 3' end side. The first tag sequence is included 5 'to the site corresponding to the mutation site in the F2 sequence in the FIP primer for the first primer. The second tag sequence is included 5 'to the site corresponding to the mutation site in the F2 sequence in the FIP primer for the second primer. The first tag sequence and the second tag sequence are different from each other by one or more bases. The BIP primer has the same sequence B1c as the sequence of the B1c region on the 5 'end side and a sequence B2 complementary to the sequence of the B2c region on the 3' end side.

  Using the primer set including the first primer, the second primer, and the third primer as described above, the nucleic acid contained in the specimen is amplified in one reaction vessel. The amplification reaction may be appropriately selected by those skilled in the art according to the amplification method to be selected and various conditions known per se.

  An example of a primer set is as follows. It is a primer set for amplifying a template sequence containing a mutation site consisting of a first base or sequence or a second base or sequence. The first primer includes a first sequence and a second sequence complementary to the template sequence on the 3 'side and 5' side of the first primer, respectively. Further, the first primer includes a first tag sequence consisting of a sequence not complementary to the template sequence between the first sequence and the second sequence. The first sequence includes a base or sequence complementary to the first base or sequence of the mutation site.

  The second primer includes a third sequence and a fourth sequence complementary to the template sequence on the 3 'side and the 5' side of the second primer, respectively. Further, the second primer includes a second tag sequence between the third sequence and the fourth sequence. The third sequence includes a base or sequence complementary to the second base or sequence at the mutation site. The first tag sequence and the second tag sequence are different from each other by one or more bases.

  The third primer includes a sequence that amplifies the template sequence in combination with the first primer or the second primer.

  In addition, one example of a primer set in the case of amplifying a template nucleic acid by the LAMP method may include a primer set selected from at least one selected from the group consisting of the following (1) to (6). First, the template sequence to be amplified in the primer set includes F3 region, F2 region, LF region and F1 region in this order from the 5 ′ end side, and B3c region, B2c region, LBc region from the 3 ′ end side. And the B1c region in this order. The mutation site is contained in the F2 region.

(1) Primer set including FIP primer and BIP primer Both the first primer and the second primer are FIP primers. The third primer is a primer set that is a BIP primer. The FIP primer has a sequence F1c complementary to the sequence of the F1 region on the 5 ′ end side, and the same sequence F2 as the sequence of the F2 region on the 3 ′ end side.

  The first tag sequence is included 5 'to the site corresponding to the mutation site in the F2 sequence in the FIP primer for the first primer. The second tag sequence is included 5 'to the site corresponding to the mutation site in the F2 sequence in the FIP primer for the second primer. The first tag sequence and the second tag sequence are different from each other by one or more bases.

  The BIP primer has the same sequence B1c as the sequence of the B1c region on the 5 'end side and a sequence B2 complementary to the sequence of the B2c region on the 3' end side.

(2) Primer set including FIP primer and BIP primer Both the first primer and the second primer are BIP primers. The third primer is an FIP primer. The BIP primer has the same sequence B1c as the sequence of the B1c region on the 5 ′ end side and a sequence B2 complementary to the sequence of the B2c region on the 3 ′ end side. The first tag sequence is included 3 ′ from the site corresponding to the mutation site in the B2 sequence in the BIP primer for the first primer. The second tag sequence is contained 3 ′ from the site corresponding to the mutation site in the B2 sequence in the BIP primer for the second primer. The first tag sequence and the second tag sequence are different from each other by one or more bases. The FIP primer has a sequence F1c complementary to the sequence of the F1 region on the 5 ′ end side, and the same sequence F2 as the sequence of the F2 region on the 3 ′ end side.

(3) Primer set including FIP primer and BIP primer and F3 primer and / or B3 primer Both the first primer and the second primer are FIP primers. The third primer is a BIP primer. In addition to the first, second and third primers, further primers are included.

  The FIP primer has a sequence F1c complementary to the sequence of the F1 region on the 5 'end side, and the same sequence F2 as the sequence of the F2 region on the 3' end side.

  The first tag sequence is included 5 'to the site corresponding to the mutation site in the F2 sequence in the FIP primer for the first primer. The second tag sequence is included 5 'to the site corresponding to the mutation site in the F2 sequence in the FIP primer for the second primer. The first tag sequence and the second tag sequence are different from each other by one or more bases.

  The BIP primer has the same sequence B1c as the sequence of the B1c region on the 5 'end side and a sequence B2 complementary to the sequence of the B2c region on the 3' end side.

  The F3 primer has the same sequence F3 as the F3 region. The B3 primer has the sequence B3 complementary to the B3c region.

(4) FIP primer and BIP primer, and primer set including F3 primer and / or B3 primer Both the first primer and the second primer are BIP primers. The third primer is an FIP primer. The BIP primer has the same sequence B1c as the sequence of the B1c region on the 5 ′ end side and a sequence B2 complementary to the sequence of the B2c region on the 3 ′ end side. The first tag sequence is included 3 ′ from the site corresponding to the mutation site in the B2 sequence in the BIP primer for the first primer. The second tag sequence is contained 3 ′ from the site corresponding to the mutation site in the B2 sequence in the BIP primer for the second primer. The first tag sequence and the second tag sequence are different from each other by one or more bases.

  The FIP primer has a sequence F1c complementary to the sequence of the F1 region on the 5 'end side, and the same sequence F2 as the sequence of the F2 region on the 3' end side. The F3 primer has the same sequence F3 as the F3 region, and the B3 primer has the sequence B3 complementary to the B3c region.

(5) Primer set including FIP primer and BIP primer, and F3 primer and / or B3 primer and / or LFc primer and / or LBc primer. Both the first primer and the second primer are FIP primers, The primer is a BIP primer. The FIP primer has a sequence F1c complementary to the sequence of the F1 region on the 5 ′ end side, and the same sequence F2 as the sequence of the F2 region on the 3 ′ end side.

  The first tag sequence is included 5 ′ from the site corresponding to the mutation site in the F2 sequence in the FIP primer for the first primer, and the second tag sequence is for the second primer. The FIP primer is contained on the 5 ′ side of the site corresponding to the mutation site in the F2 sequence, and the first tag sequence and the second tag sequence are different from each other by one or more bases.

  The BIP primer has the same sequence B1c as the sequence of the B1c region on the 5 ′ end side, the sequence B2 complementary to the sequence of the B2c region on the 3 ′ end side, and the F3 primer has the same sequence F3 as the F3 region. And the B3 primer has the sequence B3 complementary to the B3c region.

  The LFc primer has the sequence LFc complementary to the LF region, and the LBc primer has the same sequence as the LBc region.

(6) FIP primer and BIP primer, and primer set including F3 primer and / or B3 primer Both the first primer and the second primer are BIP primers. The third primer is an FIP primer. The BIP primer has the same sequence B1c as the sequence of the B1c region on the 5 ′ end side and a sequence B2 complementary to the sequence of the B2c region on the 3 ′ end side. The first tag sequence is included 3 ′ from the site corresponding to the mutation site in the B2 sequence in the BIP primer for the first primer. The second tag sequence is contained 3 ′ from the site corresponding to the mutation site in the B2 sequence in the BIP primer for the second primer. The first tag sequence and the second tag sequence are different from each other by one or more bases. The FIP primer has a sequence F1c complementary to the sequence of the F1 region on the 5 ′ end side, and the same sequence F2 as the sequence of the F2 region on the 3 ′ end side. The F3 primer has the same sequence F3 as the F3 region, and the B3 primer has the sequence B3 complementary to the B3c region. The LFc primer has the sequence LFc complementary to the LF region. The LBc primer has the same sequence as the LBc region.

  When a nucleic acid contained in a specimen is amplified using a primer set including a first primer, a second primer, and a third primer in one reaction vessel, the first primer and the second primer are converted into the third primer. The amplification reaction proceeds competitively by competing for primers. Therefore, it is possible to accurately detect not only a specimen having a genetic trait but also a heterogeneous specimen.

  For example, when the template sequence is the wild type, amplification proceeds only from the primer for amplifying the wild type. When the template sequence is a mutant type, amplification proceeds only from primers for amplifying the mutant type. In the case of the hetero type, amplification proceeds from both the primer for amplifying the wild type and the primer for amplifying the mutant type. The primer for amplifying the wild type and the primer for amplifying the mutant type may be used by mixing them in equal amounts, and the quantity ratio may be changed depending on the reaction efficiency. The amplification product may be directly hybridized with the nucleic acid probe on the DNA chip. Alternatively, the amplification product may be hybridized with the nucleic acid probe after heat denaturation and / or addition of a salt such as SSC.

  Furthermore, when the sequence of the mutation site included in the template nucleic acid can take three or more kinds of sequences, in addition to the first primer and the second primer to be used, the sequence of the region corresponding to the mutation site is changed. A modified additional primer may be used.

(C) Detection by nucleic acid probe By reacting the amplification product with the nucleic acid probe set, information on the type or sequence of the base of the mutation site of the nucleic acid contained in the specimen can be obtained.

  The nucleic acid probe set includes a first nucleic acid probe for detecting an amplification product generated by the first primer and a second nucleic acid probe for detecting the amplification product generated by the second primer.

  The design of the nucleic acid probe will be described with reference to FIG. FIG. 3 is a diagram showing a nucleic acid probe 31 in addition to the template nucleic acid 1 and its complementary strand 10, and the first primer 4 and the second primer 9 of FIG.

  The nucleic acid probe 31 includes a sequence complementary to the target sequence in the amplification product. The target sequence contains the same base or sequence at the mutation site 3 as the tag sequence 8 (see FIG. 3 (a)). Preferably, the target sequence includes the tag sequence 8, the same base or sequence at the mutation site 3, and a region derived from the template sequence (see FIG. 3 (b)).

  Furthermore, when the sequence of the mutation site included in the template nucleic acid can take three or more kinds of sequences, in addition to the first primer and the second primer to be used, the sequence of the region corresponding to the mutation site is changed. Use modified additional primers. Accordingly, the nucleic acid probe set may include additional nucleic acid probes for detecting amplification products generated by extension from additional primers. Such additional nucleic acid probes may be configured similarly to the nucleic acid probes described above.

  The chain length of the nucleic acid probe is not particularly limited, but is preferably in the range of 5 to 50 bases, more preferably in the range of 10 to 40 bases, and still more preferably in the range of 15 to 35 bases.

The nucleic acid probe is preferably immobilized on a substrate. The substrate for immobilizing the nucleic acid probe, that is, the solid phase, may be any substrate generally used as a solid phase for a DNA chip. Such a substrate may be composed of glass, silicon, nitrocellulose film, nylon film, microtiter plate, electrode, magnet, bead, plastic, latex, synthetic resin, natural resin, or optical fiber, but is not limited thereto. Not. A DNA chip may be constructed by immobilizing a plurality of types of nucleic acid probes on these substrates. It is also possible to use a DNA chip for the reaction between the nucleic acid probe and the amplification product. The substrate of the DNA chip may be any conventionally known microarray substrate such as an electrochemical detection type represented by a current detection type, a fluorescence detection type, a chemical color development type, and a radioactivity detection type.

  Any kind of microarray can be produced by a method known per se. For example, in the case of a current detection type microarray, the negative control probe immobilization region and the detection probe immobilization region may be arranged on different electrodes.

  An example of a DNA chip is shown in FIG. 4 as a schematic diagram, but is not limited thereto. The DNA chip 100 includes an immobilization region 102 on a base 101. The nucleic acid probe 103 is immobilized on the immobilization region 102. Such a DNA chip 100 can be manufactured by a method well known in the art. A person skilled in the art can appropriately change the design of the number and the arrangement of the immobilization regions 102 arranged on the base 101 as necessary. Such a DNA chip may be suitably used for a detection method using fluorescence.

  Another example of the DNA chip is shown in FIG. A DNA chip 200 in FIG. 5 includes an electrode 202 on a base body 201. The nucleic acid probe 203 is immobilized on the electrode 202. The electrode 202 is connected to the pad 204. Electrical information from the electrode 202 is acquired via the pad 204. Such a DNA chip 200 can be manufactured by a method well known in the art. A person skilled in the art can appropriately change the design of the number and arrangement of the electrodes 202 arranged on the base body 201 as needed. Furthermore, the DNA chip of this example may include a reference electrode and a counter electrode as necessary.

  The electrode is composed of a simple metal such as gold, gold alloy, silver, platinum, mercury, nickel, palladium, silicon, germanium, gallium or tungsten, or an alloy thereof, or carbon such as graphite or glassy carbon, or a material thereof. Although an oxide or a compound can be used, it is not limited to them.

  The DNA chip as in this example may be suitably used for an electrochemical detection method.

  The amplification reaction as described above and the hybridization reaction in the DNA chip may be performed continuously in one device. In that case, a primer set may be immobilized in the vicinity of the nucleic acid probe immobilization region of the DNA chip. In addition to the primer set, an amplification reagent necessary for the amplification reaction may be immobilized in the same region as and / or in the vicinity of the region where the primer set is immobilized. An example of such a device is shown in FIG. This device is also referred to as an amplification reagent integrated DNA chip.

  6A is a plan view of the amplification reagent integrated DNA chip 300, and FIG. 6B is a cross-sectional view of the amplification reagent integrated DNA chip 300 in FIG. is there. The amplification reagent integrated DNA chip 300 includes a DNA chip 301a and a lid 301b. The DNA chip 301a includes a nucleic acid probe immobilization region 302, and a primer set immobilization region 303 in the vicinity of the nucleic acid probe immobilization region 302 or in the same device. A nucleic acid probe 304 is immobilized in the nucleic acid probe immobilization region 302. A primer set 305 is releasably immobilized in the primer immobilization region 302. In the vicinity of the primer immobilization region 302, an amplification reagent immobilization region 306 is disposed. The amplification reagent 307 is releasably immobilized in the amplification reagent immobilization region 306. The amplification reagent 307 may be, for example, an enzyme, a substrate, or buffers necessary for amplification.

  The primer set 305 is a primer set for amplifying a target nucleic acid detected by the nucleic acid probe 304. After amplification, it is detected directly with the nucleic acid probe 304, or after amplification, the product is heat denatured and mixed with a salt such as SSC and then detected with the nucleic acid probe 304. The primer set 305 may be immobilized with a gelling agent such as agarose, agar, or gelatin. The immobilization may be performed by a heat drying method, a vacuum drying method, freeze drying, or the like.

  By using such an amplification reagent integrated DNA chip 300, a liquid containing a template is injected into the amplification reagent integrated DNA chip, and amplification to detection are performed in the same reaction vessel or the same substrate.

  When the amplification reagent integrated DNA chip 300 performs electrochemical detection, the nucleic acid probe 304 may be immobilized on the electrode 308. The electrode 308 is exposed in the nucleic acid probe immobilization region 302. The other surface of the DNA chip 301a is covered with an insulator 320. The electrode 308 is connected to the pad 309. Electrical information from the electrode 308 is acquired via the pad 309.

  In the amplification reagent integrated DNA chip 300, an electrode 308 and a pad 309 are formed on the surface of the substrate 311 by using an appropriate conductor, and the electrode 308 and the pad 309 are connected so as to be energized. Thereafter, the insulator 320 covers the surface of the substrate 311 excluding the circular window defining the nucleic acid probe immobilization region 302 and the pad 3. The nucleic acid probe 304 is immobilized on the electrode 308 in the nucleic acid probe immobilization region 302, and the primer set 305 is releasably immobilized in the primer immobilization region 303. Furthermore, reaction reagents other than the primer set 305 are immobilized in the reagent immobilization region 306. This is referred to as a DNA chip 301a. Next, all the nucleic acid probes, primer sets, and amplification reagents immobilized on the DNA chip 301a are covered and fixed by the lid 301b, which is a hollow rectangular parallelepiped with one side opened. For the reaction, a reaction solution containing the sample nucleic acid is added to the reaction unit 312 constituted by the DNA chip 301a and the lid 301b. This addition may be performed, for example, by puncturing the lid 301b by means such as a syringe with a needle. First, the primer set 305 and the amplification reagent 307 are released by the addition of the reaction solution, and react with the nucleic acid (not shown) in the reaction solution. This reaction is performed under conditions suitable for the amplification reaction. Next, the resulting amplification product is reacted with the nucleic acid probe 304. The amount of hybridization produced thereby is detected. The type of nucleic acid contained in the specimen may be determined from the detected amount of hybridization.

  The hybridization temperature may be set as appropriate. A salt may be added to increase the reaction efficiency. In addition, when an amplification reagent integrated DNA chip is used, a salt may be fixed in the vicinity of the DNA chip, and the salt may be mixed in the solution after gene amplification. The reaction efficiency may be increased by stirring or shaking.

  A washing solution for washing the DNA chip after hybridization has an ionic strength in the range of 0.01 to 5, and a buffer solution in the range of pH 5 to 9 is preferably used. The cleaning liquid preferably contains a salt and a surfactant. For example, an SSC solution, a Tris-HCl solution, a Tween 20 solution, or an SDS solution is preferably used. The washing temperature is, for example, in the range of 10 ° C to 70 ° C. The washing solution passes or stays on the surface of the probe-immobilized substrate or the region where the nucleic acid probe is immobilized.

  The detection of the hybrid produced by the hybridization process can utilize a fluorescence detection method and an electrochemical detection method.

(A) Fluorescence detection method Detection is performed using a fluorescent labeling substance. Primers used in the nucleic acid amplification step may be labeled with a fluorescently active substance such as a fluorescent dye such as FITC, Cy3, Cy5, or rhodamine. Alternatively, a second probe labeled with these substances may be used. A plurality of labeling substances may be used simultaneously. The label in the labeled sequence or the secondary probe is detected by the detection device. Use an appropriate detection device depending on the label used. For example, when a fluorescent substance is used as a label, it is detected using a fluorescence detector.

(B) Electrochemical detection method A double-stranded recognition substance well known in the art is used. The double-stranded recognizing substance may be selected from Hoechst 33258, acridine orange, quinacrine, dounomycin, metallointercalator, bisintercalator such as bisacridine, trisintercalator and polyintercalator. Further, these double strand recognition substances may be modified with an electrochemically active metal complex such as ferrocene or viologen.

  The double-stranded recognition substance varies depending on the type, but is generally used at a concentration in the range of 1 ng / mL to 1 mg / mL. In this case, a buffer solution having an ionic strength in the range of 0.001 to 5 and a pH in the range of 5 to 10 can be used.

  In the measurement, for example, a potential equal to or higher than the potential at which the double strand recognition substance reacts electrochemically is applied, and the reaction current value derived from the double strand recognition substance is measured. At this time, the potential may be applied at a constant speed, may be applied in pulses, or a constant potential may be applied. The current and voltage may be controlled using devices such as a potentiostat, a digital multimeter, and a function generator. Electrochemical detection can be performed by methods well known in the art.

2. Assay Kit This embodiment may be in the form of an assay kit. The assay kit may include a primer set for performing the nucleic acid analysis method described above, and a nucleic acid probe or a DNA chip. Furthermore, an enzyme, a substrate, a buffer, an instruction manual, etc. may be optionally included.

  The primer set included in the assay kit may include any of the first primer, the second primer, and the third primer described above.

  The assay kit is a primer for amplifying a wild type base or sequence in which the first primer is present at the mutation position, and a second primer is for amplifying the wild type base or sequence present in the mutation position. It may be a primer.

  Further, the assay kit may include a nucleic acid probe. The nucleic acid probe may be immobilized on a substrate. The nucleic acid probe is preferably included in the assay kit as a nucleic acid probe set. Such a nucleic acid probe set is immobilized on a substrate, a nucleic acid probe complementary to a first target sequence or a complementary sequence thereof including a first tag sequence and a sequence derived from a template sequence, A nucleic acid probe that is immobilized and is complementary to a second target sequence including the second tag sequence and a sequence derived from a template sequence or a complementary sequence thereof.

  Depending on the embodiment, the sequence can be revealed for mutations of several bases, such as mutations of one base or two or three consecutive bases. In the conventional method, it is difficult to accurately detect a mutation of several bases. According to the embodiment, such a mutation of several bases can be measured accurately and easily.

  Further, according to the embodiment, it is possible to solve various problems in the conventional method for analyzing single nucleotide mutations as described below.

  In the past, a restriction fragment length polymorphism method (RFLP method) was used as a method for detecting a single nucleotide mutation or a single nucleotide polymorphism. This method is complicated and requires a long time, and may not be analyzed depending on the base sequence near the SNP site. According to this embodiment, it is possible to easily analyze a target nucleic acid in a shorter time.

  In recent years, the Invader method, TaqMan PCR method, single base extension method, pyrosequencing method, exonuclease cycling assay, allele specific PCR, and a method using a DNA chip have been developed. allele-specific PCR is the cheapest and most convenient method. However, this method has low amplification specificity and low accuracy. In addition, there has been proposed a method for artificially adding one more mismatch to this method. This requires strict condition setting, and even if strict temperature control is performed, non-specific amplification (that is, miss amplification) occurs depending on the sequence. According to this embodiment, it is possible to easily analyze a target nucleic acid with high accuracy and without non-specific amplification.

  As a simple and inexpensive isothermal amplification method, a LAMP method (Loop-mediated thermal amplification method) has been proposed. In this method, the SNP is positioned in a region where the forward primer (FIP primer) and the reverse primer (BIP primer) overlap on the 3 'end side (starting point of synthesis). There is also a proposal to increase specificity by introducing block nucleic acids. However, these methods still result in non-specific amplification (ie, miss amplification). When analyzing a plurality of single nucleotide mutations or single nucleotide polymorphisms, it is necessary to change the reaction tank and reaction conditions for each single nucleotide mutation or single nucleotide polymorphism. Analyzing multiple mutations or polymorphisms takes time and effort. According to this embodiment, in a small reaction system (for example, in one tube), it is possible to analyze a target nucleic acid more specifically and easily without nonspecific amplification. .

  In the method using a DNA chip, a plurality of single nucleotide mutations or single nucleotide polymorphisms can be analyzed at a time. In this method, non-specific hybridization may occur due to a target having a different single base, and detection with high accuracy is difficult. According to this embodiment, it is possible to easily analyze a target nucleic acid with high accuracy and without non-specific amplification.

[Example]
Example 1 Examination of tag insertion position enabling specific amplification The tag insertion position enabling specific amplification was examined.

<Primer>
The amplification method used was the LAMP method. A primer set for detecting polymorphism C-13T in the promoter region of the human SAA1 gene was prepared. The primer set consists of FIP primer, F3 primer, B3 primer, LPF primer and BIP primer. Of these primers, the BIP primer was used as the first primer and the second primer. That is, the influence on the amplification efficiency by incorporating the tag into the 3 ′ end of the BIP primer was examined. At the same time, when incorporating a tag into the BIP primer, it was examined whether the basic primer position into which the tag is incorporated affects the amplification efficiency. The sequences of FIP primer, F3 primer, B3 primer and LPF primer are shown in Table 1. Here, 20 bases from the 3 ′ end of the FIP primer have a sequence complementary to the template nucleic acid, and serve as a primer for amplification in the first stage (first) of the LAMP method. The 3 'end 20 bases of the FIP primer are the F2 region, and the 5' end 19 bases are the F1 region.

The sequences of the BIP primers used are shown in Table 2. The region of B2 of the BIP primer was designed to contain a base corresponding to the mutation site of the template nucleic acid.

  The BIP primers of SEQ ID NO: 5 and SEQ ID NO: 6 are basic primers that do not contain a tag. The positions of bases surrounded by white squares in SEQ ID NOs: 5 and 6 correspond to the positions of mutation sites in the template nucleic acid. In this mutation site, SEQ ID NO: 5 has a base “G” complementary to the wild-type base “C” of the template nucleic acid. In this mutation site, SEQ ID NO: 6 has a base mutation type base “A” complementary to the mutation type base “T” of the template nucleic acid. Except for the base of this mutation site, SEQ ID NOs: 5 and 6 have the same sequence.

  Furthermore, a BIP primer having a tag sequence “TTTTTT” introduced into the basic primer was prepared. Their sequences are shown in SEQ ID NOs: 7-22. The tag sequence was introduced at the 3rd base, 4th base, 5th base, 6th base, 7th base, 8th base, 9th base, 10th base from the 3 'end of the BIP primer. The number of bases of the tag sequence incorporated was 5 bases (TTTTTT).

  About these primers, the primer for wild type amplification and the primer for mutation type amplification were designed, respectively. Amplification was performed in separate reaction vessels using a primer set for wild type amplification and a primer set for mutation type amplification, respectively. That is, each of SEQ ID NOs: 1 to 4 and any one of SEQ ID NOs: 5 to 22 was used as one primer set, and an amplification reaction was performed independently without causing a competitive reaction.

<Amplification>
An amplification reagent was prepared with the composition shown in Table 3.

  Two types of human genomes, C / C type and T / T type, which were identified by PCR-RFLP analysis and sequence analysis, were used for amplification at N = 2. The amplification device used was a real-time turbidity measurement device (manufactured by Teramex), and the rise time of turbidity was used as an index of amplification rate.

<Result>
The results are shown in Table 4.

  As shown in Table 4, when no tag sequence was inserted, there was almost no difference in amplification rate between perfect match and single base mismatch. From this result, it was shown that specific amplification cannot be performed in the case of a primer which does not insert a tag sequence. When the tag sequence was inserted at the third base from the 3 'end of FIP, amplification was slow. In the case of the 9th base and the 10th base from the 3 'end of FIP, it was shown that the specificity decreased. The result of 8 bases from the 4th base from the 3 'end of FIP was good. The difference in amplification rate between the perfect match and the single nucleotide mismatch at the fifth and sixth bases from the 3 'end of FIP was 30 minutes or more.

Example 2 Examination of mutation position Mutation positions capable of specific amplification were examined.

<Primer>
Primers listed in Table 5 were used. The primer was designed so that the mutation site was included at the first base, second base, third base, fourth base or fifth base from the 3 ′ end of the BIP primer. The tag sequence was positioned at the 6th and 8th bases from the 3 ′ end of the FIP primer. In the same manner as in Example 1, a wild type amplification primer and a mutation type amplification primer were designed. About these, amplification reaction was performed independently, without carrying out competitive reaction.

<Amplification>
The same method as in Example 1 was used.

<Result>
As shown in Table 6, the position corresponding to the mutation site of the primer resulted in low specificity at the 5th base from the 3 ′ end of the FIP primer. Preferred results were obtained from the first base to the fourth base from the 3 ′ end of the FIP primer. The higher specificity was at the second or third base from the 3 ′ end of the FIP primer.

Example 3 Examination of base number of tag sequence The number of bases of the tag sequence that can be inserted into the primer was examined.

<Primer>
The primers listed in Table 7 were used. The number of bases in the tag sequence was examined from 3 to 9 bases. The position of the tag sequence was the sixth base from the 3 ′ end of the BIP primer. The mutation site was designed to be the second base from the 3 ′ end of the BIP primer. In the same manner as in Example 1, a wild type amplification primer and a mutation type amplification primer were designed and amplified independently without causing a competitive reaction.

<Amplification>
The same method as in Example 1 was used.

<Result>
As shown in Table 8, amplification became unstable when the number of bases in the tag sequence was 9 bases. When the number of bases in the tag sequence was 3 to 8 bases, amplification was stable and specificity was high.

Example 4 Examination of tag sequence The tag sequence was examined.

<Primer>
Primers for detecting human SAA1 C-13T, NAT2 590A, and MTHFR C677T shown in Table 9, Table 10, and Table 11 were prepared. The tag sequence was inserted at the sixth base from the 3 ′ end of the FIP primer or BIP primer. The mutation site was the second base from the 3 ′ end of the FIP primer or BIP primer. The tag sequences were designed as eight tag sequences having different sequences of 4 bases or more (1. CCTTG, 2. TGACC, 3. GTGCA, 4. CCTCT, 5. AGTGG, 6. GACGT, 7. GCAAG, 8 ACGTC). This was used as a tag sequence.

<Amplification>
The same method as in Example 1 was used.

<Result>
From the results shown in Table 12, Table 13, and Table 14, a primer having high specificity was selected from among the 8 types of tag sequences. For SAA1 C-13T, tag 1 was selected as the tag primer for C-type amplification, and tag 2 was selected as the tag primer for T-type amplification. For NAT2 G590A, tag 6 was selected as the tag primer for G-type amplification, and tag 3 was selected as the tag primer for A-type amplification. For MTHFR C677T, tag 1 was selected as the tag primer for C-type amplification, and tag 2 was selected as the tag primer for A-type amplification.

Example 5 Competitive allele specific reaction Results of a competing allele specific reaction <Amplification>
Using the primers selected in Example 4, competitive amplification was performed with the compositions described in Tables 15 and 16.

<Electrophoresis>
After amplification, RFLP was performed and the amplified product was analyzed. SAA1 C-13T was treated with MwoI, NAT2 G590A was treated with a mixture of TaqI and TfiI, and MTHFR C677T was treated with a mixture of SpeI and PleI.

<Result>
The results are shown in FIG. As a result of LAMP-RFLP analysis, it was confirmed that specific amplification was achieved in the wild homozygote, mutant homozygote, and heterozygote for all three genes.

Example 6 Detection with amplification reagent integrated DNA chip <Production of amplification reagent integrated DNA chip>
Nucleic acid probes for detecting SAA1 C-13T, NAT2 G590A, MTHFR C677T are listed in Table 17.

As a negative control, a nucleic acid probe showing a sequence unrelated to the above three genes was prepared. The above nucleic acid probe was thiol-modified on the 3 'end side for immobilization on a gold electrode. The nucleic acid probe was immobilized using the strong covalent bond between thiol and gold. A 3 μM nucleic acid probe solution containing a final concentration of 100 μM mercaptohexanol solution was spotted 100 nL at a time on a gold electrode and dried. The same probe was spotted on each of the four electrodes. After drying, it was washed with ultrapure water and air-dried to obtain a probe-immobilized electrode. Furthermore, the primer and the enzyme were spotted on the same substrate of the DNA chip and immobilized by a heat drying method. Primers and enzyme were immobilized so as to have the concentrations shown in Tables 15 and 16 when completely eluted in 4 μL of the solution.

<Amplification to detection>
A solution containing a primer, an amplification reagent other than an enzyme, and a template was injected onto the DNA chip, and an amplification reaction was performed at 62 ° C. for 1 hour. Thereafter, heat was applied at 95 ° C. for 5 minutes. Thereafter, the mixture was allowed to stand at 55 ° C. for 10 minutes to carry out a hybridization reaction between the LAMP product and the nucleic acid probe. Thereafter, the nucleic acid probe-immobilized electrode was washed with 0.2 × SSC solution, and immersed in PIPES buffer containing 50 μM Hoechst 33258 solution as an intercalating agent for 1 minute. Thereafter, the oxidation current response of Hoechst 33258 solution was measured.

<Result>
As shown in FIG. 8, as a result of chip detection, clear type discrimination was possible.

Claims (12)

A method for analyzing nucleic acid,
(A) preparing a specimen containing nucleic acid;
(B) a step of preparing a primer set for amplifying a template sequence comprising a mutation site comprising the first base or sequence or the second base or sequence, wherein the primer set comprises the first primer and the second primer The first primer includes a first sequence and a second sequence complementary to the template sequence on the 3 ′ side and the 5 ′ side of the first primer. Each of the first primers includes a first tag sequence between the first sequence and the second sequence, the first tag sequence comprising a sequence that is different from the template sequence and is not complementary; The sequence includes a base or sequence complementary to the first base or sequence of the mutation site, and the second primer includes a third sequence and a fourth sequence complementary to the template sequence. 2 primers A second tag comprising a sequence different from the template sequence and not complementary between the third sequence and the fourth sequence. The third sequence includes a base or sequence complementary to the second base or sequence of the mutation site, and the third primer includes the first primer or the second primer. Comprising a sequence for amplifying said template sequence in a combination of:
(C) bringing all the primer sets prepared in (b) into one reaction tank, performing an amplification reaction on the sample, and obtaining an amplification product;
(D) a nucleic acid probe that is immobilized on a substrate and includes a first target sequence including the first tag sequence and a sequence derived from a template sequence or a complementary sequence thereof, and is immobilized on the substrate; Reacting a nucleic acid probe complementary to a second target sequence comprising a second tag sequence and a sequence derived from a template sequence or a complementary sequence thereof, and the amplification product obtained in (c);
(E) analyzing the nucleic acid contained in the specimen by detecting the amount of hybridization produced in (d);
Including methods.   2. The method according to claim 1, wherein the first tag sequence and the second tag sequence are different sequences. The analysis method according to claim 1, wherein the amplification reaction is a LAMP method, and the template sequence includes an F3 region, an F2 region, an LF region, and an F1 region in this order from the 5 ′ end side thereof, and The B3c region, the B2c region, the LBc region, and the B1c region are included in this order from the 3 ′ end side, the mutation site is included in the F2 region, and the primer set is selected from the group consisting of: A method comprising a set;
(1) A primer set comprising a FIP primer and a BIP primer, wherein both the first primer and the second primer are the FIP primer, and the third primer is the BIP primer, and the FIP primer is The sequence F1c complementary to the sequence of the F1 region on the 5 ′ end side thereof, the sequence F2 identical to the sequence of the F2 region on the 3 ′ end side thereof, and the first tag sequence comprising: The FIP primer for the first primer is included 5 ′ from the site corresponding to the mutation site in the F2 sequence, and the second tag sequence is the FIP primer for the second primer. F1 sequence is contained on the 5 ′ side of the site corresponding to the mutation site, and the first tag sequence and the second tag sequence are different from each other, and the BIP A primer set having the same sequence B1c as the sequence of the B1c region on the 5 ′ end side and a sequence B2 complementary to the sequence of the B2c region on the 3 ′ end side;
(2) A primer set comprising a FIP primer and a BIP primer, wherein the first primer and the second primer are both BIP primers, and the third primer is an FIP primer, The 5 ′ end side has the same sequence B1c as the B1c region sequence, the 3 ′ end side has a sequence B2 complementary to the B2c region sequence, and the first tag sequence is the first tag sequence The B2 primer for the primer is included 3 ′ from the site corresponding to the mutation site in the B2 sequence, and the second tag sequence is the B2 sequence in the BIP primer for the second primer In which the first tag sequence and the second tag sequence are different from each other, and the FIP protocol The primer has a sequence F1c complementary to the sequence of the F1 region on the 5 ′ end side, and a primer set having the same sequence F2 as the sequence of the F2 region on the 3 ′ end side,
(3) A primer comprising an FIP primer and a BIP primer, and an F3 primer and / or a B3 primer, wherein both the first primer and the second primer are the FIP primer, and the third primer is the BIP primer The FIP primer has a sequence F1c complementary to the sequence of the F1 region on the 5 ′ end side thereof, and has the same sequence F2 as the sequence of the F2 region on the 3 ′ end side thereof. 1 tag sequence is included 5 ′ from the site corresponding to the mutation site in the F2 sequence in the FIP primer for the first primer, and the second tag sequence is the second tag sequence The FIP primer for a primer is contained on the 5 ′ side of the site corresponding to the mutation site in the F2 sequence, the first The tag sequence and the second tag sequence are different from each other, and the BIP primer has the same sequence B1c as the sequence of the B1c region on the 5 ′ end side and is complementary to the sequence of the B2c region on the 3 ′ end side. A primer set having the sequence B2, wherein the F3 primer has the same sequence F3 as the F3 region, and the B3 primer has a sequence B3 complementary to the B3c region.
(4) A primer set comprising an FIP primer and a BIP primer, and an F3 primer and / or a B3 primer, wherein both the first primer and the second primer are BIP primers, and the third primer is an FIP primer. And the BIP primer has the same sequence B1c as the sequence of the B1c region on the 5 ′ end side, the sequence B2 complementary to the sequence of the B2c region on the 3 ′ end side, The tag sequence is included 3 ′ from the site corresponding to the mutation site in the B2 sequence in the BIP primer for the first primer, and the second tag sequence is that of the second primer. In the BIP primer for 3 ′ side of the site corresponding to the mutation site in the B2 sequence, The FIP primer has a sequence F1c complementary to the sequence of the F1 region at the 5 ′ end and the same sequence as the sequence of the F2 region at the 3 ′ end. A primer set having the sequence F2, the F3 primer having the same sequence F3 as the F3 region, and the B3 primer having a sequence B3 complementary to the B3c region;
(5) FIP primer and BIP primer, and F3 primer and / or B3 primer and / or LFc primer and / or LBc primer, both the first primer and the second primer are the FIP primer, 3 is a primer set in which the BIP primer is the primer set, and the FIP primer has a sequence F1c complementary to the sequence of the F1 region on the 5 ′ end side, and the sequence of the F2 region on the 3 ′ end side. And the first tag sequence is included 5 ′ from the site corresponding to the mutation site in the F2 sequence in the FIP primer for the first primer, and the first tag sequence is included in the FIP primer for the first primer. 2 tag sequence is the F2 sequence in the FIP primer for the second primer. The first tag sequence and the second tag sequence are different from each other, and the BIP primer has a sequence of the B1c region on the 5 ′ end side thereof. Having the same sequence B1c, having a sequence B2 complementary to the sequence of the B2c region on the 3 ′ end side, the F3 primer having the same sequence F3 as the F3 region, and the B3 primer being a B3c region A primer set having a sequence B3 complementary to the LF region, the LFc primer having a sequence LFc complementary to the LF region, and the LBc primer having the same sequence as the LBc region.
(6) A primer set comprising an FIP primer and a BIP primer, and an F3 primer and / or a B3 primer, wherein both the first primer and the second primer are BIP primers, and the third primer is an FIP primer. And the BIP primer has the same sequence B1c as the sequence of the B1c region on the 5 ′ end side, the sequence B2 complementary to the sequence of the B2c region on the 3 ′ end side, The tag sequence is included 3 ′ from the site corresponding to the mutation site in the B2 sequence in the BIP primer for the first primer, and the second tag sequence is that of the second primer. In the BIP primer for 3 ′ side of the site corresponding to the mutation site in the B2 sequence, The FIP primer has a sequence F1c complementary to the sequence of the F1 region at the 5 ′ end and the same sequence as the sequence of the F2 region at the 3 ′ end. Having the sequence F2, the F3 primer having the same sequence F3 as the F3 region, the B3 primer having the sequence B3 complementary to the B3c region, and the LFc primer being a sequence complementary to the LF region A primer set comprising LFC, wherein the LBc primer has the same sequence as the LBc region.   4. The method according to claim 1, wherein each of the first tag sequence and the second tag sequence is contained in the fourth to eighth bases from the 3 ′ end of each of the first primer and the second primer. 2. The method according to item 1.   4. The method according to claim 1, wherein each of the first tag sequence and the second tag sequence is contained at the fifth base or the sixth base from the 3 ′ end of each of the first primer and the second primer. 2. The method according to item 1.   The base or sequence complementary to the mutation site is contained in any one of the first base to the fourth base from the 3 ′ end of each of the first primer and the second primer. The method described.   The base or sequence complementary to the mutation site is contained in the second base or the third base from the 3 'end of each of the first primer and the second primer. The method described.   The method according to any one of claims 1 to 7, wherein the base length of each of the first tag sequence and the second tag sequence is 3 to 7 bases.   9. The method according to claim 1, wherein the first primer amplifies a wild-type base or sequence present at the mutation position, and the second primer amplifies a wild-type base or sequence present at the mutation position. The method according to claim 1.   An assay kit comprising a primer set used in the method according to any one of claims 1 to 9, wherein the nucleic acid to be analyzed consists of a first base or sequence or a second base or sequence A nucleic acid comprising a mutation site, wherein the primer set comprises a first primer, a second primer, and a third primer, wherein the first primer comprises a first sequence complementary to the template sequence; A second sequence on the 3 ′ side and 5 ′ side of the first primer, respectively, and the first primer is different from the template sequence between the first sequence and the second sequence, and A first tag sequence comprising a non-complementary sequence, the first sequence comprising a base or sequence complementary to the first base or sequence of the mutation site, and the second primer comprising: A third sequence and a fourth sequence complementary to the template sequence are included on the 3 ′ side and the 5 ′ side of the second primer, respectively, and the second primer further includes the third sequence and the fourth sequence. A second tag sequence is included between the sequences, the third sequence includes a base or sequence complementary to the second base or sequence of the mutation site, and the third primer includes the first primer An assay kit comprising a sequence for amplifying the template sequence in combination with the primer or the second primer.   A nucleic acid probe which is immobilized on the substrate and which is complementary to the first target sequence including the first tag sequence and a sequence derived from the template sequence or a complementary sequence thereof, and is immobilized on the substrate; The assay kit according to claim 9, further comprising a nucleic acid probe complementary to a second target sequence including the second tag sequence and a sequence derived from a template sequence or a complementary sequence thereof.   The first primer is a primer for amplifying a wild type base or sequence present at the mutation position, and the second primer is for amplifying a wild type base or sequence present at the mutation position. The assay kit according to any one of claims 9 to 11, which is a primer.

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