JP2010124823A - Primer typing gene polymorphism of obesity gene and competitive probe - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a typing method for gene polymorphism of β3AR gene, UCP1 gene, and β2AR gene which have been difficult to simply detect in a conventional sequence method or PCR-RFLP method, rapidly in a simple operation. <P>SOLUTION: The primer is used for typing polymorphism of obesity related genes by amplification of a nucleic acid, wherein the obesity related genes are at least one gene selected from the group consisting of the β3AR gene, UCP1 gene, and β2AR gene, and the primer amplifies a base sequence of the 3215 to 3325 positions of the β3AR gene, a base sequence of the -3888 to -3739 positions of the UCP1 gene, and a base sequence of the 216 to 338 positions of the β2AR gene, and a nucleic acid sequence of the target domain selected from their complimentary sequence. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

The present invention is a simple and rapid genetic diagnosis method comprising one or more gene polymorphisms selected from the group consisting of gene polymorphisms of β3AR gene, UCP1 gene, and β2AR gene. Provide health support information based on polymorphism information.

In recent years, it is said to be an era of satiety, and various foods are being supplied in excess. In addition, there are many opportunities to eat nutritionally biased meals such as fast food. Under these circumstances, so-called modern and lifestyle-related diseases such as “obesity” and “diabetes” due to excessive intake of nutrients, bias of nutrients, lack of exercise, etc. are increasing among modern people. ing.

Obesity is a condition in which fat is accumulated excessively in the subcutaneous and internal organs, and is said to be a risk factor for lifestyle-related diseases such as hypertension. Diabetes is a disease with symptoms such as persistent hyperglycemia and sugar excretion into the urine. Diabetes patients are said to have more than twice the mortality rate compared to healthy individuals, and diabetes is one of the lifestyle-related diseases that is widespread in modern society. In order to prevent or ameliorate such obesity and diabetes, efforts are being made to improve life through dietary restrictions and dieting. In particular, in 2008, the Ministry of Health, Labor and Welfare started making metabolic screening mandatory in Japan as part of these lifestyle-related disease countermeasures. Metabolic health checkup (specific health checkup and specific health guidance) is a medical checkup that focuses on metabolic syndrome (visceral fat syndrome). This is a new medical checkup aimed at reducing medical costs, which are increasing, and socially, obesity prevention has become a serious problem.

On the other hand, with the development of genetic and physiological research in recent years, not only acquired factors such as excessive intake of nutrients but also innate factors such as genetic polymorphisms of individual genes are the cause of obesity and diabetes. I have found out that it is influencing. Here, the gene polymorphism indicates an individual difference in the DNA base sequence constituting the gene. Due to this difference in DNA base sequence, differences in constitution and body type occur among individuals. For example, for obesity, 30 to 70% of the cause is said to be due to genetic factors. In recent years, many genes that cause obesity and diabetes have been discovered by a large-scale genome project and the like, and detailed studies thereof have been made.

Therefore, by analyzing the user's genetic polymorphism and suggesting a lifestyle improvement program such as dieting based on this genetic polymorphism information, personalized health support information that matches the individual personal constitution is provided It becomes possible to do. As a result, it is possible to improve the constitution more efficiently as compared with the case where dieting or the like is simply performed. Specifically, information on gene polymorphisms of the obesity-related gene β3 adrenergic receptor gene (hereinafter referred to as β3AR gene), uncoupling protein gene (hereinafter referred to as UCP1 gene), β2 adrenergic receptor gene (hereinafter referred to as β2AR gene). If a person is examined by a DNA test or the like and life guidance is given together with life information such as exercise that a user performs everyday, the behavior changes because obesity is thin, and the effect is improved.

However, in the conventional method, determining the gene polymorphisms of β3AR gene, UCP1 gene, and β2AR gene requires a complicated detection operation from clinical samples, and costs including time and labor costs are required. It is in a situation where inspection is not possible unless it is a specific institution. For example, as a conventional method for diagnosing gene polymorphisms of β3AR gene, UCP1 gene, and β2AR gene, PCR-RFLP method and PCR-sequence method are generally used. Specifically, the PCR-RFLP method involves purifying genomic DNA from clinical samples such as blood, amplifying the target region by the PCR method, and then polymorphic portions of β3AR gene, UCP1 gene, and β2AR gene. Treat with the recognizing restriction enzyme and confirm the product by agarose electrophoresis.
PCR amplification reactions and agarose electrophoresis take a long time, and if the number of samples increases, complicated gel electrophoresis is performed, which requires a great deal of labor and time, and is an obstacle to efficient detection. In the sample processing, since the operation is complicated, there is a high possibility of misdiagnosis due to a sample mistake. Also, complex temperature control is indispensable and dedicated reactors must be used and can only be implemented in a well-equipped laboratory. The same can be said for the PCR-sequencing method.

As one method for solving these problems, the present inventors have developed a Smart Amplification Process method (Nature Methods. 2007 Mar; 4 (3): 257-62.) Which is an isothermal amplification method. The isothermal amplification method will be described by taking a method using an asymmetric primer set and a method using a symmetric primer set as examples. The asymmetric primer set is, for example, an asymmetric primer set (hereinafter referred to as “asymmetric primer set”) in which a pair of primers have different forms of one primer and the other.
The symmetric primer set is, for example, a symmetric primer set (hereinafter referred to as “symmetric primer set”) in which one pair has the same form as the other primer. The asymmetric primer set is suitable for the Smart Amplification Process method, for example, and the symmetric primer set is a Nucleic Acid Res. By LAMP method (Loop medicated Amplification). , 2000, Vol. 28, no. 12, e63.

The present invention relates to a method for simply and rapidly typing gene polymorphisms of β3AR gene, UCP1 gene, and β2AR gene.

As a result of intensive studies to solve the above problems, the present inventor selectively hybridizes to the peripheral sequence of the gene polymorphism in order to type the gene polymorphism of the β3AR gene, UCP1 gene, and β2AR gene. The present invention was completed by finding that a polymorphism was specifically detected from a sample by preparing an oligonucleotide and amplifying the oligonucleotide by using the Smart Amplification Process method or LAMP method as a primer or a competitive probe.

The primer of the present invention comprises
Primers used for typing polymorphisms of obesity-related genes by nucleic acid amplification,
The obesity-related gene is at least one gene selected from the group consisting of a β3AR gene, a UCP1 gene, and a β2AR gene, a sequence from numbers 3215 to 3325 in the base sequence of the β3AR gene, and −3888 in a base sequence of the UCP1 gene It is a primer for amplifying a target region nucleic acid sequence selected from the sequence No. -3739, the sequence Nos. 216 to 338 in the base sequence of the β2AR gene, and their complementary sequences.

同時に二種類以上の遺伝子について増幅やタイピングを行う場合は、これらβ３ＡＲ用プライマー、ＵＣＰ遺伝子用プライマーおよびβ２ＡＲ用プライマーのうち、いずれか二種類を併用してもよいし、三種類全てを併用してもよい。また、各遺伝子を増幅するための本発明のプライマーとしては、例えば、一種類でもよいし、二種類以上を併用してもよい。The primer is
Preferably, at least one base sequence selected from the group consisting of the base sequences represented by SEQ ID NOs: 1 to 27 and at least one base sequence complementary thereto, preferably at least a continuous 7-15 base sequence, more preferably The primer according to claim 1, which has a continuous base sequence of 10 to 15 bases, more preferably a continuous base sequence of 15 bases.

When two or more genes are amplified or typed at the same time, any two of these β3AR primer, UCP gene primer and β2AR primer may be used in combination, or all three types may be used in combination. Also good. Moreover, as a primer of this invention for amplifying each gene, 1 type may be sufficient, for example, and 2 or more types may be used together.

The primer is
Oligonucleotide consisting of a base sequence complementary to at least one selected from the group consisting of base sequences represented by SEQ ID NOs: 2 to 4-Oligonucleotide consisting of any base sequence having 0 to 30 bases-SEQ ID NO: A primer capable of amplifying a β3AR gene comprising an oligonucleotide consisting of at least one base sequence selected from the group consisting of base sequences represented by 3 to 5;
Oligonucleotide consisting of a base sequence complementary to at least one selected from the group consisting of base sequences represented by SEQ ID NOs: 11 to 13-Oligonucleotide consisting of any base sequence having 0 to 30 bases-SEQ ID NO: A primer capable of amplifying a UCP1 gene composed of an oligonucleotide consisting of at least one base sequence selected from the group consisting of base sequences represented by 12 to 14, or
Oligonucleotide consisting of a base sequence complementary to at least one selected from the group consisting of base sequences represented by SEQ ID NOs: 20 to 22-Oligonucleotide consisting of any base sequence having 0 to 30 bases-SEQ ID NO: It is preferably a primer capable of amplifying a B2AR gene composed of at least one oligonucleotide selected from the group consisting of base sequences represented by 21 to 23.
In the present invention, the primer capable of amplifying the β3AR gene, the primer capable of amplifying the UCP1 gene, and the primer capable of amplifying the B2AR gene are, for example, primers composed of the above-mentioned three types (one of which is arbitrary), for example. It may be a primer including the three types of oligonucleotides. Each of the primers preferably has, for example, the three types of oligonucleotides arranged in this order. In each of the primers, the three kinds of oligonucleotides may or may not have a base sequence further interposed between the oligonucleotides, for example.

The primer according to claims 1 to 3, wherein the nucleic acid amplification method is a Smart Amplification Process method or a LAMP method.

The competitive probe of the present invention is
A competitive probe used for typing polymorphisms of obesity-related genes by nucleic acid amplification,
The obesity-related gene is at least one gene selected from the group consisting of a β3AR gene, a UCP1 gene, and a β2AR gene, a sequence from numbers 3215 to 3325 in the base sequence of the β3AR gene, and −3888 in a base sequence of the UCP1 gene Sequence from number -3739,
Competing with primers for typing gene polymorphisms in the target region nucleic acid sequence selected from the sequences 216 to 338 in the base sequence of the β2AR gene and their complementary sequences, and included to suppress non-specific amplification It is a competitive probe.

At least one base sequence selected from the group consisting of the base sequences represented by SEQ ID NOs: 28 to 33 and a base sequence complementary thereto, which is modified and designed so that the competing probe does not undergo an extension reaction from the 3 ′ end. It is preferable that it is a competitive probe of Claim 5 which has a base sequence of at least 10 bases in at least one of these.

The competitive probe according to claim 6, wherein the modification is amination, dideoxylation or phosphorylation of the 3′-terminal hydroxyl group.

Preferably, the nucleic acid amplification method is a competitive probe from claim 5 which is a Smart Amplification Process method or a LAMP method.

The amplification method of the present invention is an amplification method of obesity-related genes,
The obesity-related gene is at least one gene selected from the group consisting of a β3AR gene, a UCP1 gene, and a β2AR gene, and the amplification method is a Smart Amplification Process method or a LAMP method, and any one of claims 1 to 4 It is characterized by using at least one of the primer according to claim 1 and the competitive probe according to any one of claims 5 to 8.

The typing method of the present invention comprises:
A method of typing a polymorphism of an obesity-related gene by nucleic acid amplification, wherein the obesity-related gene is at least one gene selected from the group consisting of β3AR gene, UCP1 gene and β2AR gene, and the amplification method comprises: It is a Smart Amplification Process method or a LAMP method, characterized by using at least one of the primer according to any one of claims 1 to 4 and the competitive probe according to any one of claims 5 to 8. To do.

The determination method of the present invention is a method for determining an obesity constitution by amplification of an obesity-related gene, wherein the obesity-related gene is at least one gene selected from the group consisting of a β3AR gene, a UCP1 gene, and a β2AR gene. A step of amplifying a gene by the method for amplifying an obesity-related gene according to claim 9 and a step of determining an obesity constitution by detecting the amplification are characterized.

The kit of the present invention comprises
A kit used for typing a polymorphism of an obesity-related gene by nucleic acid amplification, wherein the obesity-related gene is at least one gene selected from the group consisting of a β3AR gene, a UCP1 gene, and a β2AR gene, It contains at least one of the primer according to any one of 1 to 4 and the competitive probe according to any one of claims 5 to 8.

Furthermore, it is preferable that it is a kit of Claim 12 containing a strand displacement type | mold nucleic acid synthetase and a substrate.

The device of the present invention
The analysis for performing the typing method according to claim 10, wherein at least one of the primer according to any one of claims 1 to 4 and the competitive probe according to any one of claims 5 to 8 is used. An apparatus comprising: a reaction unit that performs an amplification reaction; a temperature control unit that controls a temperature of the reaction unit; and a detection unit that detects the reaction.

Samples for typing polymorphisms of β3AR gene, UCP1 gene, and β2AR gene by nucleic acid amplification include clinical test materials such as blood, feces, vomit, urine, tissue, hair, nails, oral mucosal cells, and wiped samples But it ’s okay. In order to use these specimens as samples for the Smart Amplification Process method, operations such as concentration and separation of genomic DNA present in the specimen and release or concentration of nucleic acids from clinical test materials can be performed as pretreatment. As the method, an enzyme, a surfactant, an alkali, a heat treatment or the like is known, and can be appropriately selected from the methods.

In the present invention, the target sequence means a base sequence of a polynucleotide to be amplified. In general, the base sequence of a polynucleotide describes the base sequence of the sense strand from the 5 ′ side to the 3 ′ side.
In the present invention, the continuous synthesis of new target base sequences is called amplification. Furthermore, in the present invention, providing a starting point for complementary strand synthesis means that the 3 ′ end of a polynucleotide that functions as a primer necessary for complementary strand synthesis is hybridized to a template polynucleotide. In the present invention, the 3 ′ end or the 5 ′ end means not only one base at any end but also a region including one base at the end and located at the end. In the present invention, a primer set refers to a combination of primers used simultaneously.

  Among the nucleic acid amplification methods, the Smart Amplification Process method, for example, can amplify a target nucleic acid sequence with excellent specificity. For example, by nucleic acid amplification, for example, mutation in a gene, that is, presence or absence of base deletion, substitution, or insertion can be detected. In particular, it is possible to determine single nucleotide polymorphism (presence or absence of single nucleotide mutation) and the like.

  As described above, the asymmetric primer set is a primer set having a pair of asymmetric primers in which the form of one primer and the form of the other primer are different, and among them, the primer set is applied to the Smart Amplification Process method. It is preferable. Hereinafter, this primer set is also referred to as “a primer set for Smart Amplification Process”.

  As a specific example of the primer set for the Smart Amplification Process, for example, a pair of asymmetric primers includes a first primer and a second primer, and the first primer is 3 ′ of the target nucleic acid sequence. A sequence (B ′) comprising a sequence (Ac ′) hybridizing to the sequence (A) of the terminal portion at the 3 ′ terminal portion and existing 5 ′ to the sequence (A) in the target nucleic acid sequence A sequence (B ′) that hybridizes to the complementary sequence (Bc) is included on the 5 ′ side of the sequence (Ac ′), and the second primer is a 3 ′ end portion of the complementary sequence of the target nucleic acid sequence A fold containing a sequence (Cc ′) that hybridizes to the sequence (C) in the 3 ′ end portion and two nucleic acid sequences that hybridize to each other on the same strand Sequence (D-Dc ') on the 5' side of the sequence (Cc ').

  The action mechanism of nucleic acid synthesis by the first primer is schematically shown in FIG. First, a target nucleic acid sequence in a nucleic acid to be a template is determined, and a sequence (A) at the 3 ′ end portion of the target nucleic acid sequence and a sequence (B) existing on the 5 ′ side of the sequence (A) are determined. . The first primer includes a sequence (Ac ′) and further includes a sequence (B ′) on the 5 ′ side thereof. The sequence (Ac ′) hybridizes to the sequence (A), and the sequence (B ′) hybridizes to the complementary sequence (Bc) of the sequence (B). Here, the first primer may include an intervening sequence that does not affect the reaction between the sequence (Ac ′) and the sequence (B ′). When such a primer is annealed to the template nucleic acid, the sequence (Ac ′) in the primer is hybridized to the sequence (A) of the target nucleic acid sequence (FIG. 22 (a)). When a primer extension reaction occurs in this state, a nucleic acid containing a complementary sequence of the target nucleic acid sequence is synthesized. Then, the sequence (B ′) present on the 5 ′ end side of the synthesized nucleic acid hybridizes to the sequence (Bc) present in the nucleic acid, and thereby the stem in the 5 ′ end portion of the synthesized nucleic acid. -A loop structure is formed. As a result, the sequence (A) on the template nucleic acid becomes a single strand, and another primer having the same sequence as the first primer hybridizes to this portion (FIG. 22 (b)). Thereafter, an extension reaction from the newly hybridized first primer occurs by the strand displacement reaction, and at the same time, the previously synthesized nucleic acid is separated from the template nucleic acid (FIG. 22 (c)).

  In the above mechanism of action, the phenomenon in which the sequence (B ′) hybridizes to the sequence (Bc) typically occurs due to the presence of complementary regions on the same strand. In general, when a double-stranded nucleic acid is dissociated into a single strand, partial dissociation starts from its terminal or other relatively unstable part. In the double-stranded nucleic acid generated by the extension reaction using the first primer, the base pair of the terminal portion is in an equilibrium state of dissociation and binding at a relatively high temperature, and the double strand is maintained as a whole. In such a state, when a sequence complementary to the dissociated portion of the terminal is present on the same strand, a stem-loop structure can be formed as a metastable state. Although this stem-loop structure does not exist stably, the same other primer binds to the complementary strand part (sequence (A) on the template nucleic acid) exposed by the formation of the structure, and the polymerase immediately extends. By performing the above, a previously synthesized strand is replaced and released, and at the same time, a new double-stranded nucleic acid can be generated.

  The design criteria for the first primer in a preferred embodiment of the present invention are as follows. First, in order for a new primer to efficiently anneal to the template nucleic acid after the complementary strand of the template nucleic acid is synthesized by extension of the primer, the template is formed by forming a stem-loop structure at the 5 ′ end of the synthesized complementary strand. The part of the sequence (A) on the nucleic acid needs to be a single strand. For that purpose, the difference (X−Y) between the base number X of the sequence (Ac) and the base number Y of the region sandwiched between the sequence (A) and the sequence (B) in the target nucleic acid sequence relative to X The ratio (XY) / X is important. However, it is not necessary to make a single strand up to the portion which is present 5 ′ from the sequence (A) on the template nucleic acid and is not related to primer hybridization. In addition, in order for a new primer to efficiently anneal to a template nucleic acid, it is also necessary to efficiently perform the above-described stem-loop structure formation. For efficient stem-loop structure formation, ie, efficient hybridization between the sequence (B ′) and the sequence (Bc), the distance between the sequence (B ′) and the sequence (Bc) (X + Y) is important. In general, the optimum temperature for the primer extension reaction is at most around 72 ° C., and at such a low temperature, it is difficult for the extended strand to dissociate over a long region. Therefore, in order for the sequence (B ′) to efficiently hybridize to the sequence (Bc), it is considered preferable that the number of bases between both sequences is small. On the other hand, in order for the sequence (B ′) to hybridize to the sequence (Bc) and to make the portion of the sequence (A) on the template nucleic acid a single strand, the sequence (B ′) has a larger number of bases. Is considered preferable.

  From the above viewpoints, the first primer according to a preferred embodiment of the present invention is the (XY) when no intervening sequence exists between the sequence (Ac) and the sequence (B ′) constituting the primer. ) / X is, for example, -1.00 or more, preferably 0.00 or more, more preferably 0.05 or more, more preferably 0.10 or more, and for example, 1.00 or less, preferably 0.00. It is designed to be 75 or less, more preferably 0.50 or less, and even more preferably 0.25 or less. Further, (X + Y) is preferably 15 or more, more preferably 20 or more, more preferably 30 or more, and preferably 50 or less, more preferably 48 or less, and further preferably 42 or less.

Further, there may be an intervening sequence (the number of bases is Y ′) between the sequence (Ac) and the sequence (B ′) constituting the primer, and 0 between the sequence (Ac) and the sequence (B ′). To any oligonucleotide consisting of 30 bases. Furthermore, the primer may be labeled especially for detection. In the case where an intervening sequence (the number of bases is Y ′) exists between the sequence (Ac) and the sequence (B ′) constituting the primer, the first primer according to a preferred embodiment of the present invention is represented by {X− (YY ')} / X is, for example, -1.00 or more, preferably 0.00 or more, more preferably 0.05 or more, more preferably 0.10 or more, and for example, 1.00 In the following, it is designed to be preferably 0.75 or less, more preferably 0.50 or less, and further preferably 0.25 or less. Further, (X + Y + Y ′) is preferably 15 or more, more preferably 20 or more, more preferably 30 or more, and is preferably 100 or less, more preferably 75 or less, and even more preferably 50 or less.

  The first primer has a chain length that allows base pairing with a target nucleic acid while maintaining the required specificity under given conditions. The primer has a chain length of preferably 15 to 100 nucleotides, more preferably 20 to 60 nucleotides. Moreover, the lengths of the sequence (Ac) and the sequence (B ′) constituting the first primer are preferably 5 to 50 nucleotides, more preferably 7 to 30 nucleotides, respectively. If necessary, an arbitrary oligonucleotide consisting of 0 to 30 bases, which is an intervening sequence that does not affect the reaction, may be inserted between the sequence (Ac) and the sequence (B ′).

  As described above, the second primer included in the primer set according to the present invention is the 3 ′ end portion of the complementary sequence of the target nucleic acid sequence (the strand opposite to the strand to which the first primer hybridizes). A folded sequence (D-Dc ′) containing a sequence (Cc ′) that hybridizes to the sequence (C) at the 3 ′ end portion and two nucleic acid sequences that hybridize to each other on the same strand is defined as the sequence (Cc It is included on the 5 'side of'). The structure of such a second primer is, for example, as shown in FIG. 23, but is not limited to the sequence or the number of nucleotides shown in FIG. The length of the sequence (Cc ′) constituting the second primer is preferably 5 to 50 nucleotides, more preferably 10 to 30 nucleotides. The length of the folded sequence (D-Dc ′) is preferably 2 to 1000 nucleotides, more preferably 2 to 100 nucleotides, still more preferably 4 to 60 nucleotides, and even more preferably 6 to 40 nucleotides. The number of nucleotides of base pairs formed by hybridization within the sequence is preferably 2 to 500 bp, more preferably 2 to 50 bp, still more preferably 2 to 30 bp, and further preferably 3 to 20 bp. The nucleotide sequence of the folded sequence (D-Dc ′) may be any sequence and is not particularly limited, but is preferably a sequence that does not hybridize to the target nucleic acid sequence. If necessary, an intervening sequence that does not affect the reaction may be inserted between the sequence (Cc ′) and the folded sequence (D-Dc ′).

A possible mechanism of action for the nucleic acid amplification reaction by the first primer and the second primer will be described with reference to FIGS. 24 and 25. FIG. In FIG. 24 and FIG. 25, the two hybridizing sequences are complementary to each other for the sake of simplicity, but the present invention is not limited to this. First, the first primer hybridizes to the sense strand of the target nucleic acid, and an extension reaction of the primer occurs (FIG. 24 (a)). Next, a stem-loop structure is formed on the extended strand (−), whereby a new first primer hybridizes to the sequence (A) on the target nucleic acid sense strand that has become a single strand (FIG. 24 ( b)), the extension reaction of the primer occurs, and the previously synthesized extended strand (-) is desorbed. Next, the second primer hybridizes to the sequence (C) on the released extended strand (−) (FIG. 24 (c)), the extension reaction of the primer occurs, and the extended strand (+) is synthesized. (FIG. 24D). A stem-loop structure is formed at the 3 ′ end of the generated extended strand (+) and the 5 ′ end of the extended strand (−) (FIG. 24 (e)), and the extended strand (+) which is the free 3 ′ end. At the same time, an extension reaction takes place from the tip of the loop, and the extension chain (-) is detached (FIG. 24F). By the extension reaction from the loop tip, a hairpin-type double-stranded nucleic acid in which the extension strand (−) is bound to the 3 ′ side of the extension strand (+) via the sequence (A) and the sequence (Bc) is generated, The first primer hybridizes to the sequence (A) and sequence (Bc) (FIG. 24 (g)), and an extended strand (−) is generated by the extension reaction (FIG. 24 (h) and FIG. 25 (i). )).
In addition, a free 3 ′ end is provided by the folded sequence present at the 3 ′ end of the hairpin double-stranded nucleic acid (FIG. 24 (h)), and by an extension reaction therefrom (FIG. 25 (i)), A single-stranded nucleic acid having folded sequences at both ends and alternately containing extended strands (+) and extended strands (−) via the sequences derived from the first and second primers is generated (FIG. 25 (j )). In this single-stranded nucleic acid, since the free 3 ′ end (starting point of complementary strand synthesis) is provided by the folded sequence present at the 3 ′ end (FIG. 25 (k)), the same extension reaction is repeated, The chain length is doubled per extension reaction (FIGS. 25 (l) and (m)). In addition, in the extended strand (-) from the first primer desorbed in FIG. 25 (i), the free 3 'end (the complementary strand synthesis origin) is provided by the folded sequence present at the 3' end. Therefore (FIG. 25 (n)), stem-loop structures are formed at both ends by the extension reaction therefrom, and the extension strand (+) and the extension strand (-) are alternately included via the sequence derived from the primer. Single-stranded nucleic acid is generated (FIG. 25 (o)). Also in this single-stranded nucleic acid, since the complementary strand synthesis starting point is sequentially provided by the loop formation at the 3 ′ end, the extension reaction occurs successively. The single-stranded nucleic acid thus automatically extended contains sequences derived from the first primer and the second primer between the extended strand (+) and the extended strand (−). Therefore, it is possible for each primer to hybridize to cause an extension reaction, and thereby the sense strand and the antisense strand of the target nucleic acid are significantly amplified.

  In addition, the primer set for the Smart Amplification Process may include a third primer in addition to the first primer and the second primer. The third primer is, for example, a primer that hybridizes to the target nucleic acid sequence or its complementary sequence and does not compete with other primers for hybridization to the target nucleic acid sequence or its complementary sequence. In the present invention, “non-competing” means that the primer does not interfere with the provision of the complementary strand synthesis starting point by hybridizing to the target nucleic acid.

  When the target nucleic acid sequence is amplified by the first primer and the second primer, as described above, the amplification product has the target nucleic acid sequence and its complementary sequence alternately. A folded sequence or a loop structure exists at the 3 'end of the amplified product, and extension reactions occur one after another from the complementary strand synthesis starting point provided thereby. The third primer is preferably a primer that can be annealed to the target sequence present in the single-stranded portion when such an amplification product is partially in a single-stranded state. As a result, a new complementary strand synthesis starting point is provided in the target nucleic acid sequence in the amplification product, and an extension reaction takes place therefrom, so that the nucleic acid amplification reaction is performed more rapidly.

  The third primer is not limited and may be one type. For example, two or more types of the third primer may be used simultaneously in order to improve the speed and specificity of the nucleic acid amplification reaction. These third primers are typically composed of different sequences from the first primer and the second primer, but hybridize to partially overlapping regions as long as they do not compete with these primers. It is good. The chain length of the third primer is preferably 2 to 100 nucleotides, more preferably 5 to 50 nucleotides, and even more preferably 7 to 30 nucleotides.

  The third primer mainly has an auxiliary function for proceeding more quickly with the nucleic acid amplification reaction by the first primer and the second primer, for example. Therefore, the third primer preferably has a Tm lower than the Tm of each 3 'end of the first primer and the second primer. Further, the amount of the third primer added to the amplification reaction solution is preferably smaller than, for example, the amount of each of the first primer and the second primer.

  As the third primer, for example, a primer having a structure capable of forming a loop as described in International Publication No. 02/24902 pamphlet, which gives a starting point for synthesis of complementary strands to the loop portion is used. However, the present invention is not limited to this. That is, for example, a complementary strand synthesis starting point may be added to any site within the target nucleic acid sequence.

  In the primer set for the Smart Amplification Process, for example, one of the first primer and the second primer, or both the primers are labeled nucleic acids labeled with, for example, a fluorescent dye. Alternatively, the third primer may be the labeled nucleic acid. In addition, either one or both of the first primer and the second primer and the third primer may be the labeled nucleic acid.

  Moreover, when this Smart Amplification Process method is applied to, for example, a mutation determination method, it is preferable to design the primer for the Smart Amplification Process as follows. That is, the primer set for the Smart Amplification Process includes a nucleic acid sequence having a mutation at a detection site (hereinafter referred to as “mutant nucleic acid sequence”) or a nucleic acid sequence having no mutation at the detection site (hereinafter referred to as “wild-type nucleic acid sequence”). The target nucleic acid sequence is a target nucleic acid sequence, and the target site causing the target mutation is included in the sequence (A), the sequence (B) or the sequence (C), or the sequence (A) and the sequence (B) It is preferable to design the primer set so as to be arranged between or between sequence (A) and sequence (C).

  When a primer set designed with a target nucleic acid sequence having a mutation at the target site (mutant nucleic acid sequence) as the target nucleic acid sequence is used as the primer set, for example, the presence of the amplification product after the nucleic acid amplification reaction indicates that the mutant nucleic acid The presence of the sequence is indicated and the absence or reduction of the amplification product indicates the absence of the mutated nucleic acid sequence. On the other hand, when using a primer set designed with a target nucleic acid sequence (wild-type base sequence) having no mutation at the target site of interest as the target nucleic acid sequence, for example, the presence of the amplification product after the nucleic acid amplification reaction is The absence of the sequence indicates the absence or reduction of the amplification product, indicating the presence of the mutant base sequence. Here, “decrease in amplification product” means that the amount of amplification product obtained is reduced compared to the amount of amplification product obtained when the target nucleic acid sequence is present in the test nucleic acid in the nucleic acid sample. Means that

  As the primer set, for example, a primer set designed so that the target site of interest is included in the sequence (A) is preferable. With such a primer set, for example, when the target nucleic acid sequence (for example, wild-type base sequence) is included in the test nucleic acid in the nucleic acid sample, the first primer anneals to the sequence (A) in the nucleic acid amplification reaction. As a result, an amplification product is obtained. On the other hand, when the test nucleic acid in the nucleic acid sample contains a nucleic acid sequence (for example, a mutant base sequence) different from the target nucleic acid sequence, the first primer anneals to the sequence (A) in the nucleic acid amplification reaction. Because of difficulty, no amplification product is obtained, or the amount of amplification product obtained is significantly reduced. The sequence (Ac) contained in the first primer is preferably a sequence complementary to the sequence (A).

  Moreover, as the primer set, for example, a primer set designed so that the target site of interest is included in the sequence (C) is preferable. With such a primer set, for example, when the target nucleic acid sequence (for example, a wild-type base sequence) is contained in the test nucleic acid in the nucleic acid sample, the second primer is the sequence (C) in the nucleic acid amplification reaction. An amplification product is obtained. On the other hand, when the test nucleic acid in the nucleic acid sample contains a nucleic acid sequence (for example, a mutant base sequence) different from the target nucleic acid sequence, the second primer anneals to the sequence (C) in the nucleic acid amplification reaction. Because of difficulty, no amplification product is obtained or the amount of amplification product obtained is significantly reduced. The sequence (Cc ′) contained in the second primer is preferably a sequence complementary to the sequence (C).

  The primer is preferably a primer set designed so that the target site of interest is included in the sequence (B), for example. With such a primer set, for example, when the target nucleic acid sequence (for example, wild type base sequence) is contained in the test nucleic acid in the nucleic acid sample, the first primer is the sequence (A ) And the extension reaction is performed, and then the sequence (B ′) contained in the primer hybridizes to the sequence (Bc) on the extended strand. For this reason, a stem-loop structure is formed efficiently. By forming this efficient stem-loop structure, the other first primer can be annealed to the template, and the action mechanism shown in FIG. 22 proceeds efficiently, so that an amplification product is obtained. It is done. On the other hand, when the test nucleic acid in the nucleic acid sample contains a nucleic acid sequence (for example, a mutant base sequence) different from the target nucleic acid sequence, it is difficult to form the stem-loop structure in the nucleic acid amplification reaction. The action mechanism shown in FIG. 22 is hindered, and no amplification product is obtained, or the amount of amplification product obtained is significantly reduced. The sequence (B ′) contained in the first primer is preferably the same sequence as the sequence (B).

  Moreover, as the primer set, for example, a primer set designed so that the target site of interest is disposed between the sequence (A) and the sequence (B) is preferable. According to such a primer set, when the target nucleic acid sequence (for example, wild-type base sequence) is contained in the test nucleic acid in the nucleic acid sample, the first primer is added to the sequence (A) in the nucleic acid amplification reaction. After the annealing and extension reaction, the sequence (B ′) contained in the primer hybridizes to the sequence (Bc) on the extended strand, so that a stem-loop structure is efficiently formed. The formation of this efficient stem-loop structure allows the other first primer to anneal to the template, and the mechanism of action shown in FIG. 22 proceeds efficiently, resulting in an amplification product. . On the other hand, when the test nucleic acid in the nucleic acid sample contains a nucleic acid sequence different from the target nucleic acid sequence (for example, a mutant base sequence), the sequence (B ′) contained in the first primer and the extended strand Since the appropriate distance from the sequence (Bc) is not maintained, it is difficult to form the stem-loop structure in the nucleic acid amplification reaction. This is the case when there is an insertion or deletion of a long sequence between the sequence (A) and the sequence (B). Therefore, in this case, the mechanism of action shown in FIG. 22 is hindered, and no amplification product is obtained or the amount of amplification product obtained is significantly reduced.

  Further, the primer set is preferably a primer set designed so that the target site of interest is disposed between the sequence (A) and the sequence (C). According to such a primer set, when the target nucleic acid sequence is contained in the test nucleic acid in the nucleic acid sample (for example, wild type base sequence), the first primer is converted into the sequence (A) in the nucleic acid amplification reaction. After the annealing and extension reaction, the sequence (B ′) contained in the primer hybridizes to the sequence (Bc) on the extended strand, so that a stem-loop structure is efficiently formed. The formation of this efficient stem-loop structure allows the other first primer to anneal to the template, and the mechanism of action shown in FIGS. 22, 24, and 25 proceeds efficiently. An amplification product is obtained. On the other hand, when the test nucleic acid in the nucleic acid sample contains a nucleic acid sequence different from the target nucleic acid sequence (for example, a mutant base sequence), no amplification product is obtained or the amount of amplification product obtained is Remarkably reduced. For example, when the nucleic acid sample contains a nucleic acid sequence different from the target nucleic acid sequence due to insertion of a long sequence between the sequence (A) and the sequence (C), the speed (efficiency) of nucleic acid amplification is significantly reduced. Therefore, no amplification product is obtained or the amount of amplification product obtained is significantly reduced. Further, due to the deletion of the sequence between the sequence (A) and the sequence (C), a nucleic acid sequence different from the target nucleic acid sequence is contained in the nucleic acid sample, and the deletion of the sequence (B) When part or all is lost, since the sequence (B ′) contained in the first primer cannot hybridize on the extended strand, it becomes impossible or difficult to form a stem-loop structure. . For this reason, the mechanism of action shown in FIG. 22, FIG. 24, and FIG. 25 is hindered, and no amplification product is obtained, or the amount of amplification product obtained is significantly reduced. Furthermore, a nucleic acid sequence different from the target nucleic acid sequence is contained in the nucleic acid sample due to the deletion of the sequence between the sequence (A) and the sequence (C), and the portion of the sequence (B) due to this deletion Even if there is no spontaneous deletion, the speed (efficiency) of nucleic acid amplification is reduced, so that no amplification product is obtained or the amount of amplification product obtained is significantly reduced.

Competing probes are also called competing oligonucleotides and are described in Japanese Patent Application No. 2006-357509 and Biologicals. 2008 Jul; 36 (4): 234-8. In the method for detecting a gene using the isothermal amplification method, an oligonucleotide that can specifically anneal to a non-target base sequence is used. A method for detecting a gene, which comprises preventing complementary strand synthesis.

The effect of preventing complementary strand synthesis in the non-target base sequence can be obtained by using the oligonucleotide that can specifically anneal to the non-target base sequence by the competitive probe or the competitive oligonucleotide. The competitive probe method can be applied to a wide range of amplification and detection fields from infectious diseases to SNP and mutation.

The oligonucleotide of a competitive probe or a competitive oligonucleotide prevents complementary strand synthesis from the non-target base sequence by using an oligonucleotide that can specifically anneal to the non-target base sequence. Further, in the gene mutation detection method, complementary strand synthesis from the non-target base sequence is prevented by using an oligonucleotide that can specifically anneal to the mutation site in the non-target base sequence. More specifically, by using an oligonucleotide that can specifically anneal to a mutation site in a non-target base sequence (competitive oligonucleotide), the 3 ′ end of a primer or amplification product that can anneal to the target base sequence is non-targeted. Prevents annealing to the base sequence. These oligonucleotides may contain a sequence complementary to a 5 to 100 base long region including a specific site on the non-target base sequence, particularly a 10 to 30 base long region, for specific annealing. preferable.

The oligonucleotide is designed such that no extension reaction occurs from the 3 'end. More specifically, the oligonucleotide is modified so that extension reaction does not occur from its 3 ′ end, or is non-homologous to the target base sequence so that extension reaction does not occur from the 3 ′ end of the oligonucleotide. It is a thing with. The site of the modified base is not particularly limited, but is preferably a 3 'terminal base. The method for modifying the base is not particularly limited, and is preferably amination, dideoxylation or phosphorylation, but is not limited thereto. For example, the base may be modified by biotinylation, fluorescent dye, phosphorylation, thiolation, amination, phosphorothioate, halogenation, or the like.

When modifying the 3 'end of the oligonucleotide, the mutation site is preferably present near the center of the competing oligonucleotide, but is not limited thereto.

Competing probes or competing oligonucleotides reliably prevent complementary strand synthesis from non-target nucleotide sequences even in various nucleic acid sequences in gene mutation detection methods using isothermal amplification. More specifically, an oligonucleotide that can specifically anneal to a non-target base sequence is used to prevent complementary strand synthesis in the non-target base sequence. More specifically, by using a competitive oligonucleotide that can specifically anneal to a non-target base sequence, a primer that is not specific to the non-target base sequence is annealed to prevent non-specific complementary strand synthesis.

In the present invention, a competitive oligonucleotide composed of peptide nucleic acids can use peptide nucleic acid (PNA) as all or part of competing oligonucleotides. PNA has a structure in which the deoxyribose backbone of an oligonucleotide is replaced with a peptide backbone. Examples of the peptide main chain include repeating units of N- (2-aminoethyl) glycine linked by an amide bond. Bases to be bound to the peptide backbone of PNA include naturally occurring bases such as thymine, cytosine, adenine, guanine, inosine, uracil, 5-methylcytosine, thiouracil and 2,6-diaminopurine, bromothymine, azaadenine and azaguanine Examples thereof include, but are not limited to, artificial bases such as

The PNA can be reacted according to a known general peptide synthesis method after producing a monomer having each nucleobase bonded thereto by a known production method. According to the solid phase synthesis method, an automatic synthesizer may be used to synthesize the desired base sequence. In the above method, commercially available reagents and raw materials can be used. Commercially available reagents are appropriately derivatized according to conventional methods, for example, N-terminal, C-terminal, and side-chain functional groups that are not involved in the reaction of the starting amino acid. Etc. can be used after carrying out a protective reaction. The obtained nucleobase-bound PNA can be easily isolated and purified by ordinary separation means such as high performance liquid chromatography, electrophoresis chromatography, solvent extraction, salting out and the like.

Since PNA has a stronger binding force to nucleic acids than ordinary oligonucleotides, amplification of mutation sites can be more reliably suppressed depending on the design conditions of the oligos. When PNA is used as a competing oligonucleotide, the sequence complementary to the mutation site on the non-target nucleotide sequence may be its 3 'end, 5' end or near the center. The length of the competing oligonucleotide composed of PNA is preferably 5 to 50 bases, more preferably 7 to 25 bases, but is not limited thereto.

LNA is a nucleic acid having two circular structures in which the 2'-position oxygen atom and the 4'-position carbon atom of ribose are linked by a methylene bridge in the sugar-phosphate skeleton. When an oligo containing LNA anneals to DNA, the double-stranded conformation changes and the thermal stability increases. Since LNA has a stronger binding force to nucleic acids than ordinary oligonucleotides, depending on the design conditions of the oligo, it is possible to block mutation sites more reliably. When LNA is used as a competing oligonucleotide, the sequence complementary to the mutation site on the non-target base sequence may be its 3 'end, 5' end or near the center. Further, the length of the block oligo composed of LNA is preferably 5 to 50 bases, more preferably 7 to 25 bases, but is not limited thereto. Competitive oligonucleotides of the present invention include oligonucleotides synthesized only with LNA, including oligonucleotides containing LNA in part, and oligonucleotides that combine multiple combinations of LNA, PNA, DNA, RNA, and other artificial nucleic acids. It can be used as

According to a preferred embodiment of the competing probe or competing oligonucleotide, the nucleic acid amplification reaction in the genotyping of the gene polymorphism according to the present invention is carried out in the presence of the competing oligonucleotide or mismatch recognition protein, thereby more accurately mutating. It becomes possible to detect.

LAMP method As described above, the symmetric primer set is a primer set having a pair of symmetric primers in which the form of one primer and the form of the other primer are the same. It is preferable to apply. Hereinafter, this primer set is also referred to as “LAMP primer set”.

  In the LAMP method, for example, four types of primers are required, and the target gene can be amplified by recognizing six regions. That is, in this method, first, the first primer anneals to the template strand to cause an extension reaction. Next, the extended strand by the first primer is separated from the template strand by the strand displacement reaction by the second primer designed on the upstream side of the first primer. At this time, a stem-loop structure is formed at the 5 'end portion of the extended strand due to the structure of the first primer extension product that has been removed. A similar reaction is performed on the other strand of the double-stranded nucleic acid or on the 3 'end side of the stripped first primer extension product. Then, by repeating these reactions, the target nucleic acid sequence is amplified. The template in the LAMP method includes, for example, a region composed of a base sequence complementary to each of the terminal regions at the 3 ′ end and the 5 ′ end on the same strand, and when the complementary base sequences are annealed, A template in which a loop capable of base pairing is formed between them (also referred to as “dumbbell-type template nucleic acid”). The LAMP method can be performed according to, for example, International Publication No. 00/28082, International Publication No. 01/034838, etc.

(Non-isothermal amplification method)
PCR method As described above, the PCR method is performed by changing the reaction temperature, for example, by dissociating the double-stranded nucleic acid, annealing the primer to the dissociated single strand, and synthesizing the nucleic acid from the primer. Amplification can be performed. The conditions for the PCR method are not particularly limited, and can be appropriately set by those skilled in the art.

  The first determination method of the present invention will be described below with an example in which double-stranded DNA is used as a test nucleic acid (template nucleic acid).

  First, a reaction solution containing double-stranded DNA that is a test nucleic acid, a primer, Aac MutS, DNA polymerase, and dNTP is prepared. The type of the primer to be used is not particularly limited, and can be set according to, for example, the type of nucleic acid amplification reaction or the type of target nucleic acid sequence to be amplified, and one or more types may be used. Moreover, you may use 1 type, or 2 or more types of primer sets used as a pair.

  Furthermore, the amplification product obtained by the nucleic acid amplification reaction is detected, and the presence or absence of amplification is confirmed. The detection of the amplification product may be performed, for example, over time during the reaction or after a certain time has elapsed from the start of the reaction. The former is so-called real-time detection, and may be, for example, continuous detection or intermittent detection. In the latter case, for example, it is preferable to detect the amplification product at the start of the reaction and after a lapse of a certain time, and confirm the presence or absence of amplification from the fluctuation.

  The detection method of the amplification product is not particularly limited, and conventionally known methods as shown below can be used.

  Examples of the detection method of the amplification product include a method of detecting an amplification product of a specific size by general gel electrophoresis. For example, the amplification product can be detected by a fluorescent substance such as ethidium bromide or cyber green. Alternatively, a probe labeled with a labeling substance can be used and hybridized with the amplification product for detection. An example of the labeling substance is biotin. The biotin can be detected, for example, by binding with avidin bound to an enzyme such as fluorescently labeled avidin or peroxidase. Furthermore, there is a method using immunochromatography, for example, a method using a chromatographic medium using a label detectable with the naked eye (immunochromatography method). Specifically, for example, the amplification product and a labeled probe are hybridized, and this is brought into contact with a chromatographic medium on which a complementary probe capable of hybridizing to the amplification product is immobilized at a site different from the probe. Then, the hybrid of the amplification product and the labeled probe can be trapped by the supplementary probe fixed to the chromatographic medium. As a result, for example, the amplification product can be easily detected with the naked eye. Furthermore, in the present invention, for example, by detecting pyrophosphate which is a by-product of amplification, it is also possible to detect the amplification product indirectly. In particular, since the above-mentioned Smart Amplification Process method has very high amplification efficiency, indirect detection with pyrophosphate is also preferable. In such a method, for example, the white turbidity of the reaction solution is visually or optically observed by utilizing the fact that magnesium in the reaction solution and the generated pyrophosphate are combined to form a white precipitate of magnesium pyrophosphate. By doing so, the presence or absence of amplification can be detected. There is also a method utilizing the fact that pyrophosphoric acid strongly binds to a metal ion such as magnesium to form an insoluble salt, and the magnesium ion concentration in the reaction solution is remarkably reduced. In such a method, for example, a metal indicator whose color tone changes according to the magnesium ion concentration (for example, Erochrome Black T, Hydroxy Naphthol Blue, etc.) is added to the reaction solution, and the color change of the reaction solution is performed. The presence or absence of amplification can be detected by visual observation or observation with an optical method. Further, for example, even when a fluorescent dye such as Calcein is used, an increase in fluorescence accompanying the amplification reaction can be observed visually or by an optical method, so that an amplification product can be detected in real time.

  In the present invention, for example, the presence or absence of the amplification product can be detected by observing the aggregation of the solid phase carrier resulting from the generation of the amplification product. In such a method, for example, it is preferable that at least one kind of primer used in the present invention includes, for example, a solid phase carrier or a site or group capable of binding to a solid phase carrier. The solid phase carrier or the site capable of binding to the solid phase carrier may be introduced into any region such as 3 ′ end region, 5 ′ end region, central region, etc., preferably 5 ′ end of the primer. It is an area. Further, the substrate such as deoxynucleotide (dNTP) used in the amplification reaction may contain, for example, a solid phase carrier or a site capable of binding to the solid phase carrier.

  The solid phase carrier is not particularly limited, and is, for example, a carrier that is insoluble in the reaction solution used for the amplification reaction, a phase transition carrier whose properties change from a liquid phase to a solid phase (gel phase) before and after amplification, or In addition, a phase transition carrier or the like whose properties change from a solid phase (gel phase) to a liquid phase before and after amplification can be used. Preferred solid phase carriers include, for example, water-insoluble organic polymer carriers, water-insoluble inorganic polymer carriers, synthetic polymer carriers, phase transition carriers, metal colloids, magnetic particles, solvent-insoluble organic polymer carriers, solvent-insoluble inorganic polymers. Examples thereof include molecular carriers, solvent-soluble polymer carriers, gel polymer carriers, and the like. Examples of the water-insoluble organic polymer include silicon-containing substances such as porous silica, porous glass, diatomaceous earth, and celite; cross-linked polysaccharides such as nitrocellulose, hydroxyapatite, agarose, dextran, cellulose, and carboxymethylcellulose; Cross-linked products of proteins such as methylated albumin, gelatin, collagen, casein; gel-like particles, dye sols and the like. Examples of the water-insoluble inorganic polymer include aluminum oxide, titanium oxide, and ceramic particles. Examples of the synthetic polymer include polystyrene, poly (meth) acrylate, polyvinyl alcohol, polyacrylonitrile or a copolymer thereof, styrene-styrene sulfonic acid copolymer, vinyl acetate-acrylic acid ester copolymer, and the like. It is done. Examples of the metal colloid include gold colloid. Examples of the magnetic particles include magnetic iron oxide beads, particles having finely pulverized magnetic iron oxide particles on the surface, superparamagnetic particles (Japanese Patent Publication No. 4-501959), and superparaffins covered with a polymerizable silane coating. Examples thereof include magnetically responsive particles having magnetic iron oxide (Japanese Patent Publication No. 7-6986), finely powdered magnetizable particles encapsulated in an organic polymer, and the like. A magnetized solid phase carrier can easily separate a solid and a liquid using a magnetic force, for example. The shape of the solid phase carrier is not particularly limited, and examples thereof include particles, membranes, fibers, filters, etc. Among them, particles are preferable, and the surface thereof is, for example, either porous or non-porous. May be. Particularly preferable solid phase carriers include, for example, latex in which a synthetic polymer carrier is uniformly dispersed in water, metal colloid particles such as gold colloid, magnetic particles such as magnet beads, and the like.

  The method for immobilizing the primer or the substrate on the solid phase carrier is not particularly limited. The immobilization can be performed, for example, by a method known to those skilled in the art, and may be a method using either physical bonding or chemical bonding. The immobilization can be performed, for example, by using a combination of a substance capable of labeling an oligonucleotide such as a primer or a probe and a solid phase carrier bound with a substance capable of binding thereto. The combination of the substances is not particularly limited, and those known in the art can be used.For example, a combination of biotin and avidin or streptavidin, a combination of an antigen and an antibody that can bind thereto, a ligand and this And a combination of two nucleic acids that hybridize with each other. Specifically, for example, a primer or a substrate labeled with biotin is bound to a solid phase carrier whose surface is coated with avidin or streptavidin, whereby the primer or the substrate can be immobilized on the solid phase carrier. Examples of the antigen include haptens such as FITC, DIG, and DNP. Examples of antibodies that can bind to these include antibodies such as anti-FITC antibody, anti-DIG antibody, and anti-DNP antibody. These antibodies may be, for example, either monoclonal antibodies or polyclonal antibodies. In particular, the binding between biotin and streptavidin is particularly preferable because, for example, it has high specificity and good binding efficiency. Labeling substances such as biotin, hapten, and ligand may be known means (for example, Japanese Patent Application Laid-Open Nos. 59-93099 and 59-148798), either alone or in combination as necessary. Gazette and Japanese Patent Application Laid-Open No. 59-204200).

  The site or group capable of binding to the solid phase carrier can be appropriately selected according to, for example, the method for immobilizing the primer or substrate to the solid phase carrier described above. For this reason, the site or group may be, for example, one that allows physical binding to the solid phase carrier or one that allows chemical binding, but allows specific binding. Are preferred. Examples of the site capable of binding to the solid phase carrier include biotin, avidin, streptavidin, antigen, antibody, ligand, receptor, nucleic acid, protein and the like, as described above, and preferably biotin or streptavidin. Yes, more preferably biotin. By using a primer or substrate having such a site, for example, after the amplification reaction, the solid phase carrier can be bound to the generated amplification product. In this case, it is preferable that the solid phase carrier includes, for example, a binding partner of the site included in the primer or the substrate, if necessary. The binding partner in the solid phase carrier only needs to be present in a state capable of binding to the site in the primer or the substrate, for example, preferably present on the surface of the solid phase carrier, more preferably Is applied on the surface of the solid phase carrier.

  In the present invention, for example, a primer set as described above is prepared for each of a plurality of target nucleic acid sequences, and the plurality of primer sets are fixed to the solid phase carrier in a form that can be distinguished from each other. The amplification reaction may be performed using the plurality of immobilized primer sets. According to such a method, it is possible to amplify a plurality of target nucleic acid sequences at the same time, and to identify and detect an amplification product of each target nucleic acid sequence. The amplification product can be detected using, for example, an intercalator. Specifically, for example, if the plurality of primers are respectively immobilized at specific positions on a planar solid support, the position where the amplification product is detected after the amplification reaction and detection of the amplification product. Can identify the amplified target nucleic acid sequence. As the solid phase carrier in such a method, not only the planar solid phase carrier but also, for example, mutually distinguishable bead surfaces (US Pat. No. 6,046,807 and US Pat. No. 6,057,107), A quasi-flat plate carrier (Japanese Patent Laid-Open No. 2000-245460) prepared by bundling a fibrous carrier with each primer set solid-phased and cutting it into thin pieces can be used in the art. .

  In addition to these, as an amplification product detection method, for example, using an intercalator that intercalates with a double-stranded nucleic acid, the so-called intercalator method that determines the presence or absence of amplification by fluorescence generated by excitation light irradiation Can be given. Further, a method using a fluorescent substance and a quencher can be employed, and examples thereof include TaqMan (trademark) probe method and cycling probe method. Moreover, it is preferable to determine the presence or absence of amplification using a probe or primer having a compound disclosed in International Publication No. WO2008 / 111485. According to this method, when the probe or primer and the amplification product form a double-stranded nucleic acid, fluorescence is emitted by irradiation with excitation light. Therefore, the presence or absence of amplification can be determined by detecting the fluorescence. According to this method, for example, an unpurified nucleic acid sample or a nucleic acid sample with a low degree of purification is particularly preferable because the increase in background can be reduced. These methods are preferably applied to, for example, so-called real-time detection.

  Alternatively, the 5 'end of the primer may be immobilized on a solid phase such as a chip, and an amplification reaction may be performed on the solid phase. In this case, for example, a fluorescent substance that emits light by double strand formation may be added to the primer, or the amplification reaction may be performed in a state where the probe to which the fluorescent substance is added is added to the reaction solution. Thereby, for example, it is possible to detect the amplification product in real time while performing the amplification reaction on the solid phase such as the chip.

  Then, it is determined from the presence or absence of amplification whether the target site in the test nucleic acid sequence is a wild type or a mutant type. As a primer, for example, when a primer that is completely complementary to a predetermined region containing the target site in the wild-type nucleic acid sequence is used, when amplification is confirmed, the target site is a wild type, It can be determined that there is no mutation. When amplification is not confirmed, it can be determined that the target site is a mutant type and that a mutation exists. On the other hand, if, for example, a primer that is completely complementary to a predetermined region including the target site in the mutant nucleic acid sequence is used as a primer, the amplification is confirmed, the target site is a mutant type. When it can be determined that there is a mutation and amplification is not confirmed, it can be determined that the target site is a wild type and that there is no mutation.

  The determination method of the present invention is not limited unless otherwise specified. Specifically, in place of the “target primer” in the first determination method of the present invention, a second determination method using the target probe, and further using a primer for amplifying the target nucleic acid sequence Can be performed in the same manner as in the first determination method.

  The target probe can be the same as the target primer in the first determination method, for example, the type of nucleic acid or base constituting the probe. Although the length in particular of the said probe is not restrict | limited, For example, it is 5-40 bases, More preferably, it is 15-25 bases. The annealing conditions of the probe with respect to the test nucleic acid are not particularly limited, but it is preferable to hybridize in the range of 20 to 80 ° C., for example. The probe may have, for example, an active group such as a label or an amino group at one or both ends.

  In the first determination method of the present invention, for example, in the step (I ′), if a target probe capable of hybridizing to the region where the base of the target site is mutated is used, in the step (II) When amplification is confirmed, it can be determined that the base of the target site is a mutant type, and when amplification is not confirmed, it can be determined that the base of the target site is a normal type. On the other hand, for example, in the step (I ′), if a target probe capable of hybridizing to the region where the base of the target site is a wild type is used, amplification is confirmed in the step (II). When the base of the target site is determined to be normal and amplification is not confirmed, it can be determined that the base of the target site is mutant.

  As the method for suppressing non-specific amplification and the method for nucleic acid amplification of the present invention, an amplification reaction can be performed in the presence of Taq MutS or Aac MutS, and other conditions are not particularly limited. About these specific methods, it is the same as that of the determination method of the variation | mutation of this invention mentioned above.

  The determination reagent of the present invention may further contain reagents such as the above-mentioned additives such as ADP, other enzymes such as MutS, primers and polymerase, dNTPs, buffers, melting temperature adjusting agents, enzyme stabilizers and the like. The addition ratio of each component in the determination reagent of the present invention is not particularly limited, but for example, when added to the reaction solution of the amplification reaction, it is preferable to add the concentration as described above. Further, the determination reagent of the present invention may be, for example, a determination kit for use in the determination method of the present invention. In this case, for example, it is preferable to further include instructions for use. Moreover, in the determination reagent and determination kit of the present invention, each component may be stored alone in a container, for example, or may be combined in an appropriate combination and stored in each container. The form and material of the container are not particularly limited.

β3AR (β3-adrenergic receptor) is a gene that exists in human chromosome 8 at 8p12 to 8p11.2 and encodes β3 adrenergic receptor, which is a G protein-coupled receptor protein. This β3AR has a mutation in which the 64th tryptophan from the N-terminal is replaced with arginine (hereinafter referred to as Trp64Arg), and its relation to obesity has been pointed out. Accordingly, there are three types of genotypes of wild type, hetero type, and homo type as gene polymorphism information of the β3AR gene.
Here, the wild type is a type in which there is no gene mutation in any allele, the hetero type is a type in which one allele has mutation, and the homo type is a type in which both alleles have mutation. The heterozygous and homozygous persons having this Trp64Arg have a daily metabolic rate of about 200 kcal lower in both types compared to the wild type. Therefore, these genotypes are less likely to lose weight than wild-types. Moreover, it is said that a person having a mutation in this gene has a constitution in which fat is easily attached to the lower body and buttocks, and subcutaneous fat is more easily accumulated than visceral fat.

UCP1 (Uncoupling protein 1) is a gene associated with proton transfer in mitochondria that exists in human chromosome 4 at 4q28-4q31.
This UCP1 has a point mutation (hereinafter referred to as A-3826G) in which the -3826th adenine from the 5 ′ end is mutated to guanine. Therefore, there are three types of genotypes of wild type, hetero type and homo type as genetic polymorphism information of UCP1 gene. The homozygous person having this A-3826G has a metabolic rate at rest about 100 kcal lower than that of the wild type or hetero type. Therefore, people with this genotype have a constitution that is less likely to lose weight than wild-type and hetero-type people. Moreover, it is said that a person having a mutation in this gene has a constitution in which fat is easily attached to the lower body and buttocks, and subcutaneous fat is more easily accumulated than visceral fat.

The β2AR gene (β2-adrenergic receptor) is a gene that exists in human chromosome 5 at 5q31 to 5q32 and encodes a β2 adrenergic receptor that is a G protein-coupled receptor protein. This β2AR gene has a mutation in which the 16th arginine from the N-terminus is replaced with glycine (hereinafter referred to as Arg16Gly).
Therefore, there are three types of genotypes of wild type, hetero type, and homo type as gene polymorphism information of the β2AR gene. The homozygous person having this Arg16Gly has a metabolic rate at rest of about 200 kcal higher than that of the wild type or hetero type. Therefore, people with this genotype are generally leaner than wild and heterozygous people. In addition, it is said that a person having a mutation in this gene has a constitution that makes it difficult to attach fat and muscle.

A table showing the relationship between this genotype and the basal metabolic rate per day is shown below. The numerical values in the table indicate the daily basal metabolism based on the wild type, and the unit is “kcal”. For example, the homozygous form of β3AR gene indicates that the daily basal metabolic rate is about −200 kcal less than the wild type.

Regarding the sampling method for clinical specimens, a biological cell acquisition kit including a cotton swab is sent from a laboratory to the user's home, and after the user acquires the biological cell at home, the cotton swab is placed in a predetermined storage container. It may be stored and returned to the inspection organization by mail or the like. In this case, since it becomes possible to acquire a cell at home, it is not necessary to go to the inspection institution, and convenience for the user is improved.

The cells to be obtained are not limited to the mucosal cells as described above, but have a nucleus containing genetic information, such as blood cells such as leukocytes contained in blood, hair matrix cells or nail cells in hair roots, etc. Any nucleated cell may be used. In addition, the method of acquiring cells is not limited to the method of acquiring using a cotton swab as described above, and it is acquired with a brush or the like, or by performing oral cleaning with 10 mM physiological saline or the like. It may be a method of acquiring blood with a syringe or the like.

A DNA test step is performed in which DNA is extracted from the biological cells obtained in the biological cell obtaining step and the polymorphism information of the obesity gene is obtained. As a method for extracting DNA from a living cell, a known method in the field of life science is used.

Hereinafter, an example of a method for extracting DNA from living cells and obtaining information on the gene polymorphism will be specifically described, but the present invention is not limited thereto. First, the tip of a cotton swab to which living cells are attached is immersed and suspended in 300 μL of PBS buffer. Subsequently, centrifugation is performed at 10,000 rpm for 5 minutes, and the supernatant is removed. Next, a 10 mM phosphate buffer solution of pH 7.0 containing a predetermined unit of protease K is mixed and stirred in this solution, and incubated at 55 ° C. for 30 minutes. Thereafter, 200 μL of a phenol / chloroform (1: 1) solution is mixed in the reaction solution, stirred for 5 minutes, and centrifuged at 10,000 rpm for 5 minutes. An aqueous phase after centrifugation is obtained, 3M sodium acetate and 400 μL of ethanol are mixed, and 1000 rpm is performed for 10 minutes. The obtained precipitate is washed with ethanol and dried with an evaporator to obtain a DNA sample. The DNA sample can be stored for a long time in this dry state.

In addition, if it is a specimen such as blood, it can be used as a template for isothermal amplification simply by mixing blood and an alkaline reagent and heating them as described in the experimental examples described later.

The health support information provision of the present invention has been described by giving an example of providing health support information to the user based on the genetic polymorphism information of the obesity gene. It is not limited to genetic polymorphism information of obesity genes. For example, health support information can be provided based on gene polymorphism information of genes related to various diseases and constitutions such as tumor genes such as p53 and RAS and genes related to alcohol degradation such as ALDH2.

  Next, examples of the present invention will be described. However, the present invention is not limited by the following examples.

また非特異増幅を抑制するため、競合オリゴヌクレオチドとして、３’末端をアミノ化修飾した長さ１７塩基の以下のオリゴヌクレオチドＣＯ（ｍ）を野生型プライマーセットに、３’末端をアミノ化修飾した長さ１５塩基の以下のオリゴヌクレオチドＣＯ（ｗ）を変異型プライマーセットに加えて用いた。

Example 1
Detection of gene polymorphism of human UCP1-3826 (A> G) The following was used as a primer set for detecting gene polymorphism of human UCP1-3826 (A> G). The positions of the primers are as shown in FIG.

In order to suppress non-specific amplification, the following oligonucleotide CO (m) having a length of 17 bases aminated at the 3 ′ end as a competing oligonucleotide was wild-type primer set and the 3 ′ end was aminated. The following oligonucleotide CO (w) having a length of 15 bases was used in addition to the mutant primer set.

The nucleic acid sample was prepared as follows. Genomic DNA (concentration 10 ng / μL) having UCP1-3826A (wild type) homozygous, genomic DNA (concentration 10 ng / μL) having UCP1-3826G (mutant) homozygous and UCP1-3826A / G (heterotype) A genomic DNA (concentration: 10 ng / μL) was heat-treated at 98 ° C. for 3 minutes and then ice-cooled to obtain a nucleic acid sample. The gene polymorphism of the genomic DNA used here was confirmed by a sequencing method.

１μＬ核酸試料、２．０μＭ ＦＰ、２．０μＭ ＴＰ、１．０μＭ ＢＰ、０．２５μＭ ＯＰ１、０．２５μＭ ＯＰ２、１０μＭ ＣＯで反応を行った。上記の反応組成で６０℃、４０分間反応を行った。反応はＭＸ−３０００Ｐ（ＳＴＲＡＴＡＧＥＮＥ）を用い、経時的に蛍光強度を測定しながら行った。Amplification by the SMAP method was performed as follows. The composition of the reaction system (25 μL) is as follows.
20 mM Tris-HCl (pH 8.0), 1.4 mM dNTPs, 10 mM KCl, 10 mM (NH 4) 2 SO 4, 8 mM

The reaction was performed with 1 μL nucleic acid sample, 2.0 μM FP, 2.0 μM TP, 1.0 μM BP, 0.25 μM OP1, 0.25 μM OP2, 10 μM CO. The reaction was performed at 60 ° C. for 40 minutes using the above reaction composition. The reaction was performed using MX-3000P (STRATAGENE) while measuring the fluorescence intensity over time.

The results are as shown in FIGS. When three types of genomic DNA were amplified using the UCP1-3826A (wild type) amplification primer set, amplification was confirmed from the wild type homozygous containing the wild type gene (-3826A) and the heterozygous genomic DNA. No amplification from genomic DNA was confirmed. When three types of genomic DNA were amplified using the UCP1-3826G (mutant) amplification primer set, amplification was confirmed from the mutant homozygous containing the mutant gene (-3826G) and the heterozygous genomic DNA. No amplification from genomic DNA was confirmed. This result was in complete agreement with the analysis by the sequencing method.

また非特異増幅を抑制するための競合オリゴヌクレオチドとして、３’末端をアミノ化修飾した長さ１５塩基の以下のオリゴヌクレオチドＣＯ（ｍ）を野生型プライマーセットに、３’末端をアミノ化修飾した長さ１５塩基の以下のオリゴヌクレオチドＣＯ（ｗ）を変異型プライマーセットに加えて用いた。

Example 2
Detection of gene polymorphism of human β3AR (Trp64Arg) The following was used as a primer set for detecting gene polymorphism of human β3AR (Trp64Arg). The positions of the primers are as shown in FIG.

In addition, as a competing oligonucleotide for suppressing non-specific amplification, the following oligonucleotide CO (m) having a length of 15 bases aminated at the 3 ′ end was modified with a wild type primer set and the 3 ′ end was aminated The following oligonucleotide CO (w) having a length of 15 bases was used in addition to the mutant primer set.

The nucleic acid sample was prepared as follows. β3AR Trp64 (wild type) homologous genomic DNA, β3AR Arg64 (mutant type) genomic DNA and β3AR Trp64 / Arg64 (heterotype) genomic DNA, heat-treated at 98 ° C. for 3 minutes, and then ice-cooled Was used as a nucleic acid sample. The genetic polymorphism of the genomic DNA used here was confirmed by sequencing after extracting the genomic DNA.

ポリメラーゼ、１μＬ核酸試料、２．０μＭ ＦＰ、２．０μＭ ＴＰ、１．０μＭ ＢＰ、０．２５μＭ ＯＰ、１０μＭ ＣＯで反応を行った。上記の反応組成で６０℃、４０分間反応を行った。反応はＭＸ−３０００Ｐ（ＳＴＲＡＴＡＧＥＮＥ）を用い、経時的に蛍光強度を測定しながら行った。The amplification reaction by the SMAP reaction was performed as follows. The composition of the reaction system (25 μL) is as follows. 20 mM Tris-HCl (pH 8.0), 1.4 mM dNTPs, 5% DMSO, 10 mM KCl, 10 mM

The reaction was performed with polymerase, 1 μL nucleic acid sample, 2.0 μM FP, 2.0 μM TP, 1.0 μM BP, 0.25 μM OP, 10 μM CO. The reaction was performed at 60 ° C. for 40 minutes using the above reaction composition. The reaction was performed using MX-3000P (STRATAGENE) while measuring the fluorescence intensity over time.

The results are as shown in FIGS. When three types of genomic DNA were amplified using the β3AR Trp64 (wild type) amplification primer set, amplification was confirmed from the wild type heterozygous DNA containing the wild type gene (β3AR Trp64) and the heterozygous genomic DNA. Amplification from genomic DNA was not confirmed. When three types of genomic DNA were amplified using the β3AR Arg64 (mutant) amplification primer set, amplification was confirmed from mutant and heterozygous genomic DNA containing the mutant gene (β3AR Arg64). Amplification from genomic DNA was not confirmed. This result was in complete agreement with the analysis by the sequencing method.

また非特異増幅を抑制するための競合オリゴヌクレオチドとして、３’末端をアミノ化修飾した長さ１５塩基の以下のオリゴヌクレオチドＣＯ（ｍ）を野生型プライマーセットに、３’末端をアミノ化修飾した長さ１５塩基の以下のオリゴヌクレオチドＣＯ（ｗ）を変異型プライマーセットに加えて用いた。

Example 3
Detection of human polymorphism of human β2AR (Arg16Gly) As a primer set for detecting the polymorphism of human β2AR (Arg16Gly), the following was used. The positions of the primers are as shown in FIG.

In addition, as a competing oligonucleotide for suppressing non-specific amplification, the following oligonucleotide CO (m) having a length of 15 bases aminated at the 3 ′ end was modified with a wild type primer set and the 3 ′ end was aminated The following oligonucleotide CO (w) having a length of 15 bases was used in addition to the mutant primer set.

The nucleic acid sample was prepared as follows. Genomic DNA having homology with β2AR Arg16 (wild type), genomic DNA having homology with β2AR Gly16 (mutant type) and genomic DNA of β2AR Arg16 / Gly16 (heterotype) were heat-treated at 98 ° C. for 10 minutes and then ice-cooled. This was used as a nucleic acid sample. The genetic polymorphism of the genomic DNA used here was confirmed by sequencing after extracting the genomic DNA.

ポリメラーゼ、１μＬ核酸試料、２．０μＭ ＦＰ、２．０μＭ ＴＰ、１．０μＭ ＢＰ、０．２５μＭ ＯＰ、１０μＭ ＣＯで反応を行った。上記の反応組成で６０℃、４０分間反応を行った。反応はＭＸ−３０００Ｐ（ＳＴＲＡＴＡＧＥＮＥ）を用い、経時的に蛍光強度を測定しながら行った。The amplification reaction by the SMAP reaction was performed as follows. The composition of the reaction system (25 μL) is as follows. 20 mM Tris-HCl (pH 8.0), 1.4 mM dNTPs, 5% DMSO, 10 mM KCl, 10 mM

The reaction was performed with polymerase, 1 μL nucleic acid sample, 2.0 μM FP, 2.0 μM TP, 1.0 μM BP, 0.25 μM OP, 10 μM CO. The reaction was performed at 60 ° C. for 40 minutes using the above reaction composition. The reaction was performed using MX-3000P (STRATAGENE) while measuring the fluorescence intensity over time.

The results are as shown in FIGS. When three types of genomic DNA were amplified using the β2AR Arg16 (wild type) amplification primer set, amplification was confirmed from the wild type heterozygous DNA containing the wild type gene (β2AR Arg16) and the heterozygous type DNA. Amplification from genomic DNA was not confirmed (FIG. 11). When three types of genomic DNA were amplified using the β2AR Gly16 (mutant) amplification primer set, amplification was confirmed from the mutant and heterozygous genomic DNA containing the mutant gene (β2AR Gly16). Amplification from genomic DNA was not confirmed (FIG. 12). This result was in complete agreement with the analysis by the sequencing method.

また非特異増幅を抑制するため、競合オリゴヌクレオチドとして、３’末端をアミノ化修飾した長さ１７塩基の以下のオリゴヌクレオチドＣＯ（ｍ）を野生型プライマーセットに、３’末端をアミノ化修飾した長さ１５塩基の以下のオリゴヌクレオチドＣＯ（ｗ）を変異型プライマーセットに加えて用いた。

Example 4
Detection of gene polymorphism of human UCP1-3826 (A> G) The following was used as a primer set for detecting gene polymorphism of human UCP1-3826 (A> G). The positions of the primers are as shown in FIG.

In order to suppress non-specific amplification, the following oligonucleotide CO (m) having a length of 17 bases aminated at the 3 ′ end as a competing oligonucleotide was wild-type primer set and the 3 ′ end was aminated. The following oligonucleotide CO (w) having a length of 15 bases was used in addition to the mutant primer set.

The nucleic acid sample was prepared as follows. A blood sample having a UCP1-3826A (wild type) homozygote, a blood sample having a UCP1-3826G (mutant) homozygote and a blood sample having a UCP1-3826A / G (heterotype) homogenized at 98 ° C. for 3 minutes, The cooled sample was used as a nucleic acid sample. The gene polymorphism of the blood sample used here was confirmed by the sequencing method.

１μＬ核酸試料、２．０μＭ ＦＰ、２．０μＭ ＴＰ、１．０μＭ ＢＰ、０．２５μＭ ＯＰ１、０．２５μＭ ＯＰ２、１０μＭ ＣＯで反応を行った。上記の反応組成で６０℃、４０分間反応を行った。反応はＭＸ−３０００Ｐ（ＳＴＲＡＴＡＧＥＮＥ）を用い、経時的に蛍光強度を測定しながら行った。Amplification by the SMAP method was performed as follows. The composition of the reaction system (25 μL) is as follows.
20 mM Tris-HCl (pH 8.0), 1.4 mM dNTPs, 10 mM KCl, 10 mM (NH 4) 2 SO 4, 8 mM

The reaction was performed with 1 μL nucleic acid sample, 2.0 μM FP, 2.0 μM TP, 1.0 μM BP, 0.25 μM OP1, 0.25 μM OP2, 10 μM CO. The reaction was performed at 60 ° C. for 40 minutes using the above reaction composition. The reaction was performed using MX-3000P (STRATAGENE) while measuring the fluorescence intensity over time.

The results are as shown in FIGS. When three types of blood samples were amplified using the UCP1-3826A (wild type) amplification primer set, amplification was confirmed from wild type and heterozygous blood samples containing the wild type gene (-3826A). No amplification from the blood sample was confirmed. When three types of blood samples were amplified using the UCP1-3826G (mutant) amplification primer set, amplification was confirmed from the mutant homozygous and heterozygous blood samples containing the mutant gene (-3826G). No amplification from the blood sample was confirmed. This result was in complete agreement with the analysis by the sequencing method.

また非特異増幅を抑制するための競合オリゴヌクレオチドとして、３’末端をアミノ化修飾した長さ１５塩基の以下のオリゴヌクレオチドＣＯ（ｍ）を野生型プライマーセットに、３’末端をアミノ化修飾した長さ１５塩基の以下のオリゴヌクレオチドＣＯ（ｗ）を変異型プライマーセットに加えて用いた。

Example 5
Detection of gene polymorphism of human β3AR (Trp64Arg) The following was used as a primer set for detecting gene polymorphism of human β3AR (Trp64Arg). The positions of the primers are as shown in FIG.

In addition, as a competing oligonucleotide for suppressing non-specific amplification, the following oligonucleotide CO (m) having a length of 15 bases aminated at the 3 ′ end was modified with a wild type primer set and the 3 ′ end was aminated The following oligonucleotide CO (w) having a length of 15 bases was used in addition to the mutant primer set.

The nucleic acid sample was prepared as follows. β3AR Trp64 (wild type) homozygous blood sample, β3AR Arg64 (mutant type) homogenous blood sample and β3AR Trp64 / Arg64 (heterotype) blood sample, heat-treated at 98 ° C. for 3 minutes, and then ice-cooled Was used as a nucleic acid sample. The gene polymorphism of the blood sample used here was confirmed by a sequencing method after extracting the blood sample.

ポリメラーゼ、１μＬ核酸試料、２．０μＭ ＦＰ、２．０μＭ ＴＰ、１．０μＭ ＢＰ、０．２５μＭ ＯＰ、１０μＭ ＣＯで反応を行った。上記の反応組成で６０℃、４０分間反応を行った。反応はＭＸ−３０００Ｐ（ＳＴＲＡＴＡＧＥＮＥ）を用い、経時的に蛍光強度を測定しながら行った。The amplification reaction by the SMAP reaction was performed as follows. The composition of the reaction system (25 μL) is as follows. 20 mM Tris-HCl (pH 8.0), 1.4 mM dNTPs, 5% DMSO, 10 mM KCl, 10 mM

The reaction was performed with polymerase, 1 μL nucleic acid sample, 2.0 μM FP, 2.0 μM TP, 1.0 μM BP, 0.25 μM OP, 10 μM CO. The reaction was performed at 60 ° C. for 40 minutes using the above reaction composition. The reaction was performed using MX-3000P (STRATAGENE) while measuring the fluorescence intensity over time.

The results are as shown in FIGS. When three types of blood samples were amplified using the β3AR Trp64 (wild type) amplification primer set, amplification was confirmed from wild type homozygous and heterozygous blood samples containing the wild type gene (β3AR Trp64). No amplification from blood samples was confirmed. When three types of blood samples were amplified using the β3AR Arg64 (mutant) amplification primer set, amplification was confirmed from mutant and heterozygous blood samples containing the mutant gene (β3AR Arg64). No amplification from blood samples was confirmed. This result was in complete agreement with the analysis by the sequencing method.

また非特異増幅を抑制するための競合オリゴヌクレオチドとして、３’末端をアミノ化修飾した長さ１５塩基の以下のオリゴヌクレオチドＣＯ（ｍ）を野生型プライマーセットに、３’末端をアミノ化修飾した長さ１５塩基の以下のオリゴヌクレオチドＣＯ（ｗ）を変異型プライマーセットに加えて用いた。

Example 6
Detection of human polymorphism of human β2AR (Arg16Gly) As a primer set for detecting the polymorphism of human β2AR (Arg16Gly), the following was used. The positions of the primers are as shown in FIG.

In addition, as a competing oligonucleotide for suppressing non-specific amplification, the following oligonucleotide CO (m) having a length of 15 bases aminated at the 3 ′ end was modified with a wild type primer set and the 3 ′ end was aminated The following oligonucleotide CO (w) having a length of 15 bases was used in addition to the mutant primer set.

The nucleic acid sample was prepared as follows. A blood sample having β2AR Arg16 (wild type) homozygote, a blood sample having β2AR Gly16 (mutant type) homozygote and a blood sample of β2AR Arg16 / Gly16 (heterotype) were heat-treated at 98 ° C. for 10 minutes and then ice-cooled. This was used as a nucleic acid sample. The gene polymorphism of the blood sample used here was confirmed by a sequencing method after extracting the blood sample.

ポリメラーゼ、１μＬ核酸試料、２．０μＭ ＦＰ、２．０μＭ ＴＰ、１．０μＭ ＢＰ、０．２５μＭ ＯＰ、１０μＭ ＣＯで反応を行った。上記の反応組成で６０℃、４０分間反応を行った。反応はＭＸ−３０００Ｐ（ＳＴＲＡＴＡＧＥＮＥ）を用い、経時的に蛍光強度を測定しながら行った。The amplification reaction by the SMAP reaction was performed as follows. The composition of the reaction system (25 μL) is as follows. 20 mM Tris-HCl (pH 8.0), 1.4 mM dNTPs, 5% DMSO, 10 mM KCl, 10 mM

The reaction was performed with polymerase, 1 μL nucleic acid sample, 2.0 μM FP, 2.0 μM TP, 1.0 μM BP, 0.25 μM OP, 10 μM CO. The reaction was performed at 60 ° C. for 40 minutes using the above reaction composition. The reaction was performed using MX-3000P (STRATAGENE) while measuring the fluorescence intensity over time.

The results are as shown in FIGS. When three types of blood samples were amplified using the β2AR Arg16 (wild type) amplification primer set, amplification was confirmed from wild type and heterozygous blood samples containing the wild type gene (β2AR Arg16). No amplification from blood samples was confirmed. When three types of blood samples were amplified using the β2AR Gly16 (mutant) amplification primer set, amplification was confirmed from the mutant and heterozygous blood samples containing the mutant gene (β2AR Gly16). No amplification from blood samples was confirmed. This result was in complete agreement with the analysis by the sequencing method.

It is the figure which showed the position of the primer which detects the gene polymorphism of human UCP1-3826 (A> G). It is a figure at the time of amplifying UCP1-3826 wild type homogenous genomic DNA using human UCP1-3826 amplification primer set. It is a figure at the time of amplifying UCP1-3826 mutant | homogeneous genomic DNA using the human UCP1-3826 amplification primer set. It is a figure at the time of amplifying UCP1-3826 heterotype genomic DNA using a human UCP1-3826 amplification primer set. It is the figure which showed the position of the primer which detects the gene polymorphism of human (beta) 3AR (Trp64Arg). It is a figure at the time of amplifying the genomic DNA of (beta) 3AR (Trp64Arg) wild type homology using a human (beta) 3AR (Trp64Arg) amplification primer set. It is a figure at the time of amplifying the genomic DNA of (beta) 3AR (Trp64Arg) mutant type | mold homology using a human (beta) 3AR (Trp64Arg) amplification primer set. It is a figure at the time of amplifying (beta) 3AR (Trp64Arg) heterotype genomic DNA using a human (beta) 3AR (Trp64Arg) amplification primer set. It is the figure which showed the position of the primer which detects the gene polymorphism of human (beta) 2AR (Arg16Gly). It is a figure at the time of amplifying the genomic DNA of (beta) 2AR (Arg16Gly) wild type homology using a human (beta) 2AR (Arg16Gly) amplification primer set. It is a figure at the time of amplifying (beta) 2AR (Arg16Gly) mutant | homogeneous genomic DNA using a human (beta) 2AR (Arg16Gly) amplification primer set. It is a figure at the time of amplifying (beta) 2AR (Arg16Gly) heterogenous genomic DNA using a human (beta) 2AR (Arg16Gly) amplification primer set. It is a figure at the time of amplifying the blood sample of UCP1-3826 wild type homology using human UCP1-3826 amplification primer set. It is a figure at the time of amplifying the blood sample of UCP1-3826 mutant | homogeneous homology using the human UCP1-3826 amplification primer set. It is a figure at the time of amplifying the blood sample of UCP1-3826 heterotype using human UCP1-3826 amplification primer set. It is a figure at the time of amplifying the blood sample of (beta) 3AR (Trp64Arg) wild type homology using a human (beta) 3AR (Trp64Arg) amplification primer set. It is a figure at the time of amplifying the blood sample of (beta) 3AR (Trp64Arg) mutant type | mold using a human (beta) 3AR (Trp64Arg) amplification primer set. It is a figure at the time of amplifying the blood sample of (beta) 3AR (Trp64Arg) hetero type using the human (beta) 3AR (Trp64Arg) amplification primer set. It is a figure at the time of amplifying the blood sample of (beta) 2AR (Arg16Gly) wild type homology using a human (beta) 2AR (Arg16Gly) amplification primer set. It is a figure at the time of amplifying the blood sample of (beta) 2AR (Arg16Gly) mutant type | mold homology using a human (beta) 2AR (Arg16Gly) amplification primer set. It is a figure at the time of amplifying the blood sample of (beta) 2AR (Arg16Gly) heterotype using a human (beta) 2AR (Arg16Gly) amplification primer set. It is the figure which showed the action mechanism of the nucleic acid synthesis by a 1st primer. It is the figure which showed the example of the 2nd primer contained in the primer set by this invention. It is the figure which showed the action mechanism (ah) of the nucleic acid amplification reaction by a 1st primer and a 2nd primer. It is the figure which showed the action mechanism (i-o) of the nucleic acid amplification reaction by a 1st primer and a 2nd primer.

Claims (14)

Primers used for typing polymorphisms of obesity-related genes by nucleic acid amplification,
The obesity-related gene is at least one gene selected from the group consisting of a β3AR gene, a UCP1 gene, and a β2AR gene, a sequence from numbers 3215 to 3325 in the base sequence of the β3AR gene, and −3888 in a base sequence of the UCP1 gene A primer for amplifying a target region nucleic acid sequence selected from the sequence from No. -3739, the sequence from No. 216 to 338 in the base sequence of the β2AR gene, and their complementary sequences. The primer is
The at least one base sequence selected from the group consisting of the base sequences represented by SEQ ID NOs: 1 to 27 and at least one base sequence complementary to the base sequence and at least one base sequence complementary to the base sequence of at least 7 bases. Primer.

The primer is
Oligonucleotide consisting of a base sequence complementary to at least one selected from the group consisting of base sequences represented by SEQ ID NOs: 2 to 4-Oligonucleotide consisting of any base sequence having 0 to 30 bases-SEQ ID NO: A primer capable of amplifying a β3AR gene comprising an oligonucleotide consisting of at least one base sequence selected from the group consisting of base sequences represented by 3 to 5;
Oligonucleotide consisting of a base sequence complementary to at least one selected from the group consisting of base sequences represented by SEQ ID NOs: 11 to 13-Oligonucleotide consisting of any base sequence having 0 to 30 bases-SEQ ID NO: A primer capable of amplifying a UCP1 gene composed of an oligonucleotide consisting of at least one base sequence selected from the group consisting of base sequences represented by 12 to 14, or
Oligonucleotide consisting of a base sequence complementary to at least one selected from the group consisting of base sequences represented by SEQ ID NOs: 20 to 22-Oligonucleotide consisting of any base sequence having 0 to 30 bases-SEQ ID NO: The primer according to claim 2, which is a primer capable of amplifying a B2AR gene composed of an oligonucleotide having at least one base sequence selected from the group consisting of base sequences represented by 21 to 23 The primer according to claims 1 to 3, wherein the nucleic acid amplification method is a Smart Amplification Process method or a LAMP method. A competitive probe used for typing a polymorphism of an obesity-related gene by nucleic acid amplification, wherein the obesity-related gene is at least one gene selected from the group consisting of β3AR gene, UCP1 gene and β2AR gene, and β3AR Selected from sequences 3215 to 3325 in the nucleotide sequence of the gene, sequences -3888 to −3739 in the nucleotide sequence of the UCP1 gene, sequences 216 to 338 in the nucleotide sequence of the β2AR gene, and complementary sequences thereof Competing probes that compete with primers typing genetic polymorphisms in the targeted target nucleic acid sequence and suppress non-specific amplification. The competing probe is designed to be modified so that no extension reaction occurs from the 3 ′ end.
The base sequence of at least 10 bases in at least one base sequence selected from the group consisting of the base sequences represented by SEQ ID NOs: 28 to 33 and at least one base sequence complementary thereto, Competitive probe.

The competitive probe according to claim 6, wherein the modification is amination, dideoxylation or phosphorylation of the 3′-terminal hydroxyl group. 6. The competitive probe according to claim 5, wherein the nucleic acid amplification method is a Smart Amplification Process method or a LAMP method. A method for amplifying obesity-related genes,
The obesity-related gene is at least one gene selected from the group consisting of a β3AR gene, a UCP1 gene, and a β2AR gene, and the amplification method is a Smart Amplification Process method or a LAMP method, and any one of claims 1 to 4 An amplification method using at least one of the primer according to claim 5 and the competitive probe according to any one of claims 5 to 8. A method of typing a polymorphism of an obesity-related gene by nucleic acid amplification, wherein the obesity-related gene is at least one gene selected from the group consisting of β3AR gene, UCP1 gene and β2AR gene, and the amplification method comprises: It is a Smart Amplification Process method or a LAMP method, and a genetic polymorphism using at least one of the primer according to any one of claims 1 to 4 and the competitive probe according to any one of claims 5 to 8. Typing method. The method for judging an obesity constitution by amplification of an obesity-related gene, wherein the obesity-related gene is at least one gene selected from the group consisting of a β3AR gene, a UCP1 gene, and a β2AR gene, A method for determining an obesity constitution, comprising a step of amplifying a gene by an amplification method of an obesity-related gene, and a step of judging an obesity constitution by detecting the amplification. A kit used for typing a polymorphism of an obesity-related gene by nucleic acid amplification, wherein the obesity-related gene is at least one gene selected from the group consisting of a β3AR gene, a UCP1 gene, and a β2AR gene, A kit comprising at least one of the primer according to any one of 1 to 4 and the competitive probe according to any one of claims 5 to 8. The kit according to claim 12, further comprising a strand displacement nucleic acid synthase and a substrate. The analysis for performing the typing method according to claim 10, wherein at least one of the primer according to any one of claims 1 to 4 and the competitive probe according to any one of claims 5 to 8 is used. An analysis apparatus comprising: a reaction unit that performs an amplification reaction; a temperature control unit that controls a temperature of the reaction unit; and a detection unit that detects the reaction.

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