JP6006814B2 - Nucleic acid amplification primer design method, nucleic acid amplification primer production method, nucleic acid amplification primer, primer set, and nucleic acid amplification method - Google Patents

Description

  The present invention relates to a method for designing a primer for nucleic acid amplification, a method for producing a primer for nucleic acid amplification, a primer for nucleic acid amplification, a primer set, and a method for amplifying nucleic acid.

  An isothermal nucleic acid amplification method using a primer set including a turnback primer (TP) and a DNA polymerase having strand displacement ability is used in a wide range of fields such as medicine and molecular biology (Non-Patent Documents 1-5). ). TP is a primer capable of forming a stem-loop structure having a sequence annealed to a target nucleic acid sequence and a sequence that can be annealed to a sequence extended from the target nucleic acid sequence on the 5 ′ side. In addition to TP, a folding primer (FP), a boost primer (BP), and two outer primers (OP1 and OP2) having a sequence that anneals to the target nucleic acid sequence and a sequence that can form a folded structure inside the primer on the 5 ′ side Can also be used. The nucleic acid amplification method using these primers does not require a special device such as a thermal cycler, and has an excellent effect that it does not require time for temperature setting. The nucleic acid amplification method can also be preferably applied to early diagnosis of diseases.

Nature Methods 4 (2007) 257-262. Clin. Cancer Res. 13 (2007) 4974-4983. BioTechniques 43 (2007) 479-484. Biologicals 36 (2008) 234-238. Clin. Chem. 10 (2009) 1373.

  In an isothermal nucleic acid amplification method using a primer set containing TP, improvement in nucleic acid amplification rate and improvement in nucleic acid amplification specificity are required.

  Therefore, the present invention is a nucleic acid amplification primer used in an isothermal nucleic acid amplification method using a primer set containing TP, and a method for designing a nucleic acid amplification primer capable of improving the nucleic acid amplification rate and nucleic acid amplification specificity, An object of the present invention is to provide a method for producing a primer for nucleic acid amplification, a primer for nucleic acid amplification, a primer set, and a method for amplifying nucleic acid.

The method for designing a primer for nucleic acid amplification of the present invention comprises:
A method of designing a primer used for amplification of a target nucleic acid sequence, comprising the following steps (D1), (D2) and (D3).
(D1) A step of selecting a sequence (A) in the target nucleic acid sequence and determining a sequence (Ac ′) complementary to the sequence (A) as an annealing sequence on the 3 ′ end side of the primer.
(D2) In the target nucleic acid sequence, a sequence (B) existing on the 5 ′ end side from the sequence (A) is selected, and the sequence (B) is used as a stem loop forming sequence on the 5 ′ end side of the primer. Determining the same sequence (B ′)
(D3) In the target nucleic acid sequence, a sequence (W) between the sequence (A) and the sequence (B), and a sequence complementary to the sequence (W) on the 3 ′ end side from the sequence (A) (Wc) is selected, and the same sequence (W ′) as the sequence (W) is determined as a sequence between the sequences (Ac ′) and (B ′) of the primer.

The method for producing the primer of the present invention comprises:
A method for producing a primer used for amplification of a target nucleic acid sequence,
Including primer design process and primer synthesis process,
The primer designing step is performed by the primer designing method of the present invention.

The primer set of the present invention is
A primer set used for amplification of a target nucleic acid sequence in a double-stranded template nucleic acid comprising a first template nucleic acid and a second template nucleic acid,
The primer set includes a first primer and a second primer,
The first primer is a primer produced by the production method of the present invention based on the target nucleic acid sequence in the first template nucleic acid,
The second primer includes, on the 3 ′ end side, a sequence (Ec ′) complementary to the sequence (E ′) on the 3 ′ end side of the target nucleic acid sequence in the second template nucleic acid. It includes the same sequence (F ′) as the sequence (F) present on the 5 ′ side of the sequence (E) of the target nucleic acid sequence in the second template nucleic acid.

The first nucleic acid amplification method of the present invention comprises:
The complementary nucleic acid amplification method for synthesizing a nucleic acid complementary to a target nucleic acid sequence in a template nucleic acid, comprising the following steps (S1) to (S5).
(S1) A step of producing a primer by the method for producing a primer of the present invention.
(S2) A step of providing a template nucleic acid.
(S3) A step of annealing the primer to the template nucleic acid and performing a primer extension reaction to synthesize a complementary nucleic acid containing a complementary sequence of the target nucleic acid sequence.
(S4) The sequence (B ′) present on the 5 ′ side of the complementary nucleic acid synthesized in the step (S3) is hybridized to the sequence (Bc) present on the complementary nucleic acid, thereby A step of making the portion of the sequence (A) a single strand.
(S5) Annealing another primer having the same sequence as the primer to the portion of the sequence (A) on the template nucleic acid made into a single strand by the step (S4), thereby carrying out a strand displacement reaction, A step of replacing the complementary nucleic acid synthesized in the step (S3) with a complementary nucleic acid newly synthesized by the other primer.

The second nucleic acid amplification method of the present invention comprises:
A method of amplifying a target nucleic acid sequence in a double-stranded template nucleic acid, comprising the following steps (S′1) to (S′5).
(S′1) A step of providing the primer set of the present invention.
(S′2) A step of providing a double-stranded template nucleic acid comprising a first template nucleic acid and a second template nucleic acid.
(S′3) The first and second primers containing the complementary sequences of the target nucleic acid sequences by annealing the first and second primers to the first and second template nucleic acids, respectively, and performing primer extension reactions, respectively. Synthesizing complementary nucleic acids;
(S′4) The sequences (B ′) and (F ′) present on the 5 ′ side of the first and second complementary nucleic acids synthesized by the step (S′3) are present on the complementary nucleic acids. A step of hybridizing to the sequence (Bc) and the sequence (Fc), respectively, so that the portions of the sequence (A) and the sequence (E) on the first and second template nucleic acids become single-stranded.
(S′5) The sequence (A) and the sequence (E) on the first and second template nucleic acids made into single strands by the step (S′4) have the same sequence as the primer. By performing the strand displacement reaction by annealing the other primer, the first and second complementary nucleic acids synthesized in the step (S′3) are replaced with the complementary nucleic acid newly synthesized by the other primer. Process.

The primer of the present invention is a primer used for amplification of a target nucleic acid sequence,
It is produced by the production method of the present invention and is used for the second nucleic acid amplification method of the present invention.

  In the following, the primer designing method of the present invention may be referred to as “primer designing method (0)”. The primer designing method of the present invention may be any of primer designing methods (1) to (10) described later, instead of the primer designing method (0). The primer production method of the present invention using the primer design method (0) may be referred to as “primer production method (0)” hereinafter. The primer production method of the present invention may use any of the primer design methods (1) to (10) described later instead of the primer design method (0). “Production method (1)”, “Primer production method (2)”, “Primer production method (3)”, “Primer production method (4)”, “Primer production method (5)”, “Primer production method” “Production method (6)”, “Primer production method (7)”, “Primer production method (8)”, “Primer production method (9)”, or “Primer production method (10)” is there.

  Hereinafter, the primer produced by the primer production method (0) may be referred to as “primer (0)”. In addition, the primer produced by any one of the production methods (1) to (10) of the primer is referred to as “primer (1)”, “primer (2)”, “primer (3)”, “primer (4)”, respectively. ) ”,“ Primer (5) ”,“ Primer (6) ”,“ Primer (7) ”,“ Primer (8) ”,“ Primer (9) ”, or“ Primer (10) ”. However, the primers (0) to (10) may be primers produced by any other method as long as the structures are the same.

  As described above, the primer of the present invention is the primer (0) used in the second nucleic acid amplification method of the present invention. Instead of this, the primer (8) may be the primer of the present invention. In the primer set of the present invention, as described above, the first primer is the primer (0). In the primer set of the present invention, the first primer may be the primer (8) instead of the primer (0).

  As described above, in the first nucleic acid amplification method of the present invention, in the step (S1), a primer is produced by the primer production method (0). The first nucleic acid amplification method of the present invention is any one of the primer production methods (1) to (10) described later, instead of the primer production method (0) in the step (S1). A primer may be produced by the method described above, or the primer (8) may be provided by any method.

  As described above, in the second nucleic acid amplification method of the present invention, in the step (S′1), the first primer is the primer (0) or the primer (8). Provide set. In the second nucleic acid amplification method of the present invention, in the step (S′1), instead of providing the primer set of the present invention, a method for producing a primer set of the present invention described later (a method for producing a primer set ( The primer set may be produced by any one of 0) to (10) and the primer set production method (6 ′)).

  In order to achieve the above object, the present inventors have made various studies, and as a result, between the sequences (Ac ′) and (B ′) of TP, the same as the sequence (W) of the target nucleic acid sequence. It has been found that the use of the primer (0) provided with the sequence (W ′) can improve the nucleic acid amplification rate in the isothermal nucleic acid amplification method, and the present invention has been achieved. As described above, the present invention, instead of the primer design method (0), the primer production method (0), or the primer (0), the primer design methods (1) to (10) described later, Primer production method (1) to (10) described later, or method for designing nucleic acid amplification primer using any of primers (1) to (10) described later, method for producing nucleic acid amplification primer, and nucleic acid amplification It may be a primer, a primer set, or a nucleic acid amplification method. Further, the specificity of the nucleic acid amplification reaction is improved by using the method for designing a primer for nucleic acid amplification, the method for producing a primer for nucleic acid amplification, the primer for nucleic acid amplification, the primer set, or the method for amplifying nucleic acid of the present invention.

FIG. 1A is a schematic diagram showing an example of a primer for nucleic acid amplification of the present invention. FIG. 1B is a schematic diagram showing an example of a method for designing a primer for nucleic acid amplification of the present invention. FIG. 2 is a schematic view illustrating the action mechanism of nucleic acid synthesis using the primer for nucleic acid amplification of the present invention. FIG. 3 is a schematic diagram showing another example of the action mechanism of nucleic acid synthesis using the primer for nucleic acid amplification of the present invention. FIG. 4 is a schematic diagram showing an example of a second primer (FP) included in the primer set of the present invention. FIG. 5 (A) is a schematic diagram showing an example of a basic mechanism of the nucleic acid amplification method of the present invention. FIG. 5 (B) is a schematic diagram showing an example of a basic mechanism of the nucleic acid amplification method of the present invention. FIGS. 6 a to n are schematic diagrams showing other examples of the mechanism of the nucleic acid amplification method of the present invention. FIG. 7 (A) is a schematic diagram showing the bases that serve as classification criteria for TP in Example 1 of the present invention, and FIG. 7 (B) shows the rate of nucleic acid amplification reaction for each combination of TPs classified according to the criteria. It is a graph which shows the evaluation score of specificity. FIG. 8 (A) is a schematic diagram showing bases that serve as classification criteria for TP in Example 2 of the present invention, and FIG. 8 (B) shows the rate of nucleic acid amplification reaction for each combination of TPs classified according to the criteria. It is a graph which shows the evaluation score of specificity. FIG. 9 is a diagram showing an example of the primer of the present invention. FIG. 10 is a diagram showing another example of the primer of the present invention. FIG. 11 is a diagram showing still another example of the primer of the present invention. FIG. 12 is a diagram showing still another example of the primer of the present invention. FIG. 13 is a diagram showing still another example of the primer of the present invention. FIG. 14 is a diagram showing still another example of the primer of the present invention. FIG. 15 is a diagram showing still another example of the primer of the present invention. FIG. 16 is a diagram showing still another example of the primer of the present invention. FIG. 17 is a diagram showing still another example of the primer of the present invention. FIG. 18 is a diagram showing still another example of the primer of the present invention.

  Next, the present invention will be described in detail.

The primer (0) is
A sequence (Ac ′) complementary to the sequence (A) on the 3 ′ end side of the target nucleic acid sequence on the 3 ′ end side of the primer (0);
The 5 ′ end side of the primer (0) includes the same sequence (B ′) as the sequence (B) present 5 ′ side of the sequence (A) of the target nucleic acid sequence,
In the target nucleic acid sequence, there is a sequence (W) between the sequence (A) and the sequence (B), and a sequence complementary to the sequence (W) at the 3 ′ end side from the sequence (A) ( Wc) exists,
Furthermore, the sequence (W ′) is the same as the sequence (W) of the target nucleic acid sequence between the sequences (Ac ′) and (B ′) of the primer. Specifically, for example, as shown in FIG. 1A. In the figure, “TP” indicates the primer (0). In addition, the design method and the production method of the primer (0) having such a structure are not particularly limited, but the primer is designed by the primer design method (0) of the present invention or the primer production method (0) It is preferable to manufacture by. In addition, hereinafter, when “TP” is simply described, it represents any of the primers (0) to (10) used in the present invention unless otherwise specified.

  In the present invention, the “target nucleic acid” or “target nucleic acid sequence” means not only the nucleic acid to be amplified or the sequence itself, but also a complementary sequence or a nucleic acid having the sequence.

  In the present invention, the number of bases of the sequence (W ′) is not particularly limited, but is, for example, 1 or more, preferably 2 or more, more preferably 3 or more. That is, the base number of the sequence (W) of the target nucleic acid sequence is, for example, 1 or more, preferably 2 or more, more preferably 3 or more. The sequence (W) of the target nucleic acid sequence may be included in at least one of the sequence (A) and the sequence (B).

  The mechanism of the amplification reaction by TP 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. . Further, in the target nucleic acid sequence, a sequence (W) existing between the sequence (A) and the sequence (B), and a sequence (Wc) complementary to the sequence (W) is the sequence (W The sequence (W) that exists at the 3 ′ end side from A) is determined. TP comprises a sequence (Ac ′), and further comprises a sequence (B ′) on the 5 ′ side thereof. Furthermore, the same sequence (W ′) as the sequence (W) of the target nucleic acid sequence is included between the sequences (Ac ′) and (B ′) of the primer. The sequence (Ac ′) hybridizes to the sequence (A), the sequence (B ′) hybridizes to the complementary sequence (Bc) of the sequence (B), and the sequence (W ′) , Which hybridizes to the complementary sequence (Wc) of the sequence (W). Here, TP is an intervening sequence that does not affect the reaction between the sequence (W ′) and the sequence (B ′) or between the sequence (W ′) and the sequence (Ac ′). May be included. When such a primer is annealed to the template nucleic acid, the sequences (Ac ′) and (W ′) (referred to as (Ac′-W ′) in the primer, the same applies hereinafter) in the primer are converted into the sequence of the target nucleic acid sequence (A−). It is in a hybridized state with Wc) (FIG. 2 (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′-W ′) present on the 5 ′ end side of the synthesized nucleic acid hybridizes to the sequence (Bc-Wc) present in the nucleic acid, and thereby the synthesized nucleic acid 5 'A stem-loop structure is formed at the end portion. As a result, the sequence (A-Wc) on the template nucleic acid becomes a single strand, and another TP having the same sequence as the previous TP hybridizes to this portion ((b) in the figure). Thereafter, an extension reaction from the newly hybridized TP occurs by the strand displacement reaction, and at the same time, the previously synthesized nucleic acid is separated from the template nucleic acid ((c) in the figure).

  In the present invention, “hybridize” means that a part of the primer according to the present invention hybridizes to a target nucleic acid under stringent conditions and does not hybridize to a nucleic acid molecule other than the target nucleic acid. Stringent conditions can be determined depending on the melting temperature Tm (° C.) of the duplex of the primer according to the present invention and its complementary strand, the salt concentration of the hybridization solution, and the like. Sambrook, E .; F. Frisch, T .; Reference can be made to Maniatis; Molecular Cloning 2nd edition, Cold Spring Harbor Laboratory (1989). For example, when hybridization is performed at a temperature slightly lower than the melting temperature of the primer to be used, the primer can be specifically hybridized to the target nucleic acid. Such a primer can be designed using commercially available primer construction software such as Primer 3 (manufactured by Whitehead Institute for Biomedical Research). According to a preferred embodiment of the present invention, a primer that hybridizes to a target nucleic acid comprises a sequence of all or part of a nucleic acid molecule complementary to the target nucleic acid.

  In the above amplification reaction, the phenomenon that the sequence (B′-W ′) hybridizes to the sequence (Bc-Wc) 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. The double-stranded nucleic acid generated by the extension reaction with TP is in a balanced state of dissociation and binding at the base portion at a relatively high temperature, and the double-stranded nucleic acid 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 portion (sequence (A-Wc) on the template nucleic acid) exposed by the formation of the structure, and the polymerase immediately By performing an elongation reaction, the previously synthesized strand is replaced and released, and at the same time, a new double-stranded nucleic acid can be generated.

  A mechanism that can improve the specificity of nucleic acid amplification in the isothermal nucleic acid amplification method by using TP is assumed as follows, for example. In general, many non-specific amplification products in nucleic acid amplification methods are considered to be caused by mishybridization of primers with sequences other than the target nucleic acid sequence (mishybridization). Further, the more sequences in the primer that do not contribute to hybridization with the target nucleic acid sequence, the more likely the primer will be mishybridized, and the fewer such sequences, the less likely the primer will be mishybridized. In this regard, in TP, a sequence (Ac′-W ′) that hybridizes to the target nucleic acid sequence (A-Wc) and a sequence (B′-W ′) that hybridizes to the target nucleic acid sequence (Bc-Wc) Are duplicates. Therefore, TP is less likely to mishybridize compared to a conventional primer having no sequence (W ′), and as a result, it is considered that the specificity of nucleic acid amplification is improved in the isothermal nucleic acid amplification method. Further, it is considered that the specificity of nucleic acid amplification is improved as the length of the sequence (W ′) is longer. However, this mechanism is speculation and does not limit the present invention.

  A mechanism that can improve the nucleic acid amplification rate in the isothermal nucleic acid amplification method by using TP is assumed as follows, for example. That is, as described above, TP is unlikely to mishybridize to sequences other than the target nucleic acid sequence, so that the efficiency of hybridization to the target nucleic acid sequence is increased, and as a result, the nucleic acid amplification rate is considered to be improved. In addition, it is considered that the longer the length of the sequence (W ′), the higher the nucleic acid amplification rate. However, this mechanism is speculation and does not limit the present invention.

  The design criteria in a preferred embodiment of TP 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. The conditions for this are as follows: the base number of the sequence (Ac ′) is X, the base number of the region between the sequence (A) and the sequence (B) in the target nucleic acid sequence is Y, the sequence (W) and Consider the number of bases in (W ′) as Z. First, the number of bases (X + Z) of the sequence (Ac'-W ') and the number of bases (Y-Z) of the region sandwiched between the sequence (A-Wc) and the sequence (B-W) in the target nucleic acid sequence The ratio (X−Y + 2Z) / X of the difference (X−Y + 2Z) to 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 which is not related to primer hybridization. In addition, in order for a new primer to efficiently anneal to a template nucleic acid, it is necessary to efficiently perform the above-described stem-loop structure formation. For efficient stem-loop structure formation, that is, efficient hybridization between the sequence (B′-W ′) and the sequence (Bc-Wc), the sequence (B′-W ′) and the sequence The distance (X + YZ) between (Bc−Wc) 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′-W ′) to efficiently hybridize to the sequence (Bc-Wc), it is considered preferable that the number of bases between both sequences is small. On the other hand, in order for the sequence (B′-W ′) to hybridize to the sequence (Bc-Wc) to make the portion of the sequence (A) on the template nucleic acid single-stranded, the sequence (B′-W It is considered that a larger number of bases between ') and the sequence (Bc-Wc) is preferable.

  From the above viewpoints, in a preferred embodiment of the present invention, between the sequence (W ′) and the sequence (B ′), and between the sequence (W ′) and the sequence (Ac ′). (X−Y + 2Z) / X is −1.00 or more, preferably 0.00 or more, more preferably 0.05 or more, and further preferably 0.10 or more. .50 or less, preferably 1.00 or less, more preferably 0.50 or less, and even more preferably 0.25 or less. Further, (X + Y-Z) is preferably 10 or more, more preferably 20 or more, more preferably 25 or more, and is preferably 60 or less, more preferably 50 or less, more preferably 48 or less, more preferably 45 or less, more preferably 42 or less.

  Further, an intervening sequence (the number of bases is Y ′) exists between the sequence (W ′) and the sequence (B ′) or between the sequence (W ′) and the sequence (Ac ′). In a preferred embodiment of the present invention, {X- (Y-Y ') + 2Z} / X is -1.00 or more, preferably 0.00 or more, more preferably 0.05 or more, more preferably Is designed to be 0.10 or more, 1.50 or less, preferably 1.00 or less, more preferably 0.50 or less, and even more preferably 0.25 or less. Further, (X + Y + Y′−Z) is preferably 10 or more, more preferably 20 or more, more preferably 25 or more, and preferably 100 or less, more preferably 75 or less, and even more preferably 50 or less. .

  The TP has a chain length that allows base pairing with a target nucleic acid while maintaining the necessary specificity under given conditions. The chain length of the TP is preferably 15 to 100 nucleotides, more preferably 30 to 60 nucleotides. The lengths of the sequence (Ac ′) and the sequence (B ′) constituting the TP are each preferably 5 to 50 nucleotides, more preferably 10 to 30 nucleotides. If necessary, an intervening sequence that does not affect the reaction may be inserted between the sequence (W ′) and the sequence (B ′) or between the sequence (W ′) and the sequence (Ac ′). Good.

The primer design method (0) (nucleic acid amplification primer design method) of the present invention includes the following steps (D1), (D2) and (D3) as shown in FIG. 1B. And
(D1) A step of selecting a sequence (A) in the target nucleic acid sequence and determining a sequence (Ac ′) complementary to the sequence (A) as an annealing sequence on the 3 ′ end side of the primer.
(D2) In the target nucleic acid sequence, a sequence (B) existing on the 5 ′ end side from the sequence (A) is selected, and the sequence (B) is used as a stem loop forming sequence on the 5 ′ end side of the primer. Determining the same sequence (B ′)
(D3) In the target nucleic acid sequence, a sequence (W) between the sequence (A) and the sequence (B), and a sequence complementary to the sequence (W) on the 3 ′ end side from the sequence (A) (Wc) is selected, and the same sequence (W ′) as the sequence (W) is determined as a sequence between the sequences (Ac ′) and (B ′) of the primer.

  In the step (D3), the number of bases of the sequence (W) of the target nucleic acid sequence is, for example, 1 or more, preferably 2 or more, more preferably 3 or more. In the step (D3), the sequence (W) of the target nucleic acid sequence may be included in at least one of the sequence (A) and the sequence (B).

The primer production method of the present invention is a method for producing a primer used for amplification of a target nucleic acid sequence,
Including primer design process and primer synthesis process,
The primer designing step is performed by the primer designing method of the present invention, and is not particularly limited in other respects. As described above, the primer production method (0) of the present invention may be used as the primer production method of the present invention, or alternatively, the primer design methods (1) to (10) described later may be used. Either of them may be used.

  The primer production method of the present invention may be any method that can be used for oligonucleotide synthesis, and is not particularly limited. For example, the phosphoramidite method, the phosphate triester method, the H-phosphonate method, the thiophosphonate Law.

  The primer produced by the primer production method of the present invention is composed of deoxynucleotides and / or ribonucleotides. In the present invention, “ribonucleotide” (sometimes simply referred to as “N”) refers to ribonucleotide triphosphate such as ATP, UTP, CTP, GTP, and the like. Furthermore, ribonucleotides include these derivatives, for example, ribonucleotides (α-thio-ribonucleotides) in which the oxygen atom of the phosphate group at the α-position is replaced with a sulfur atom.

  Primers produced by the primer production method of the present invention include oligonucleotide primers composed of unmodified deoxynucleotides and / or modified deoxynucleotides, and oligonucleotide primers composed of unmodified ribonucleotides and / or modified ribonucleotides Also included are chimeric oligonucleotide primers containing unmodified deoxynucleotides and / or modified deoxynucleotides and unmodified ribonucleotides and / or modified ribonucleotides.

The primer set of the present invention, as described above,
A primer set used for amplification of a target nucleic acid sequence in a double-stranded template nucleic acid comprising a first template nucleic acid and a second template nucleic acid,
The primer set includes a first primer and a second primer,
The first primer is the primer (0) of the present invention or the primer (8) of the present invention,
The second primer includes, on the 3 ′ end side, a sequence (Ec ′) complementary to the sequence (E ′) on the 3 ′ end side of the target nucleic acid sequence in the second template nucleic acid. It includes the same sequence (F ′) as the sequence (F) present on the 5 ′ side of the sequence (E) of the target nucleic acid sequence in the second template nucleic acid. The primer (8) of the present invention will be described later.

  In the primer set of the present invention, each of the two primers of the pair of primers may be TP. That is, the second primer may be any one of the primers (0) to (10).

  The mechanism of action considered for a nucleic acid amplification reaction using a primer set in which two of the pair of primers are both TP (TP-TP) will be described with reference to FIG. However, this figure only shows an example of a possible mechanism of action for a part of the steps of the nucleic acid amplification reaction, and does not limit the present invention. In addition, in the same figure, for simplification of explanation, the two sequences to be hybridized are mutually complementary sequences, but the present invention is not limited thereby. First, TP hybridizes to the sense strand of the target nucleic acid, and the primer extension reaction occurs ((a) in the figure). Next, a stem-loop structure is formed on the extended strand (-), and thereby, a new TP hybridizes to the sequence (A) on the target nucleic acid sense strand that has become a single strand ((b) in the figure). The primer extension reaction occurs, and the previously synthesized extension strand (-) is released. Next, the second TP hybridizes to the sequence (E) on the extended chain (−) that has been desorbed ((c) in the same figure), the primer extension reaction occurs, and the extended chain (+) is synthesized. ((D) in the figure). A stem-loop structure is formed at the 3 ′ end of the generated extended strand (+) and the 5 ′ end of the extended strand (−), and a new second TP is added to the sequence (E) on the extended strand (−). Are hybridized, and the extended chain (-) is removed, and an intermediate composed of the extended chain (+) is formed ((f) in the figure). TP hybridizes to the sequence (A) and sequence (Bc) (FIG. (G)), and an extended chain (−) is generated by the extension reaction (FIG. (H)). Thereafter, loop formation, extension reaction, etc. occur one after another, and the target nucleic acid sequence is amplified.

The second primer may be a primer produced by the same production method as the primer (0). That is,
The second primer is produced by a production method including a primer design step and a primer synthesis step,
The primer designing step may include the following steps (D′ 1), (D′ 2), and (D′ 3).
(D′ 1) In the target nucleic acid sequence in the second template nucleic acid, the sequence (E) is selected, and the sequence complementary to the sequence (E) (Ec) is used as the annealing sequence on the 3 ′ end side of the primer. Step to determine ').
(D′ 2) In the target nucleic acid sequence in the second template nucleic acid, a sequence (F) present on the 5 ′ end side from the sequence (E) is selected, and the stem on the 5 ′ end side of the primer Determining the same sequence (F ′) as the sequence (F) as a loop-forming sequence.
(D′ 3) In the target nucleic acid sequence in the second template nucleic acid, the sequence (V) between the sequence (E) and the sequence (F), and on the 3 ′ end side from the sequence (E) The sequence (V) and the complementary sequence (Vc) are selected, and the same sequence (V ′) as the sequence (V) as the sequence between the sequences (Ec ′) and (F ′) of the primer Determining.

  In the primer set of the present invention, the first primer included in the primer set is the nucleic acid amplification primer of the present invention, and the second primer included in the primer set is a complementary sequence of the target nucleic acid sequence. A folded sequence (H-Hc ′) containing a sequence (Gc ′) that hybridizes to the sequence (G) of the 3 ′ end portion at the 3 ′ end portion and two nucleic acid sequences that hybridize to each other on the same strand May be a primer (hereinafter referred to as FP) containing 5 ′ of the sequence (Gc ′).

  As described above, the FP contained in the primer set of the present invention hybridizes to the sequence (G) of the 3 ′ end portion of the complementary sequence of the target nucleic acid sequence (the strand opposite to the strand to which TP hybridizes). A folded sequence (H-Hc ′) containing a sequence (Gc ′) that soybeans at the 3 ′ end portion and two nucleic acid sequences that hybridize to each other on the same strand is located on the 5 ′ side of the sequence (Gc ′). Is included. Such an FP structure is, for example, as shown in FIG. 4, but is not limited to the sequence or the number of nucleotides shown in the figure. The length of the sequence (Gc ′) constituting the FP is preferably 5 to 50 nucleotides, more preferably 10 to 30 nucleotides. The length of the folded sequence (H-Hc ′) 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 (H-Hc ′) 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 (Gc ′) and the folded sequence (H-Hc ′).

  A possible mechanism of action for the nucleic acid amplification reaction by TP and FP will be described with reference to FIG. 5 (FIGS. 5A and 5B). In the figure, for simplification of description, the two sequences to be hybridized are complementary to each other, but the present invention is not limited thereby. First, TP hybridizes to the sense strand of the target nucleic acid, and the primer extension reaction occurs ((a) in the figure). Next, a stem-loop structure is formed on the extended strand (-), whereby a new TP is hybridized to the sequence (A) on the target nucleic acid sense strand that has become a single strand ((b) in the figure) An extension reaction of the primer occurs, and the previously synthesized extension strand (−) is eliminated. Next, FP hybridizes to the sequence (G) on the detached extended strand (−) (FIG. (C)), the primer extension reaction occurs, and the extended strand (+) is synthesized (same as above). (D). A stem-loop structure is formed at the 3 ′ end of the generated extended strand (+) and the 5 ′ end of the extended strand (−) (FIG. (E)), and the extended strand (+) which is the free 3 ′ end. At the same time, an extension reaction takes place from the loop tip of the above, and the extension chain (-) is detached ((f) in the figure). 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, TP hybridizes to the sequence (A) and sequence (Bc) (FIG. (G)), and an extended chain (−) is generated by the extension reaction (FIG. (H) and (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. (H)), and by an extension reaction therefrom (FIG. (I)), A single-stranded nucleic acid having folding sequences at both ends and alternately containing extended strands (+) and extended strands (−) is generated via sequences derived from TP and FP ((j) in the figure). In this single-stranded nucleic acid, the free 3 ′ end (starting point of complementary strand synthesis) is provided by the folded sequence present at the 3 ′ end (FIG. (K)), so that the same extension reaction is repeated, The chain length is doubled per extension reaction ((l) and (m) in the figure). In addition, in the extended strand (−) from TP desorbed in (i) in the same figure, the free 3 ′ end (starting point of complementary strand synthesis) is provided by the folded sequence present at the 3 ′ end (same as the complementary strand synthesis origin). FIG. (N)), a single-stranded chain in which stem-loop structures are formed at both ends by extension reaction therefrom, and alternately include extended strands (+) and extended strands (−) via a sequence derived from a primer. Nucleic acids are produced ((o) in the figure). 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 a sequence derived from TP and FP between the extended strand (+) and the extended strand (−), so that each primer hybridizes. It is possible to cause an extension reaction by soy, and this significantly amplifies the sense strand and the antisense strand of the target nucleic acid.

  The primer set according to the present invention may include a boost primer (hereinafter referred to as BP) in addition to TP and FP. BP 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 is amplified by TP and FP, the amplification product has the target nucleic acid sequence and its complementary sequence alternately as described above. 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. BP 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.

  BP is not necessarily limited to one type, and two or more types of BP may be used at the same time in order to improve the speed and specificity of the nucleic acid amplification reaction. These BPs are typically composed of sequences different from TP and FP, but may hybridize to partially overlapping regions as long as they do not compete with these primers. The chain length of BP is preferably 2 to 100 nucleotides, more preferably 5 to 50 nucleotides, and even more preferably 7 to 30 nucleotides.

  The main purpose of BP is to assist the nucleic acid amplification reaction by TP and FP more rapidly. Therefore, BP preferably has a Tm value lower than the Tm value at the 3 ′ end of each of TP and FP. Further, the addition amount of BP to the amplification reaction solution is preferably smaller than the addition amounts of TP and FP.

  Examples of BP include those having a structure capable of forming a loop as described in WO 02/24902 pamphlet and giving a starting point for complementary strand synthesis to the loop portion. It is not limited to. That is, as long as it is within the target nucleic acid sequence, it may provide a complementary strand synthesis origin at any site.

  The schematic diagram of the amplification reaction when BP is used is shown in (a) to (n) of FIG. (A) (b) (e) (f) (i) (l), (a) (b) (f) (j) (m), (a) (b) ( f) (j) (g) (k) (n), (a) (c) (g) (k) (n) or (a) (d) (h) Exists. Hereinafter, these routes will be described in order. First, a first route having the main routes of (a), (b), (e), (f), (i), and (l) will be described. In this pathway, first, the first primer (TP) is hybridized to the sense strand of the target nucleic acid (FIG. (A)), and the TP elongation reaction occurs to produce an extended strand tp_1 (FIG. (B)). )). Next, BP hybridizes to the extended chain tp_1 ((b) in the figure), and an extension reaction of the BP occurs to generate an extended chain elBP ((e) in the same figure). In addition, the second primer (FP) hybridizes to the extended chain tp_1 ((b) in the figure), and the FP extension reaction occurs to generate an extended chain fp_tp_1 ((f) in the same figure). Next, the extended chain elBP hybridizes to the extended chain fp_tp_1 ((i) in the figure), and an extension reaction of the extended chain elBP occurs to generate an extended chain bp_fp_1 ((l) in the same figure). Furthermore, a stem-loop structure is formed at the 3 ′ end of the extended strand bp_fp_1 ((1) in the figure), and an extension reaction occurs using the 3 ′ end as a complementary strand synthesis origin. Next, a second route having the main routes of (a), (b), (f), (j), and (m) will be described. This route is common to the first route until the extended chain fp_tp_1 is generated ((f) in the figure). A stem-loop structure is formed at the 3 ′ end of the extended strand fp_tp_1 (FIG. (F)), and an extension reaction occurs using the 3 ′ end as a complementary strand synthesis origin to generate an extended strand fp_fp_2 (FIG. (J )). Next, a stem-loop structure is formed at the 3 ′ end of the extended strand fp_fp_2 ((j) in the figure), and an extension reaction occurs using the 3 ′ end as a complementary strand synthesis origin to generate an extended strand fp_fp_4 (same as above). Figure (m)). Furthermore, a stem-loop structure is formed at the 3 ′ end of the extended strand fp_fp_4 ((m) in the figure), and an extension reaction occurs using the 3 ′ end as a complementary strand synthesis origin. Subsequently, a third route having the main routes of (a), (b), (f), (j), (g), (k), and (n) will be described. This route is common to the second route until the extended chain fp_fp_2 is generated ((j) in the figure). TP hybridizes to the extended chain fp_fp_2 ((j) in the figure), and an extension reaction of the TP occurs to generate an extended chain tp_fp_1 ((g) in the same figure). A stem-loop structure is formed at the 3 ′ end of the extended strand tp_fp_1 (FIG. (G)), and an extension reaction occurs using the 3 ′ end as a complementary strand synthesis origin to generate an extended strand tp_tp_2 (FIG. (K) )). In addition, a stem-loop structure is formed at the 3 ′ end of the extended chain tp_tp_2 ((k) in the figure), and an extension reaction occurs using the 3 ′ end as a complementary chain synthesis origin to generate an extended chain tp_tp_4 (in the same figure). (N)). Furthermore, a stem-loop structure is formed at the 3 ′ end of the extended strand tp_tp — 4 ((n) in the figure), and an extension reaction occurs using the 3 ′ end as a complementary strand synthesis origin. Further, TP hybridizes to the extended chain tp_tp_4 ((n) in the figure), and the TP elongation reaction occurs. Next, a fourth route having the main routes of (a), (c), (g), (k), and (n) will be described. In this pathway, first, FP hybridizes to the antisense strand of the target nucleic acid (FIG. (A)), the FP elongation reaction occurs, and an extended strand fp_1 is produced (FIG. (C)). Next, the first primer (TP) is hybridized to the extended chain fp_1 ((c) in the figure), and the extension reaction of the TP occurs to generate an extended chain tp_fp_1 ((g) in the same figure). FIG. 9G and subsequent figures are common to the third route. Finally, a fifth route having the main routes of (a), (d), and (h) in the figure will be described. In this pathway, first, BP hybridizes to the antisense strand of the target nucleic acid (FIG. (A)), the BP elongation reaction occurs, and an elongated strand bp_1 is produced (FIG. (D)). Next, TP hybridizes to the extended chain bp_1 ((d) in the figure), and the extension reaction of the TP occurs to generate an extended chain elTP ((h) in the same figure). The above-described reaction path is an exemplification, and the present invention is not limited or limited by the reaction path.

As described above, the first nucleic acid amplification method of the present invention is a complementary nucleic acid amplification method for synthesizing a nucleic acid complementary to a target nucleic acid sequence in a template nucleic acid, and includes the following (S1) to (S5) The other steps are not particularly limited.
(S1) A step of producing a primer by any one of the production methods (0) to (10) of the primer of the present invention or providing the primer (8) of the present invention by any method.
(S2) A step of providing a template nucleic acid.
(S3) A step of annealing the primer to the template nucleic acid and performing a primer extension reaction to synthesize a complementary nucleic acid containing a complementary sequence of the target nucleic acid sequence.
(S4) The sequence (B ′) present on the 5 ′ side of the complementary nucleic acid synthesized in step (S3) is hybridized to the sequence (Bc) present on the complementary nucleic acid, thereby A step of making the portion of the sequence (A) a single strand.
(S5) Annealing another primer having the same sequence as the primer to the portion of the sequence (A) on the template nucleic acid made into a single strand by the step (S4), thereby carrying out a strand displacement reaction, A step of replacing the complementary nucleic acid synthesized in the step (S3) with a complementary nucleic acid newly synthesized by the other primer. The primer production methods (1) to (10) will be described later.

  According to a preferred embodiment of the present invention, the double-stranded nucleic acid obtained in step (S5) is repeatedly used in step (S4). That is, since the double-stranded nucleic acid obtained by the step (S5) has the same structure as that obtained by the step (S3), it is directly used in the step (S4). Thereby, a nucleic acid complementary to the target nucleic acid sequence in the template nucleic acid can be mass-produced.

  According to a preferred embodiment of the present invention, the first nucleic acid amplification method of the present invention is carried out isothermally. Therefore, according to a preferred embodiment of the present invention, the step (S3), the step (S4) and the step (S5) are performed isothermally. Here, “isothermal” refers to maintaining an approximately constant temperature condition such that the enzyme and the primer can substantially function.

  In a preferred embodiment of the present invention, in order for the primer to anneal to the target nucleic acid, for example, the reaction temperature is preferably set to a temperature close to or below the melting temperature (Tm) of the primer, The level of stringency is preferably set in consideration of the melting temperature (Tm) of the primer. Therefore, this temperature is preferably about 20 ° C to about 75 ° C, more preferably about 35 ° C to about 65 ° C.

The design method of the primer set of the present invention is:
A method for designing a primer set used for amplification of a target nucleic acid sequence in a double-stranded template nucleic acid comprising a first template nucleic acid and a second template nucleic acid,
The primer set includes a first primer and a second primer,
The first primer is designed by any one of the primer designing methods (0) to (10) of the present invention,
The 3 ′ end side of the second primer includes a sequence (Ec ′) complementary to the sequence (E) on the 3 ′ end side of the target nucleic acid sequence in the second template nucleic acid, and 5 ′ end side Further, the second template nucleic acid is designed to include the same sequence (F ′) as the sequence (F) existing 5 ′ to the sequence (E) of the target nucleic acid sequence. . The primer set design method of the present invention using any one of the primer design methods (0) to (10) of the present invention is referred to as “primer set design method (0)” and “primer set design”, respectively. "Method (1)", "Primer set design method (2)", "Primer set design method (3)", "Primer set design method (4)", "Primer set design method (5)", “Primer set design method (6)”, “Primer set design method (7)”, “Primer set design method (8)”, “Primer set design method (9)”, “Primer set design method” (10) ". The primer designing methods (1) to (10) of the present invention will be described later.

The method for producing the primer set of the present invention includes:
A method for producing a primer set used for amplification of a target nucleic acid sequence in a double-stranded template nucleic acid comprising a first template nucleic acid and a second template nucleic acid,
Including primer set design process and primer set synthesis process,
The primer set design process is performed by the primer set design method of the previous basic invention. The primer set production method of the present invention using any one of the primer set design methods (0) to (10) of the present invention is referred to as "primer set production method (0)", "primer set production method", respectively. Manufacturing method (1) "," Primer set manufacturing method (2) "," Primer set manufacturing method (3) "," Primer set manufacturing method (4) "," Primer set manufacturing method (5) " , “Primer set production method (6)”, “Primer set production method (7)”, “Primer set production method (8)”, “Primer set production method (9)”, “Primer set production” Method (10) ".

As described above, the second nucleic acid amplification method of the present invention is a method of amplifying a target nucleic acid sequence in a double-stranded template nucleic acid, and includes the following steps (S′1) to (S′5): The other points are not particularly limited.
(S′1) A primer set is produced by the method for producing a primer set of the present invention, or the first primer is the primer (0) or the primer (8). Is provided by any method.
(S′2) A step of providing a double-stranded template nucleic acid comprising a first template nucleic acid and a second template nucleic acid.
(S′3) The first and second primers containing the complementary sequences of the target nucleic acid sequences by annealing the first and second primers to the first and second template nucleic acids, respectively, and performing primer extension reactions, respectively. Synthesizing complementary nucleic acids;
(S′4) The sequences (B ′) and (F ′) present on the 5 ′ side of the first and second complementary nucleic acids synthesized by the step (S′3) are present on the complementary nucleic acids. A step of hybridizing to the sequence (Bc) and the sequence (Fc), respectively, so that the portions of the sequence (A) and the sequence (E) on the first and second template nucleic acids become single-stranded.
(S′5) The sequence (A) and the sequence (E) on the first and second template nucleic acids made into single strands by the step (S′4) have the same sequence as the primer. By performing the strand displacement reaction by annealing the other primer, the first and second complementary nucleic acids synthesized in the step (S′3) are replaced with the complementary nucleic acid newly synthesized by the other primer. Process.

  According to a preferred embodiment of the present invention, the double-stranded nucleic acid obtained in step (S′5) is repeatedly used in step (S′4). That is, since the double-stranded nucleic acid obtained by the step (S′5) has the same structure as that obtained by the step (S′3), it is directly used in the step (S′4). Thereby, a nucleic acid complementary to the target nucleic acid sequence in the template nucleic acid can be mass-produced.

  According to a preferred embodiment of the present invention, the second nucleic acid amplification method of the present invention is carried out isothermally. Therefore, according to a preferred embodiment of the present invention, the step (S′3), the step (S′4) and the step (S′5) are performed isothermally. Here, “isothermal” refers to maintaining an approximately constant temperature condition such that the enzyme and the primer can substantially function.

  As in the first nucleic acid amplification method of the present invention, in order for the primer to anneal to the target nucleic acid, for example, the reaction temperature is preferably set to a temperature near the melting temperature (Tm) of the primer or lower. Furthermore, it is preferable to set the stringency level in consideration of the melting temperature (Tm) of the primer. Therefore, this temperature is preferably about 20 ° C to about 75 ° C, more preferably about 35 ° C to about 65 ° C.

  The template nucleic acid or nucleic acid sample containing the target nucleic acid sequence used in the first and second nucleic acid amplification methods of the present invention (hereinafter sometimes simply referred to as “the nucleic acid amplification method of the present invention”) is DNA or RNA Either of them may be used. DNA includes any of cDNA, genomic DNA, and synthetic DNA. RNA includes all of RNA, mRNA, rRNA, siRNA, hnRNA and synthetic RNA. These nucleic acids can be prepared, for example, from blood, tissues, cells, biological samples such as animals and plants, or microorganism-derived samples separated from biological samples, food, soil, waste water, and the like.

  Isolation of the template nucleic acid or nucleic acid sample can be performed by any method, and examples thereof include a method using a lysis treatment with a surfactant, sonication, shaking and stirring using glass beads, a French press, and the like. In addition, when an endogenous nuclease is present, it is preferable to purify the isolated nucleic acid. Nucleic acid purification can be performed, for example, by phenol extraction, chromatography, ion exchange, gel electrophoresis, density-dependent centrifugation, and the like.

  More specifically, the template nucleic acid or the nucleic acid sample is a double-stranded nucleic acid such as genomic DNA or PCR fragment isolated by the above method, or a cDNA prepared by reverse transcription from total RNA or mRNA. Any single stranded nucleic acid can be used. In the case of the above double-stranded nucleic acid, it can be used more optimally by carrying out denaturing and making it into a single strand.

  The enzyme used in the reverse transcription reaction is not particularly limited as long as it has cDNA synthesis activity using RNA as a template. For example, avian myeloblastosis virus-derived reverse transcriptase (AMV RTase), rous-related virus 2 reverse transcriptase (RAV-2 RTase), Moloney murine leukemia virus-derived reverse transcriptase (MMLV RTase), and other reverse transcriptases. In addition, a DNA polymerase having reverse transcription activity can also be used. For the purpose of the present invention, an enzyme having reverse transcription activity at a high temperature is optimal. For example, a DNA polymerase derived from Thermus bacterium (Tth DNA polymerase, etc.), a DNA polymerase derived from Bacillus bacterium, etc. can be used. As a particularly preferable enzyme, for example, as a DNA polymerase derived from a thermophilic Bacillus bacterium, B. st-derived DNA polymerase (Bst DNA polymerase); Ca-derived DNA polymerase (Bca DNA polymerase), for example, BcaBEST DNA polymerase, Bca (exo-) DNA polymerase, and the like can be mentioned. For example, Bca DNA polymerase does not require manganese ions for reaction, and can synthesize cDNA while suppressing secondary structure formation of template RNA under high temperature conditions.

  In the nucleic acid amplification method of the present invention, even when the template nucleic acid is a double-stranded nucleic acid, it can be used for the reaction as it is. However, if necessary, it can be denatured into a single-stranded nucleic acid by denaturing it. It is also possible to efficiently anneal the primer. Increasing the temperature to about 95 ° C is a preferred nucleic acid denaturation method. As another method, it is possible to denature by raising the pH, but in this case, it is necessary to lower the pH in order to hybridize the primer to the target nucleic acid.

  The polymerase used in the nucleic acid amplification method of the present invention is not particularly limited as long as it has strand displacement activity (strand displacement ability), and any of room temperature, medium temperature, and heat resistance can be suitably used. . Further, this polymerase may be either a natural body or a mutant with artificial mutation. Examples of such a polymerase include DNA polymerase. Furthermore, it is preferable that this DNA polymerase has substantially no 5 ′ → 3 ′ exonuclease activity. Examples of such a DNA polymerase include thermophilic Bacillus bacteria such as Bacillus stearothermophilus (hereinafter referred to as “B.st”) and Bacillus caldotenax (hereinafter referred to as “B.ca”). Examples include mutants lacking the 5 ′ → 3 ′ exonuclease activity of the derived DNA polymerase, Klenow fragment of E. coli derived DNA polymerase I, and the like. Examples of the DNA polymerase used in the nucleic acid amplification reaction include Vent DNA polymerase, Vent (Exo-) DNA polymerase, DeepVent DNA polymerase, DeepVent (Exo-) DNA polymerase, Φ29 phage DNA polymerase, MS-2 phage DNA polymerase, Z -Taq DNA polymerase, Pfu DNA polymerase, Pfu turbo DNA polymerase, KOD DNA polymerase, 9 ° Nm DNA polymerase, Therminater DNA polymerase and the like.

  Furthermore, in the nucleic acid amplification method of the present invention, by using a DNA polymerase having reverse transcription activity, for example, BcaBEST DNA polymerase, Bca (exo-) DNA polymerase, etc., reverse transcription reaction and cDNA from total RNA or mRNA are performed. The template DNA polymerase reaction can be carried out with one kind of polymerase. Moreover, you may use combining DNA polymerase and above-mentioned reverse transcriptases, such as MMLV reverse transcriptase.

  Other reagents used in the nucleic acid amplification method of the present invention include, for example, a catalyst such as magnesium chloride, magnesium acetate, and magnesium sulfate, a substrate such as dNTP mix, a tris hydrochloride buffer, a tricine buffer, a sodium phosphate buffer, and a phosphate. A buffer solution such as potassium buffer can be used. Furthermore, additives such as dimethyl sulfoxide (DMSO) and betaine (N, N, N-trimethylglycine), acidic substances described in International Publication No. 99/54455 pamphlet, and cationic complexes may be used.

  In the nucleic acid amplification method of the present invention, a melting temperature adjusting agent can be added to the reaction solution in order to increase the nucleic acid amplification efficiency. The melting temperature (Tm) of a nucleic acid is generally determined by the specific nucleotide sequence of the duplex forming portion in the nucleic acid. By adding a melting temperature adjusting agent to the reaction solution, this melting temperature can be changed. Therefore, under a certain temperature, the intensity of double strand formation in the nucleic acid can be adjusted. A general melting temperature adjusting agent has an effect of lowering the melting temperature. By adding such a melting temperature adjusting agent, the melting temperature of the duplex forming part between two nucleic acids can be lowered, in other words, the strength of the duplex formation can be lowered. It becomes. Therefore, when such a melting temperature adjusting agent is added to the reaction solution in the nucleic acid amplification reaction, it is efficiently used in a GC-rich nucleic acid region that forms a strong double strand or a region that forms a complex secondary structure. The double-stranded portion can be made into a single strand, and this makes it easy for the next primer to hybridize to the target region after the extension reaction with the primer is completed, so that the nucleic acid amplification efficiency can be increased. The melting temperature adjusting agent used in the present invention and its concentration in the reaction solution are appropriately selected by those skilled in the art in consideration of other reaction conditions that affect the hybridization conditions, such as salt concentration, reaction temperature, etc. The Therefore, the melting temperature adjusting agent is not particularly limited, but is preferably dimethyl sulfoxide (DMSO), betaine, formamide or glycerol, or any combination thereof, more preferably dimethyl sulfoxide (DMSO). .

  Furthermore, in the nucleic acid amplification method of the present invention, an enzyme stabilizer can be added to the reaction solution. Thereby, since the enzyme in the reaction solution is stabilized, the amplification efficiency of the nucleic acid can be increased. The enzyme stabilizer used in the present invention may be any known in the art such as glycerol, bovine serum albumin, saccharides, etc., and is not particularly limited.

  Furthermore, in the nucleic acid amplification method of the present invention, a reagent for enhancing the heat resistance of an enzyme such as DNA polymerase or reverse transcriptase can be added to the reaction solution as an enzyme stabilizer. As a result, the enzyme in the reaction solution is stabilized, so that the nucleic acid synthesis efficiency and amplification efficiency can be increased. Such a reagent may be any known in the art and is not particularly limited, but is preferably a saccharide, more preferably a mono- or oligosaccharide, more preferably trehalose, sorbitol or mannitol, or these It is set as the mixture of 2 or more types.

  The nucleic acid amplification method of the present invention can be performed isothermally. Therefore, according to a preferred embodiment of the nucleic acid amplification method, the nucleic acid amplification method comprises a step of incubating the nucleic acid amplification solution isothermally. Here, “isothermal” refers to maintaining an approximately constant temperature condition such that the enzyme and the primer can substantially function. Furthermore, the “substantially constant temperature condition” means that not only the set temperature is accurately maintained but also a temperature change that does not impair the substantial functions of the enzyme and the primer is allowed. To do.

  The nucleic acid amplification method under a certain temperature condition can be carried out by keeping the temperature at which the activity of the enzyme used can be maintained. Further, in this nucleic acid amplification method, in order for the primer to anneal to the target nucleic acid, for example, the reaction temperature is preferably set to a temperature near or below the melting temperature (Tm) of the primer, The level of stringency is preferably set in consideration of the melting temperature (Tm) of the primer. Therefore, this temperature is preferably about 20 ° C to about 75 ° C, more preferably about 35 ° C to about 65 ° C.

  In the nucleic acid amplification method of the present invention, the amplification reaction is repeated until either the enzyme is inactivated or one of the reagents including the primer is used up.

  In the nucleic acid amplification method of the present invention, a nucleic acid containing an unnatural nucleotide can be used as a template nucleic acid. As used herein, “non-natural nucleotide” means a nucleotide that contains a base other than the base (adenine, guanine, cytosine, and thymine or uracil) contained in a natural nucleotide, and that can be incorporated into a nucleic acid sequence. Examples thereof include xanthosines, diaminopyrimidines, isoG and isoC (Proc. Natl. Acad. Sci. USA 92, 6329-6333, 1995). In general, a nucleic acid amplification enzyme having no heat resistance is used for amplification of a target nucleic acid containing a non-natural nucleotide. On the other hand, since the nucleic acid amplification reaction can be performed at an isothermal temperature of, for example, about 50 ° C., the possibility that the nucleic acid amplification enzyme (DNA polymerase or the like) is inactivated is low as compared with the conventional PCR method. Therefore, the nucleic acid amplification reaction using the primer set according to the present invention is also effective for amplification of a target nucleic acid containing a non-natural nucleotide in which a nucleic acid amplification enzyme having no heat resistance is used. The enzyme used for the amplification of the nucleic acid containing the non-natural nucleotide is not particularly limited as long as it can amplify such a target nucleic acid, but Y188L / E478Q mutant HIV I reverse transcription particularly from the viewpoint of incorporation efficiency Enzymes, AMV reverse transcriptase, Klenow fragment of DNA polymerase, 9 ° N DNA polymerase, HotTub DNA polymerase and the like are suitable (Michael Sismour1 et al., Biochemistry 42, No. 28, 8598, 2003 / US Pat. No. 6,617,106). Description, Michael J. Lutz et al., Bioorganic & Medical Chemistry letters 8, 1149-1152, 1998, etc.). Furthermore, a substance that improves the heat resistance of the nucleic acid amplification enzyme, such as trehalose, can also be added to the reaction solution, whereby the target nucleic acid containing unnatural nucleotides can be more efficiently amplified.

  Next, the primer design methods (1) to (10), the primer production methods (1) to (10), the primers (1) to (10), and the like will be described.

The primer (1)
A primer used for amplification of a target nucleic acid sequence,
A sequence (Ac ′) complementary to the sequence (A) on the 3 ′ end side of the target nucleic acid sequence on the 3 ′ end side of the primer;
The 5 ′ terminal side of the primer contains the same sequence (B ′) as the sequence (B) present 5 ′ to the sequence (A) of the target nucleic acid sequence,
A sequence complementary to the sequence (A5′-3) of 3 bases from the 5 ′ end of the sequence (A) of the target nucleic acid sequence is the sequence of the target nucleic acid (A) and the sequence of the target nucleic acid (A ) And the sequence (C) between the sequence (B) and the sequence (A5′-3) of the 3 bases and the sequence (A5′-3) of the sequence (A5′-3) It is 1 or less in a sequence other than 9 bases including a 3 base sequence adjacent to the 5 ′ end side and a 3 base sequence adjacent to the 3 ′ end side of the sequence (A5′-3). ,
It is characterized by that. The features of the primer (1) other than those described above may be the same as those of the primer (0).

  For example, as shown in FIG. 9, when the complementary sequence of the primer (TP) forms a loop by folding, the ratio of the newly annealed site (A5′-3) to the single strand is large. Efficient amplification is possible. Therefore, a sequence complementary to the sequence (A5′-3) of 3 bases from the 5 ′ end of the sequence (A) of the target nucleic acid sequence is the sequence of the target nucleic acid (A) and the target nucleic acid. In the loop sequence (AC) formed by the sequence (C) between the sequence (A) and the sequence (B), the 3-base sequence (A5′-3) and the sequence (A5′- 1) in a sequence other than 9 bases, including a 3 base sequence adjacent to the 5 ′ end of 3) and a 3 base sequence adjacent to the 3 ′ end of the sequence (A5′-3) The following.

The primer design method (1) is:
A method for designing a primer used for amplification of a target nucleic acid sequence,
A sequence (Ac ′) complementary to the sequence (A) on the 3 ′ end side of the target nucleic acid sequence on the 3 ′ end side of the primer;
The 5 ′ terminal side of the primer contains the same sequence (B ′) as the sequence (B) present 5 ′ to the sequence (A) of the target nucleic acid sequence,
A sequence complementary to the sequence (A5′-3) of 3 bases from the 5 ′ end of the sequence (A) of the target nucleic acid sequence is the sequence of the target nucleic acid (A) and the sequence of the target nucleic acid (A ) And the sequence (C) between the sequence (B) and the sequence (A5′-3) of the 3 bases and the sequence (A5′-3) of the sequence (A5′-3) It is 1 or less in a sequence other than 9 bases including a 3 base sequence adjacent to the 5 ′ end side and a 3 base sequence adjacent to the 3 ′ end side of the sequence (A5′-3). To design,
It is characterized by that. The primer designing method of the present invention may be the primer designing method (1).

Moreover, the manufacturing method (1) of the said primer is:
A method for producing a primer used for amplification of a target nucleic acid sequence,
Including primer design process and primer synthesis process,
The primer designing step is performed by the primer designing method (1). The primer production method of the present invention may be the primer production method (1).

  Although the design method and production method of the primer (1) are not particularly limited, it is preferably designed by the primer design method (1) or by the primer production method (1).

The primer (2)
A primer used for amplification of a target nucleic acid sequence,
A sequence (Ac ′) complementary to the sequence (A) on the 3 ′ end side of the target nucleic acid sequence on the 3 ′ end side of the primer;
The 5 ′ terminal side of the primer contains the same sequence (B ′) as the sequence (B) present 5 ′ to the sequence (A) of the target nucleic acid sequence,
A sequence complementary to the sequence (A5′-3) of 3 bases from the 5 ′ end of the sequence (A) of the target nucleic acid sequence is the sequence (A) of the target nucleic acid sequence and the sequence (A) In a sequence (A-20) comprising a 20-base sequence adjacent to the 3 ′ end side and a 20-base sequence adjacent to the 5 ′ end side of the sequence (A), the 3-base sequence (A5 '-3), a sequence of 3 bases adjacent to the 5' end of the sequence (A5'-3), and a sequence of 3 bases adjacent to the 3 'end of the sequence (A5'-3) It is characterized by being 1 or less in the sequence other than 9 bases. The features of the primer (2) other than those described above may be the same as those of the primer (0).
For example, as shown in FIG. 10, efficient amplification is possible when the ratio of the single-stranded site where the primer (TP) anneals on the genome sequence is large. For this purpose, a sequence complementary to the sequence (A5′-3) of 3 bases from the 5 ′ end of the sequence (A) of the target nucleic acid sequence is the sequence (A) of the target nucleic acid sequence, In the sequence (A-20) comprising a 20 base sequence adjacent to the 3 ′ end of the sequence (A) and a 20 base sequence adjacent to the 5 ′ end of the sequence (A), the 3 bases Sequence (A5′-3), a sequence of 3 bases adjacent to the 5 ′ end of the sequence (A5′-3), and 3 bases adjacent to the 3 ′ end of the sequence (A5′-3) 1 or less in a sequence other than 9 bases including

The primer design method (2) is:
A method for designing a primer used for amplification of a target nucleic acid sequence,
A sequence (Ac ′) complementary to the sequence (A) on the 3 ′ end side of the target nucleic acid sequence on the 3 ′ end side of the primer;
The 5 ′ terminal side of the primer contains the same sequence (B ′) as the sequence (B) present 5 ′ to the sequence (A) of the target nucleic acid sequence,
A sequence complementary to the sequence (A5′-3) of 3 bases from the 5 ′ end of the sequence (A) of the target nucleic acid sequence is the sequence (A) of the target nucleic acid sequence and the sequence (A) In a sequence (A-20) comprising a 20-base sequence adjacent to the 3 ′ end side and a 20-base sequence adjacent to the 5 ′ end side of the sequence (A), the 3-base sequence (A5 '-3), a sequence of 3 bases adjacent to the 5' end of the sequence (A5'-3), and a sequence of 3 bases adjacent to the 3 'end of the sequence (A5'-3) It is designed so that it is 1 or less in the sequence other than 9 bases included.
It is characterized by that. The primer designing method of the present invention may be the primer designing method (2).

Moreover, the manufacturing method (2) of the said primer is:
A method for producing a primer used for amplification of a target nucleic acid sequence,
Including primer design process and primer synthesis process,
The primer designing step is performed by the above-described primer designing method (2). The primer production method of the present invention may be the primer production method (2).

  Although the design method and production method of the primer (2) are not particularly limited, it is preferably designed by the primer design method (2) or by the primer production method (2).

The primer (3) is
A primer used for amplification of a target nucleic acid sequence,
A sequence (Ac ′) complementary to the sequence (A) on the 3 ′ end side of the target nucleic acid sequence on the 3 ′ end side of the primer;
The 5 ′ terminal side of the primer contains the same sequence (B ′) as the sequence (B) present 5 ′ to the sequence (A) of the target nucleic acid sequence,
A primer characterized in that the ratio of G and C is 4 bases or more at 6 bases from the 5 ′ end of the sequence (B) of the target nucleic acid sequence. The features of the primer (3) other than those described above may be the same as those of the primer (0) of the present invention.
Specifically, for example, as shown in FIG. If the ratio of G and C is 4 bases or more in 6 bases from the 5 ′ end of the target nucleic acid sequence (B) (6 bases from the 3 ′ end of (Bc)), the hybrid in this part Since the soybean bond is strong, the loop formed by the sequence (A) shown in the figure and the sequence between sequences (A) and (B) is strong, and the amplification reaction is easily performed efficiently.

The primer design method (3) is:
A method for designing a primer used for amplification of a target nucleic acid sequence,
A sequence (Ac ′) complementary to the sequence (A) on the 3 ′ end side of the target nucleic acid sequence on the 3 ′ end side of the primer;
The 5 ′ terminal side of the primer contains the same sequence (B ′) as the sequence (B) present 5 ′ to the sequence (A) of the target nucleic acid sequence,
It is designed such that the ratio of G and C is 4 bases or more at 6 bases from the 5 ′ end of the sequence (B) of the target nucleic acid sequence. The primer designing method of the present invention may be the primer designing method (3).

Further, the production method (3) of the primer includes:
A method for producing a primer used for amplification of a target nucleic acid sequence,
Including primer design process and primer synthesis process,
The primer designing step is performed by the above-described primer designing method (3). The primer production method of the present invention may be the primer production method (3).

  Although the design method and production method of the primer (3) are not particularly limited, it is preferably designed by the primer design method (3) or by the primer production method (3).

The primer (4)
A primer used for amplification of a target nucleic acid sequence,
A sequence (Ac ′) complementary to the sequence (A) on the 3 ′ end side of the target nucleic acid sequence on the 3 ′ end side of the primer;
The 5 ′ terminal side of the primer contains the same sequence (B ′) as the sequence (B) present 5 ′ to the sequence (A) of the target nucleic acid sequence,
Among the 5 base sequences (A5′-9-13) from the 9th base to the 13th base from the 5 ′ end side of the sequence (A) of the target nucleic acid sequence, an arbitrary sequence of 3 bases (A5 The sequence complementary to '-9-13 (3)) is a 20-base sequence adjacent to the sequence (A5'-9-13 (3)) with 3 bases adjacent to the 5' end (however, Excluding the sequence (B) and the sequence adjacent to the 5 ′ end of the sequence (B)), and the sequence (A5′-9-13 (3)) adjacent to the 3 ′ end with 3 bases 1 sequence or less (excluding the sequence (Bc) complementary to the sequence (B) and the sequence adjacent to the 3 ′ end of the sequence (Bc)), or ,
Among the 5 base sequences (A3′-6-10) from the 6th base to the 10th base from the 3 ′ end side of the sequence (A) of the target nucleic acid sequence, an arbitrary sequence of 3 bases (A3 The sequence complementary to '-6-10 (3)) is a 20 base sequence adjacent to the 5' end side of the sequence (A3'-6-10 (3)) with 3 bases (provided that Excluding the sequence (B) and the sequence adjacent to the 5 ′ end of the sequence (B)), and the sequence (A3′-6-10 (3)) adjacent to the 3 ′ end of the sequence (3 bases apart) The sequence is designed to be 1 or less in the 20 base sequence (excluding the sequence (Bc) complementary to the sequence (B) and the sequence adjacent to the 3 ′ end of the sequence (Bc)). It is characterized by doing. Other features of the primer (4) may be the same as those of the primer (0).
For example, as shown in FIG. 12, when the complementary sequence of the primer (TP) forms a loop by folding, the proportion of the site (A3′-3) where the TP is newly annealed is a single strand. If it is large, efficient amplification is possible. For this reason, a sequence complementary to 3 consecutive bases arbitrarily selected from 5 bases from the 9th base to the 13th base counted from the 5 ′ end side of the sequence (A) of the target nucleic acid sequence, Up to 20 bases in total from the 4th base of the sequence counted upstream from the 5 ′ end of the 3 consecutive bases (ie, adjacent to the 5 ′ end with 3 bases apart) up to 23 bases (however, the sequence (B) and its upstream sequence are not included) and from the 4th base of the sequence counted from the 3 ′ end of the 3 consecutive bases to the downstream side (that is, adjacent to the 3 ′ end side by 3 bases) ) Up to 23 bases in total (up to 20 bases (not including the turnback sequence (Bc) and its downstream sequence)) or less, or the sequence of the target nucleic acid sequence ( A) 10th base from the 6th base counting from the 3 ′ end side From the 4 bases of the sequence counted from the 5 ′ end of the 3 consecutive bases to the upstream side (ie, 5 ′ end side) A total of 20 bases (not including sequence (B) and its upstream sequence) up to the 23rd base and 3 downstream from the 3 ′ end of the 3 consecutive bases. 20 bases in total from the 4th base of the sequence (that is, adjacent to the 3 'end side by 3 bases apart) up to the 23rd base (however, the turnback sequence (Bc) and the downstream side sequence are not included) ) Within one. In the present invention, the “upstream side” of the base sequence means the 5 ′ end side unless otherwise specified. The “downstream side” of the base sequence refers to the 3 ′ end side unless otherwise specified. Further, “base” refers to a nucleobase (sometimes referred to as “base”) unless otherwise specified.

The scientific reason why the primer (4) is effective is more specifically, for example, the following 1. ~ 3. It can be explained as follows. However, these are merely examples of theoretical considerations, and do not limit the present invention.
1. Since the end of the loop is fixed by the stem structure, it is necessary for the new TP to hybridize helically.
2. Since the 5 ′ end of the TP has a turnback region and is long, it is advantageous that the 5 ′ side first forms a nucleus and the 3 ′ side hybridizes in a spiral shape.
Since the 3.5 'end has steric hindrance of the stem structure, it is advantageous that the initial nucleation is some distance away from the 5' end.

The primer design method (4) is:
A method for designing a primer used for amplification of a target nucleic acid sequence,
A sequence (Ac ′) complementary to the sequence (A) on the 3 ′ end side of the target nucleic acid sequence on the 3 ′ end side of the primer;
The 5 ′ terminal side of the primer contains the same sequence (B ′) as the sequence (B) present 5 ′ to the sequence (A) of the target nucleic acid sequence,
Among the 5 base sequences (A5′-9-13) from the 9th base to the 13th base from the 5 ′ end side of the sequence (A) of the target nucleic acid sequence, an arbitrary sequence of 3 bases (A5 The sequence complementary to '-9-13 (3)) is a 20-base sequence adjacent to the sequence (A5'-9-13 (3)) with 3 bases adjacent to the 5' end (however, Excluding the sequence (B) and the sequence adjacent to the 5 ′ end of the sequence (B)), and the sequence (A5′-9-13 (3)) adjacent to the 3 ′ end with 3 bases 1 sequence or less (excluding the sequence (Bc) complementary to the sequence (B) and the sequence adjacent to the 3 ′ end of the sequence (Bc)), or ,
Among the 5 base sequences (A3′-6-10) from the 6th base to the 10th base from the 3 ′ end side of the sequence (A) of the target nucleic acid sequence, an arbitrary sequence of 3 bases (A3 The sequence complementary to '-6-10 (3)) is a 20 base sequence adjacent to the 5' end side of the sequence (A3'-6-10 (3)) with 3 bases (provided that Excluding the sequence (B) and the sequence adjacent to the 5 ′ end of the sequence (B)), and the sequence (A3′-6-10 (3)) adjacent to the 3 ′ end of the sequence (3 bases apart) The sequence is designed to be 1 or less in the 20 base sequence (excluding the sequence (Bc) complementary to the sequence (B) and the sequence adjacent to the 3 ′ end of the sequence (Bc)). It is characterized by doing. The primer designing method of the present invention may be the primer designing method (4).

The primer production method (4) includes:
A method for producing a primer used for amplification of a target nucleic acid sequence,
Including primer design process and primer synthesis process,
The primer designing step is performed by the above-described primer designing method (4). The primer production method of the present invention may be the primer production method (4).

  The design method and production method of the primer (4) are not particularly limited, but it is preferably designed by the primer design method (4) or by the primer production method (4).

The primer (5)
A primer used for amplification of a target nucleic acid sequence,
A sequence (Ac ′) complementary to the sequence (A) on the 3 ′ end side of the target nucleic acid sequence on the 3 ′ end side of the primer;
The 5 ′ terminal side of the primer contains the same sequence (B ′) as the sequence (B) present 5 ′ to the sequence (A) of the target nucleic acid sequence,
The sequence complementary to the 6 base sequence (Ac′3′-6) from the 3 ′ end of the primer (Ac ′) of the primer is the sequence of the reaction intermediate formed by the extension reaction of the primer. Thus, it is designed so that it does not exist (zero) in the sequence excluding the primer sequence and its complementary sequence portion. The features of the primer (5) other than those described above may be the same as those of the primer (0) of the present invention.
Specifically, for example, as shown in FIG. A sequence complementary to the sequence of 6 bases from the 3 ′ end side of the primer is a sequence of a reaction intermediate formed by the extension reaction of the primer, except for the sequence of the primer and its complementary sequence portion. In addition, when it does not exist (0), it is easy to prevent the primer (TP) from hybridizing to a place other than the originally designed place, and the amplification reaction can be performed efficiently.

The primer design method (5) is:
A method for designing a primer used for amplification of a target nucleic acid sequence,
A sequence (Ac ′) complementary to the sequence (A) on the 3 ′ end side of the target nucleic acid sequence on the 3 ′ end side of the primer;
The 5 ′ terminal side of the primer contains the same sequence (B ′) as the sequence (B) present 5 ′ to the sequence (A) of the target nucleic acid sequence,
The sequence complementary to the 6 base sequence (Ac′3′-6) from the 3 ′ end of the primer (Ac ′) of the primer is the sequence of the reaction intermediate formed by the extension reaction of the primer. Thus, it is designed so that it does not exist (zero) in the sequence excluding the primer sequence and its complementary sequence portion. The primer designing method of the present invention may be the primer designing method (5).

Moreover, the manufacturing method (5) of the said primer is:
A method for producing a primer used for amplification of a target nucleic acid sequence,
Including primer design process and primer synthesis process,
The primer designing step is performed by the primer designing method (5) described above. The primer production method of the present invention may be the primer production method (5).

  The design method and production method of the primer (5) are not particularly limited, but it is preferably designed by the primer design method (5) or by the primer production method (5).

The primer (6)
A primer used for amplification of a target nucleic acid sequence,
A sequence (Ac ′) complementary to the sequence (A) on the 3 ′ end side of the target nucleic acid sequence on the 3 ′ end side of the primer;
The 5 ′ terminal side of the primer contains the same sequence (B ′) as the sequence (B) present 5 ′ to the sequence (A) of the target nucleic acid sequence,
A sequence complementary to the 3 base sequence (Ac′3′-3) from the 3 ′ end side of the sequence (Ac ′) of the primer does not exist (0) in the primer. . Other characteristics of the primer (6) may be the same as those of the primer (0).
The sequence of the three bases is, for example, as shown in the schematic diagram of FIG. Thus, the absence of a sequence complementary to the sequence of 3 bases from the 3 ′ end side of the primer in the sequence of the same type of primer prevents the same type of primer from causing an undesirable amplification reaction. Cheap.

The primer design method (6) is:
A method for designing a primer used for amplification of a target nucleic acid sequence,
A sequence (Ac ′) complementary to the sequence (A) on the 3 ′ end side of the target nucleic acid sequence on the 3 ′ end side of the primer;
The 5 ′ terminal side of the primer contains the same sequence (B ′) as the sequence (B) present 5 ′ to the sequence (A) of the target nucleic acid sequence,
The primer is designed so that a sequence complementary to the 3 base sequence (Ac′3′-3) from the 3 ′ end of the sequence (Ac ′) of the primer does not exist (0) in the primer.
It is characterized by that. . The primer designing method of the present invention may be the primer designing method (6).

The primer production method (6) includes:
A method for producing a primer used for amplification of a target nucleic acid sequence,
Including primer design process and primer synthesis process,
The primer designing step is performed by the primer designing method (6). . The primer production method of the present invention may be the primer production method (6).

  The design method and production method of the primer (6) are not particularly limited, but it is preferably designed by the primer design method (6) or by the primer production method (6).

The design method of the primer set of the present invention is:
A method for designing a primer set used for amplification of a target nucleic acid sequence in a double-stranded template nucleic acid comprising a first template nucleic acid and a second template nucleic acid,
The primer set includes a first primer and a second primer,
A sequence (Ac ′) complementary to the sequence (A) on the 3 ′ end side of the target nucleic acid sequence in the first template nucleic acid on the 3 ′ end side of the first primer;
On the 5 ′ end side of the first primer, the same sequence (B ′) as the sequence (B) present on the 5 ′ side of the sequence (A) of the target nucleic acid sequence in the first template nucleic acid Including
A sequence (Ec ′) complementary to the sequence (E) on the 3 ′ end side of the target nucleic acid sequence in the second template nucleic acid on the 3 ′ end side of the second primer;
On the 5 ′ end side of the second primer, the same sequence (F ′) as the sequence (F) present on the 5 ′ side of the sequence (E) of the target nucleic acid sequence in the second template nucleic acid Including
A sequence complementary to the 3 base sequence (Ac′3′-3) from the 3 ′ end of the sequence (Ac ′) of the first primer does not exist in the second primer (0 Pieces),
A sequence complementary to the sequence of 3 bases (Ec′3′-3) from the 3 ′ end side of the sequence (Ec ′) of the second primer does not exist in the first primer (0 pieces) Design),
It may be a primer set design method characterized by this. Such a primer set design method of the present invention is referred to as “primer set design method (6 ′)”. Other characteristics of the primer set designed by such a primer set design method (6 ′) may be the same as the primer set of the present invention described above, for example. The position of the three-base sequence in the first primer is as shown in the schematic diagram of FIG. 14B, for example. Thus, since the sequence complementary to the sequence of 3 bases from the 3 ′ end side in the first primer does not exist in the sequence of the second primer, the first primer and the second primer It is easy to prevent an undesired amplification reaction from occurring with the other primers.

In the primer set design method (6 ′),
A sequence complementary to the 3 base sequence (Ac′3′-3) from the 3 ′ end of the sequence (Ac ′) of the first primer is not present in the first primer (0 Pieces),
A sequence complementary to the 3 base sequence (Ec′3′-3) from the 3 ′ end of the sequence (Ec ′) of the second primer does not exist in the second primer (0 It is preferable to design as follows. Thereby, it is easy to prevent the first primers and the second primers from causing an undesirable amplification reaction.

The method for producing the primer set of the present invention comprises:
A method for producing a primer set used for amplification of a target nucleic acid sequence in a double-stranded template nucleic acid comprising a first template nucleic acid and a second template nucleic acid,
Including primer set design process and primer set synthesis process,
The primer set design process may be performed by the primer set design method (6 ′). Such a primer set production method is referred to as “primer set production method (6 ′)”.

The primer (7)
A primer used for amplification of a target nucleic acid sequence,
A sequence (Ac ′) complementary to the sequence (A) on the 3 ′ end side of the target nucleic acid sequence on the 3 ′ end side of the primer;
The 5 ′ terminal side of the primer contains the same sequence (B ′) as the sequence (B) present 5 ′ to the sequence (A) of the target nucleic acid sequence,
The primer sequence should be designed so that there are no more than 2 sequences in the primer sequence complementary to the 3 base sequence (B'5'-3) from the 5 'end of the sequence (B') of the primer. It is characterized by. Other features of the primer (7) may be the same as those of the primer (0) of the present invention.
Specifically, for example, as shown in FIG. In this way, if the number of sequences complementary to the 3 base sequence from the 5 ′ end of any one primer is 2 or less in the same primer sequence, the same primer forms a dimer, which is not desirable. It is thought that it is easy to prevent an amplification reaction from occurring and inhibiting the function of turning back.

The primer design method (7) is:
A method for designing a primer used for amplification of a target nucleic acid sequence,
A sequence (Ac ′) complementary to the sequence (A) on the 3 ′ end side of the target nucleic acid sequence on the 3 ′ end side of the primer;
The 5 ′ terminal side of the primer contains the same sequence (B ′) as the sequence (B) present 5 ′ to the sequence (A) of the target nucleic acid sequence,
The primer sequence should be designed so that there are no more than 2 sequences in the primer sequence complementary to the 3 base sequence (B'5'-3) from the 5 'end of the sequence (B') of the primer. It is characterized by. The primer designing method of the present invention may be the primer designing method (7).

The primer production method (7) includes:
A method for producing a primer used for amplification of a target nucleic acid sequence,
Including primer design process and primer synthesis process,
The primer designing step is performed by the primer designing method (7). The primer production method of the present invention may be the primer production method (7).

  The design method and production method of the primer (7) are not particularly limited, but it is preferably designed by the primer design method (7) or by the primer production method (7).

The primer (8)
A primer used for amplification of a target nucleic acid sequence,
A sequence (Ac ′) complementary to the sequence (A) on the 3 ′ end side of the target nucleic acid sequence on the 3 ′ end side of the primer;
The 5 ′ terminal side of the primer contains the same sequence (B ′) as the sequence (B) present 5 ′ to the sequence (A) of the target nucleic acid sequence,
Complementary to an arbitrary sequence of 4 bases (A5′-4-10 (4)) among 7 bases from the 4th base to the 10th base from the 5 ′ end of the target nucleic acid sequence (A) Specific sequences include the sequence (A) of the target nucleic acid sequence, a 20-base sequence adjacent to the 3 ′ end of the sequence (A), and 20 adjacent to the 5 ′ end of the sequence (A). In the sequence (A-20) containing the base sequence, adjacent to the sequence (A5′-4-10 (4)) and the 5 ′ end side of the sequence (A5′-4-10 (4)) 2 or more in a sequence other than 10 bases, including a 3 base sequence to be sequenced and a 3 base sequence adjacent to the 3 ′ end of the sequence (A5′-4-10 (4)) It is characterized by. The primer of the present invention may be the primer (8). Other features of the primer (8) may be the same as those of the primer (0) of the present invention.
For example, as shown in FIG. 16, efficient amplification is possible when the proportion of single-stranded sites where the primer (TP) anneals is large on the genome sequence. For this purpose, a sequence complementary to 4 consecutive bases arbitrarily selected from 7 bases from the 4th base to the 10th base counted from the 5 ′ end of the sequence (A) of the target nucleic acid sequence, The sequence (A) of the target nucleic acid sequence, a 20-base sequence adjacent to the 3 ′ end of the sequence (A) of the target nucleic acid sequence, and the 5 ′ end of the target nucleic acid sequence (A) In the sequence including the 20-base sequence, the region of 10 bases in total, including the 4 bases in succession, the 3 bases on the upstream side, and the 3 bases on the downstream side, is made to be 2 or more.

The primer design method (8) is:
A method for designing a primer used for amplification of a target nucleic acid sequence,
A sequence (Ac ′) complementary to the sequence (A) on the 3 ′ end side of the target nucleic acid sequence on the 3 ′ end side of the primer;
The 5 ′ terminal side of the primer contains the same sequence (B ′) as the sequence (B) present 5 ′ to the sequence (A) of the target nucleic acid sequence,
Complementary to an arbitrary sequence of 4 bases (A5′-4-10 (4)) among 7 bases from the 4th base to the 10th base from the 5 ′ end of the target nucleic acid sequence (A) Specific sequences include the sequence (A) of the target nucleic acid sequence, a 20-base sequence adjacent to the 3 ′ end of the sequence (A), and 20 adjacent to the 5 ′ end of the sequence (A). In the sequence (A-20) containing the base sequence, adjacent to the sequence (A5′-4-10 (4)) and the 5 ′ end side of the sequence (A5′-4-10 (4)) 2 or more of the sequences other than 10 bases, including the 3 base sequences to be sequenced and the 3 base sequences adjacent to the 3 ′ end of the sequence (A5′-4-10 (4)) To design,
It is characterized by that. The primer designing method of the present invention may be the primer designing method (8).

The primer production method (8) includes:
A method for producing a primer used for amplification of a target nucleic acid sequence,
Including primer design process and primer synthesis process,
The primer designing step is performed by the primer designing method (8). The primer production method of the present invention may be the primer production method (8).

  Although the design method and production method of the primer (8) are not particularly limited, it is preferably designed by the primer design method (8) or by the primer production method (8).

In the primer set of the present invention described above, the primer for nucleic acid amplification of the present invention may be the primer (8) of the present invention. Such a primer set is also included in the primer set of the present invention. That is, the primer set of the present invention is
A primer set used for amplification of a target nucleic acid sequence in a double-stranded template nucleic acid comprising a first template nucleic acid and a second template nucleic acid,
The primer set includes a first primer and a second primer,
The first primer is the primer (8),
The second primer includes, on the 3 ′ end side, a sequence (Ec ′) complementary to the sequence (E ′) on the 3 ′ end side of the target nucleic acid sequence in the second template nucleic acid. A primer set comprising the same sequence (F ′) as the sequence (F) existing 5 ′ from the sequence (E) of the target nucleic acid sequence in the second template nucleic acid There may be.

  In the nucleic acid amplification method of the present invention described above, the primer for nucleic acid amplification of the present invention may be the primer (8) of the present invention. Such a nucleic acid amplification method is also included in the nucleic acid amplification method of the present invention.

The primer (9)
A primer used for amplification of a target nucleic acid sequence,
A sequence (Ac ′) complementary to the sequence (A) on the 3 ′ end side of the target nucleic acid sequence on the 3 ′ end side of the primer;
The 5 ′ terminal side of the primer contains the same sequence (B ′) as the sequence (B) present 5 ′ to the sequence (A) of the target nucleic acid sequence,
A sequence complementary to the 4 base sequence (B3'-4) from the 3 'end of the sequence (B) of the target nucleic acid sequence has 3 bases on the 5' end of the sequence (B3'-4). It is designed to be one or more in a sequence of 20 bases adjacent to each other and a sequence of 20 bases adjacent to each other by 3 bases on the 3 ′ end side of the sequence (B3′-4). It is characterized by. The features of the primer (9) other than those described above may be the same as those of the primer (0) of the present invention.
For example, as shown in FIG. 17, efficient amplification is possible when there is a high probability that the region where the primer (TP) turns back on the genome sequence is a single strand. For this purpose, a sequence complementary to 4 bases from the 3 ′ end of the sequence (B) of the target nucleic acid sequence starts from the 4th base of the sequence counted upstream from the 5 ′ end of the 4 bases (ie, 20 bases up to the 23rd base (adjacent to the 5 'end side with 3 bases), and the 4th base of the sequence counted from the 3' end downstream of the 4 bases (ie, on the 3 'end side) One or more of the 20 bases up to the 23rd base (adjacent to each other with 3 bases).

The primer design method (9) is:
A method for designing a primer used for amplification of a target nucleic acid sequence,
A sequence (Ac ′) complementary to the sequence (A) on the 3 ′ end side of the target nucleic acid sequence on the 3 ′ end side of the primer;
The 5 ′ terminal side of the primer contains the same sequence (B ′) as the sequence (B) present 5 ′ to the sequence (A) of the target nucleic acid sequence,
A sequence complementary to the 4 base sequence (B3'-4) from the 3 'end of the sequence (B) of the target nucleic acid sequence has 3 bases on the 5' end of the sequence (B3'-4). It is designed to be one or more in a sequence of 20 bases adjacent to each other and a sequence of 20 bases adjacent to each other by 3 bases on the 3 ′ end side of the sequence (B3′-4). It is characterized by. The primer designing method of the present invention may be the primer designing method (9).

The primer production method (9) includes:
A method for producing a primer used for amplification of a target nucleic acid sequence,
Including primer design process and primer synthesis process,
The primer designing step is performed by the primer designing method (9). The primer production method of the present invention may be the primer production method (9).

  The design method and production method of the primer (9) are not particularly limited, but it is preferably designed by the primer design method (9) or by the primer production method (9).

The primer (10)
A primer used for amplification of a target nucleic acid sequence,
A sequence (Ac ′) complementary to the sequence (A) on the 3 ′ end side of the target nucleic acid sequence on the 3 ′ end side of the primer;
The 5 ′ terminal side of the primer contains the same sequence (B ′) as the sequence (B) present 5 ′ to the sequence (A) of the target nucleic acid sequence,
Among the 8 base sequences (Ac′3′-6-13) from the 6th base to the 13th base from the 3 ′ end side of the sequence (Ac ′), an arbitrary sequence of 5 bases (Ac′− 3′-6-13 (5)) is designed so that a sequence complementary to the primer sequence does not exist in the primer sequence. Other features of the primer (10) may be the same as those of the primer (0).
Specifically, for example, as shown in FIG. As described above, when a sequence complementary to the 5 base sequence from the 7th base to the 11th base from the 3 ′ end side in any one primer is not present in the primer sequence, By maintaining a single-stranded structure in the region, hybridization nucleation is facilitated, and it is considered that this contributes to an efficient amplification reaction.

The primer design method (10) is:
A method for designing a primer used for amplification of a target nucleic acid sequence,
A sequence (Ac ′) complementary to the sequence (A) on the 3 ′ end side of the target nucleic acid sequence on the 3 ′ end side of the primer;
The 5 ′ terminal side of the primer contains the same sequence (B ′) as the sequence (B) present 5 ′ to the sequence (A) of the target nucleic acid sequence,
Among the 8 base sequences (Ac′3′-6-13) from the 6th base to the 13th base from the 3 ′ end side of the sequence (Ac ′), an arbitrary sequence of 5 bases (Ac′− 3′-6-13 (5)) is designed so that a sequence complementary to the primer sequence does not exist in the primer sequence. The primer designing method of the present invention may be the primer designing method (10).

The primer production method (10) includes:
A method for producing a primer used for amplification of a target nucleic acid sequence,
Including primer design process and primer synthesis process,
The primer designing step is performed by the primer designing method (10) described above. The primer production method of the present invention may be the primer production method (10).

  The design method and production method of the primer (10) are not particularly limited, but it is preferably designed by the primer design method (10) or by the primer production method (10).

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

Prediction of Tm value (melting temperature) Prediction of Tm value is described in Primer 3 software package (S. Rozen et al., Bioinformatics Methods and Protocols: Methods in Molecular Biology. Humana Press, Totowa, NJ, 365-386 (2000)). This was done using the “oligotm” software. In the simulation for predicting the Tm value, the ion condition was set to 10 mmol / L Na ⁺ and 8 mmol / L Mg ²⁺ .

Sequence of TP primer The sequence of TP for detecting the target nucleic acid sequence using two TPs was determined by the following method. First, for one TP, the sequence (Ac ′) is determined so that the length of the sequence (Ac ′) is 12 to 25 bases, the Tm value is 50 to 60 ° C., and the GC content is 40 to 60%. did. The sequence (B) is set 15 to 40 bases upstream of the sequence (A) in the target nucleic acid sequence corresponding to the sequence (Ac ′), and the TP (B ′) corresponding to the sequence (B) is determined. did. The TP determined by this was designated as F1 TP. Further, a sequence complementary to the sequence obtained by moving the sequence (A) in the target nucleic acid sequence corresponding to the sequence (Ac ′) of F1 TP one base upstream was defined as the sequence (Ac ′) of F2 TP. Thereafter, the sequence (Ac ′) of F3 TP to F8 TP was determined in the same manner in the same manner as Forward TP (F1 TP to F8 TP). The sequence (B ′) is the same in all Forward TPs. Further, Reverse TP (R1 TP to R8 TP) used in combination with these Forward TPs was determined so that the length of the amplified sequence was within 200 bp. The method for determining the sequence (Ac ′) and the sequence (B ′) is the same as in the case of F1 TP to F8 TP. For 21 types of target sequences, F1 TP to F8 TP and R1 TP to R8 TP were determined by the above method, and a total of 200 pairs (400 types) of TPs were designed and prepared. The target nucleic acid sequences (21 types of SNP target sequences) and the corresponding TP sequences are shown below.

Beta-2 AR (SEQ ID NO: 1) GGTCCGCCCGCTGAGGCGCCCCCAGCCAGTGCGCTCACCTGCCAGACTGCGCGCCATGGGGCAACCCGGGAACGGCAGCGCCTTCTTGCTGGCACCCAATAGAAGCCATGCGCCGGACCACGACGTCACGCAGCAAAGGGACGAGGTGTGGGTGGTGGGCATCGGCGTGTGTCGTCTCTCATCG
Beta-2 AR B2 TP-F1 (SEQ ID NO: 2) CTATTGGGTGCATGGGGCAACCCGGGAACG
Beta-2 AR B2 TP-F2 (SEQ ID NO: 3) CTATTGGGTGCCATGGGGCAACCCGGGAAC
Beta-2 AR B2 TP-F3 (SEQ ID NO: 4) CTATTGGGTGGCCATGGGGCAACCCGGGAA
Beta-2 AR B2 TP-F4 (SEQ ID NO: 5) CTATTGGGTGCGCCATGGGGCAACCCGGGA
Beta-2 AR B2 TP-F5 (SEQ ID NO: 6) CTATTGGGTGGCGCCATGGGGCAACCCGGG
Beta-2 AR B2 TP-F6 (SEQ ID NO: 7) CTATTGGGTGCGCGCCATGGGGCAACCCGG
Beta-2 AR B2 TP-F7 (SEQ ID NO: 8) CTATTGGGTGGCGCGCCATGGGGCAACCCG
Beta-2 AR B2 TP-F8 (SEQ ID NO: 9) CTATTGGGTGTGCGCGCCATGGGGCAACCC
Beta-2 AR B2 TP-R1 (SEQ ID NO: 10) AGAAGCCATGCACCCACACCTCGTCCCTTT
Beta-2 AR B2 TP-R2 (SEQ ID NO: 11) AGAAGCCATGCCACCCACACCTCGTCCCTT
Beta-2 AR B2 TP-R3 (SEQ ID NO: 12) AGAAGCCATGACCACCCACACCTCGTCCCT
Beta-2 AR B2 TP-R4 (SEQ ID NO: 13) AGAAGCCATGCACCACCCACACCTCGTCCC
Beta-2 AR B2 TP-R5 (SEQ ID NO: 14) AGAAGCCATGCCACCACCCACACCTCGTCC
Beta-2 AR B2 TP-R6 (SEQ ID NO: 15) AGAAGCCATGCCCACCACCCACACCTCGTC
Beta-2 AR B2 TP-R7 (SEQ ID NO: 16) AGAAGCCATGGCCCACCACCCACACCTCGT
Beta-2 AR B2 TP-R8 (SEQ ID NO: 17) AGAAGCCATGTGCCCACCACCCACACCTCG

Beta-3 AR (SEQ ID NO: 18) TAGCCGGGGCCCTGCTGGCGCTGGCGGTGCTGGCCACCGTGGGAGGCAACCTGCTGGTCATCGTGGCCATCGCCTGGACTCCGAGACTCCAGACCATGACCAACGTGTTCGTGACTTCGCTGGCCGCAGCCGACCTGGTGATGGGACTC
Beta-3 AR B3 TP-F1 (SEQ ID NO: 19) CAGGCGATGGCTGGCCACCGTGGGAGGCA
Beta-3 AR B3 TP-F2 (SEQ ID NO: 20) CAGGCGATGTGCTGGCCACCGTGGGAGGC
Beta-3 AR B3 TP-F3 (SEQ ID NO: 21) CAGGCGATGGTGCTGGCCACCGTGGGAGG
Beta-3 AR B3 TP-F4 (SEQ ID NO: 22) CAGGCGATGGGTGCTGGCCACCGTGGGAG
Beta-3 AR B3 TP-F5 (SEQ ID NO: 23) CAGGCGATGCGGTGCTGGCCACCGTGGGA
Beta-3 AR B3 TP-F6 (SEQ ID NO: 24) CAGGCGATGGCGGTGCTGGCCACCGTGGG
Beta-3 AR B3 TP-F7 (SEQ ID NO: 25) CAGGCGATGGGCGGTGCTGGCCACCGTGG
Beta-3 AR B3 TP-F8 (SEQ ID NO: 26) CAGGCGATGTGGCGGTGCTGGCCACCGTG
Beta-3 AR B3 TP-R1 (SEQ ID NO: 27) CTGGACTCCAGCGAAGTCACGAACACGTT
Beta-3 AR B3 TP-R2 (SEQ ID NO: 28) CTGGACTCCCAGCGAAGTCACGAACACGT
Beta-3 AR B3 TP-R3 (SEQ ID NO: 29) CTGGACTCCCCAGCGAAGTCACGAACACG
Beta-3 AR B3 TP-R4 (SEQ ID NO: 30) CTGGACTCCGCCAGCGAAGTCACGAACAC
Beta-3 AR B3 TP-R5 (SEQ ID NO: 31) CTGGACTCCGGCCAGCGAAGTCACGAACA
Beta-3 AR B3 TP-R6 (SEQ ID NO: 32) CTGGACTCCCGGCCAGCGAAGTCACGAAC
Beta-3 AR B3 TP-R7 (SEQ ID NO: 33) CTGGACTCCGCGGCCAGCGAAGTCACGAA
Beta-3 AR B3 TP-R8 (SEQ ID NO: 34) CTGGACTCCTGCGGCCAGCGAAGTCACGA

UCP1 (SEQ ID NO: 35) AATGAGAGAATTATGGGAAAGTATAGAACACTATTCAAATGCAAAGCACTGTATGATTTTTATTTAATAGGAAGACATTTTGTGCAGCGATTTCTGATTGACCACAGTTTGATCAAGTGCATTTGTTAATGTGTTCATTATTCAAAAAGGAACATGAGAATTTGTTACATTCTGATTTGTCTCCTCT
UCP1 UCP TP-F1 (SEQ ID NO: 36) CTTGATCAAACTGTAATAGGAAGACATTTTGTGCAGCG
UCP1 UCP TP-F2 (SEQ ID NO: 37) CTTGATCAAACTGTTAATAGGAAGACATTTTGTGCAGC
UCP1 UCP TP-F3 (SEQ ID NO: 38) CTTGATCAAACTGTTTAATAGGAAGACATTTTGTGCAG
UCP1 UCP TP-F4 (SEQ ID NO: 39) CTTGATCAAACTGATTTAATAGGAAGACATTTTGTGCA
UCP1 UCP TP-F5 (SEQ ID NO: 40) CTTGATCAAACTGTATTTAATAGGAAGACATTTTGTGC
UCP1 UCP TP-F6 (SEQ ID NO: 41) CTTGATCAAACTGTTATTTAATAGGAAGACATTTTGTG
UCP1 UCP TP-F7 (SEQ ID NO: 42) CTTGATCAAACTGTTTATTTAATAGGAAGACATTTTGT
UCP1 UCP TP-F8 (SEQ ID NO: 43) CTTGATCAAACTGTTTTATTTAATAGGAAGACATTTTG
UCP1 UCP TP-R1 (SEQ ID NO: 44) CAAGTGCATTTGTGTAACAAATTCTCCTTTCCTTTTTG
UCP1 UCP TP-R2 (SEQ ID NO: 45) CAAGTGCATTTGTTGTAACAAATTCTCCTTTCCTTTTT
UCP1 UCP TP-R3 (SEQ ID NO: 46) CAAGTGCATTTGTATGTAACAAATTCTCCTTTCCTTTT
UCP1 UCP TP-R4 (SEQ ID NO: 47) CAAGTGCATTTGTAATGTAACAAATTCTCCTTTCCTTT
UCP1 UCP TP-R5 (SEQ ID NO: 48) CAAGTGCATTTGTGAATGTAACAAATTCTCCTTTCCTT
UCP1 UCP TP-R6 (SEQ ID NO: 49) CAAGTGCATTTGTTGAATGTAACAAATTCTCCTTTCCT
UCP1 UCP TP-R7 (SEQ ID NO: 50) CAAGTGCATTTGTCTGAATGTAACAAATTCTCCTTTCC
UCP1 UCP TP-R8 (SEQ ID NO: 51) CAAGTGCATTTGTTCTGAATGTAACAAATTCTCCTTTC

G2_template (SEQ ID NO: 52) GGCATGGATTTTTACGTGCCCAGACAAGAGCCAATGTTCAGATGGATGAGATTCTCAGATCTGAGCACGTGGGAGGGCGATCGCAATCTGGCTCCCAGCTTTGTGAATGAAGATGGCGTCGAATGACGCCAACCCATCTGATGGGTCCGCAGCCAACCTCGTCCCAGAGGTCAATAATGAGGTTATGGCTCTGGAGCCCGTTGTTGGTGCCGCTATTGCGGCACCTGTGGCGGGCCAACAAAACGTAATTGACCCCTGGATTAGAAACAATTTTGTACAAGCCCCTGGTGGAGAGTTCACAGTGTCCCCTAGAAACGCTCCAGGTGAGATACTGTGGAGCGCGCCCTTGGGCCCTGATCTGAACCCCTATCTTTCTCATTTGTCCAGAATGTACAATGGTTATGCAGGTGG
norovirus G2 1 G2 fTP1-1 (SEQ ID NO: 53) CTCATCCATCTGGGCATGGATTTTTACGTG
norovirus G2 1 G2 fTP1-2 (SEQ ID NO: 54) TCTCATCCATCTGCATGGATTTTTACGTGC
norovirus G2 1 G2 fTP1-3 (SEQ ID NO: 55) ATCTCATCCATCCATGGATTTTTACGTGCC
norovirus G2 1 G2 fTP1-4 (SEQ ID NO: 56) AATCTCATCCATATGGATTTTTACGTGCCC
norovirus G2 1 G2 fTP1-5 (SEQ ID NO: 57) GAATCTCATCCATGGATTTTTACGTGCCCA
norovirus G2 1 G2 fTP1-6 (SEQ ID NO: 58) AGAATCTCATCCGGATTTTTACGTGCCCAG
norovirus G2 1 G2 fTP1-7 (SEQ ID NO: 59) GAGAATCTCATCGATTTTTACGTGCCCAGA
norovirus G2 1 G2 fTP1-8 (SEQ ID NO: 60) TGAGAATCTCATATTTTTACGTGCCCAGAC
norovirus G2 1 G2 rTP1-1 (SEQ ID NO: 61) GATCTGAGCACGTTCACAAAGCTGGGAGCC
norovirus G2 1 G2 rTP1-2 (SEQ ID NO: 62) ATCTGAGCACGTATTCACAAAGCTGGGAGC
norovirus G2 1 G2 rTP1-3 (SEQ ID NO: 63) TCTGAGCACGTGCATTCACAAAGCTGGGAG
norovirus G2 1 G2 rTP1-4 (SEQ ID NO: 64) CTGAGCACGTGGTCATTCACAAAGCTGGGA
norovirus G2 1 G2 rTP1-5 (SEQ ID NO: 65) TGAGCACGTGGGTTCATTCACAAAGCTGGG
norovirus G2 1 G2 rTP1-6 (SEQ ID NO: 66) GAGCACGTGGGACTTCATTCACAAAGCTGG
norovirus G2 1 G2 rTP1-7 (SEQ ID NO: 67) AGCACGTGGGAGTCTTCATTCACAAAGCTG
norovirus G2 1 G2 rTP1-8 (SEQ ID NO: 68) GCACGTGGGAGGATCTTCATTCACAAAGCT

G2_template (SEQ ID NO: 52) GGCATGGATTTTTACGTGCCCAGACAAGAGCCAATGTTCAGATGGATGAGATTCTCAGATCTGAGCACGTGGGAGGGCGATCGCAATCTGGCTCCCAGCTTTGTGAATGAAGATGGCGTCGAATGACGCCAACCCATCTGATGGGTCCGCAGCCAACCTCGTCCCAGAGGTCAATAATGAGGTTATGGCTCTGGAGCCCGTTGTTGGTGCCGCTATTGCGGCACCTGTGGCGGGCCAACAAAACGTAATTGACCCCTGGATTAGAAACAATTTTGTACAAGCCCCTGGTGGAGAGTTCACAGTGTCCCCTAGAAACGCTCCAGGTGAGATACTGTGGAGCGCGCCCTTGGGCCCTGATCTGAACCCCTATCTTTCTCATTTGTCCAGAATGTACAATGGTTATGCAGGTGG
norovirus G2 2 G2 fTP2-1 (SEQ ID NO: 69) TCATTCGACGCCGCGATCGCAATCTGGCTC
norovirus G2 2 G2 fTP2-2 (SEQ ID NO: 70) GTCATTCGACGCCGATCGCAATCTGGCTCC
norovirus G2 2 G2 fTP2-3 (SEQ ID NO: 71) CGTCATTCGACGGATCGCAATCTGGCTCCC
norovirus G2 2 G2 fTP2-4 (SEQ ID NO: 72) GCGTCATTCGACATCGCAATCTGGCTCCCA
norovirus G2 2 G2 fTP2-5 (SEQ ID NO: 73) GGCGTCATTCGATCGCAATCTGGCTCCCAG
norovirus G2 2 G2 fTP2-6 (SEQ ID NO: 74) TGGCGTCATTCGCGCAATCTGGCTCCCAGC
norovirus G2 2 G2 fTP2-7 (SEQ ID NO: 75) TTGGCGTCATTCGCAATCTGGCTCCCAGCT
norovirus G2 2 G2 fTP2-8 (SEQ ID NO: 76) GTTGGCGTCATTCAATCTGGCTCCCAGCTT
norovirus G2 2 G2 rTP2-1 (SEQ ID NO: 77) CCATCTGATGGGACCTCATTATTGACCTCT
norovirus G2 2 G2 rTP2-2 (SEQ ID NO: 78) CATCTGATGGGTAACCTCATTATTGACCTC
norovirus G2 2 G2 rTP2-3 (SEQ ID NO: 79) ATCTGATGGGTCTAACCTCATTATTGACCT
norovirus G2 2 G2 rTP2-4 (SEQ ID NO: 80) TCTGATGGGTCCATAACCTCATTATTGACC
norovirus G2 2 G2 rTP2-5 (SEQ ID NO: 81) CTGATGGGTCCGCATAACCTCATTATTGAC
norovirus G2 2 G2 rTP2-6 (SEQ ID NO: 82) TGATGGGTCCGCCCATAACCTCATTATTGA
norovirus G2 2 G2 rTP2-7 (SEQ ID NO: 83) GATGGGTCCGCAGCCATAACCTCATTATTG
norovirus G2 2 G2 rTP2-8 (SEQ ID NO: 84) ATGGGTCCGCAGAGCCATAACCTCATTATT

G2_template (SEQ ID NO: 52) GGCATGGATTTTTACGTGCCCAGACAAGAGCCAATGTTCAGATGGATGAGATTCTCAGATCTGAGCACGTGGGAGGGCGATCGCAATCTGGCTCCCAGCTTTGTGAATGAAGATGGCGTCGAATGACGCCAACCCATCTGATGGGTCCGCAGCCAACCTCGTCCCAGAGGTCAATAATGAGGTTATGGCTCTGGAGCCCGTTGTTGGTGCCGCTATTGCGGCACCTGTGGCGGGCCAACAAAACGTAATTGACCCCTGGATTAGAAACAATTTTGTACAAGCCCCTGGTGGAGAGTTCACAGTGTCCCCTAGAAACGCTCCAGGTGAGATACTGTGGAGCGCGCCCTTGGGCCCTGATCTGAACCCCTATCTTTCTCATTTGTCCAGAATGTACAATGGTTATGCAGGTGG
norovirus G2 3 G2 fTP3-1 (SEQ ID NO: 85) GAGCCATAACCTTGGGTCCGCAGCCAACCT
norovirus G2 3 G2 fTP3-2 (SEQ ID NO: 86) AGAGCCATAACCGGGTCCGCAGCCAACCTC
norovirus G2 3 G2 fTP3-3 (SEQ ID NO: 87) CAGAGCCATAACGGTCCGCAGCCAACCTCG
norovirus G2 3 G2 fTP3-4 (SEQ ID NO: 88) CCAGAGCCATAAGTCCGCAGCCAACCTCGT
norovirus G2 3 G2 fTP3-5 (SEQ ID NO: 89) TCCAGAGCCATATCCGCAGCCAACCTCGTC
norovirus G2 3 G2 fTP3-6 (SEQ ID NO: 90) CTCCAGAGCCATCCGCAGCCAACCTCGTCC
norovirus G2 3 G2 fTP3-7 (SEQ ID NO: 91) GCTCCAGAGCCACGCAGCCAACCTCGTCCC
norovirus G2 3 G2 fTP3-8 (SEQ ID NO: 92) GGCTCCAGAGCCGCAGCCAACCTCGTCCCA
norovirus G2 3 G2 rTP3-1 (SEQ ID NO: 93) CGTTGTTGGTGCTTACGTTTTGTTGGCCCG
norovirus G2 3 G2 rTP3-2 (SEQ ID NO: 94) GTTGTTGGTGCCATTACGTTTTGTTGGCCC
norovirus G2 3 G2 rTP3-3 (SEQ ID NO: 95) TTGTTGGTGCCGAATTACGTTTTGTTGGCC
norovirus G2 3 G2 rTP3-4 (SEQ ID NO: 96) TGTTGGTGCCGCCAATTACGTTTTGTTGGC
norovirus G2 3 G2 rTP3-5 (SEQ ID NO: 97) GTTGGTGCCGCTTCAATTACGTTTTGTTGG
norovirus G2 3 G2 rTP3-6 (SEQ ID NO: 98) TTGGTGCCGCTAGTCAATTACGTTTTGTTG
norovirus G2 3 G2 rTP3-7 (SEQ ID NO: 99) TGGTGCCGCTATGGTCAATTACGTTTTGTT
norovirus G2 3 G2 rTP3-8 (SEQ ID NO: 100) GGTGCCGCTATTGGGTCAATTACGTTTTGT

G2_template (SEQ ID NO: 52) GGCATGGATTTTTACGTGCCCAGACAAGAGCCAATGTTCAGATGGATGAGATTCTCAGATCTGAGCACGTGGGAGGGCGATCGCAATCTGGCTCCCAGCTTTGTGAATGAAGATGGCGTCGAATGACGCCAACCCATCTGATGGGTCCGCAGCCAACCTCGTCCCAGAGGTCAATAATGAGGTTATGGCTCTGGAGCCCGTTGTTGGTGCCGCTATTGCGGCACCTGTGGCGGGCCAACAAAACGTAATTGACCCCTGGATTAGAAACAATTTTGTACAAGCCCCTGGTGGAGAGTTCACAGTGTCCCCTAGAAACGCTCCAGGTGAGATACTGTGGAGCGCGCCCTTGGGCCCTGATCTGAACCCCTATCTTTCTCATTTGTCCAGAATGTACAATGGTTATGCAGGTGG
norovirus G2 4 G2 fTP4-1 (SEQ ID NO: 101) CAGGTGCCGCAATGAGGTTATGGCTCTGGA
norovirus G2 4 G2 fTP4-2 (SEQ ID NO: 102) ACAGGTGCCGCAGAGGTTATGGCTCTGGAG
norovirus G2 4 G2 fTP4-3 (SEQ ID NO: 103) CACAGGTGCCGCAGGTTATGGCTCTGGAGC
norovirus G2 4 G2 fTP4-4 (SEQ ID NO: 104) CCACAGGTGCCGGGTTATGGCTCTGGAGCC
norovirus G2 4 G2 fTP4-5 (SEQ ID NO: 105) GCCACAGGTGCCGTTATGGCTCTGGAGCCC
norovirus G2 4 G2 fTP4-6 (SEQ ID NO: 106) CGCCACAGGTGCTTATGGCTCTGGAGCCCG
norovirus G2 4 G2 fTP4-7 (SEQ ID NO: 107) CCGCCACAGGTGTATGGCTCTGGAGCCCGT
norovirus G2 4 G2 fTP4-8 (SEQ ID NO: 108) CCCGCCACAGGTATGGCTCTGGAGCCCGTT
norovirus G2 4 G2 rTP4-1 (SEQ ID NO: 109) CCAACAAAACGTGGGCTTGTACAAAATTGT
norovirus G2 4 G2 rTP4-2 (SEQ ID NO: 110) CAACAAAACGTAGGGGCTTGTACAAAATTG
norovirus G2 4 G2 rTP4-3 (SEQ ID NO: 111) AACAAAACGTAAAGGGGCTTGTACAAAATT
norovirus G2 4 G2 rTP4-4 (SEQ ID NO: 112) ACAAAACGTAATCAGGGGCTTGTACAAAAT
norovirus G2 4 G2 rTP4-5 (SEQ ID NO: 113) CAAAACGTAATTCCAGGGGCTTGTACAAAA
norovirus G2 4 G2 rTP4-6 (SEQ ID NO: 114) AAAACGTAATTGACCAGGGGCTTGTACAAA
norovirus G2 4 G2 rTP4-7 (SEQ ID NO: 115) AAACGTAATTGACACCAGGGGCTTGTACAA
norovirus G2 4 G2 rTP4-8 (SEQ ID NO: 116) AACGTAATTGACCCACCAGGGGCTTGTACA

G2_template (SEQ ID NO: 52) GGCATGGATTTTTACGTGCCCAGACAAGAGCCAATGTTCAGATGGATGAGATTCTCAGATCTGAGCACGTGGGAGGGCGATCGCAATCTGGCTCCCAGCTTTGTGAATGAAGATGGCGTCGAATGACGCCAACCCATCTGATGGGTCCGCAGCCAACCTCGTCCCAGAGGTCAATAATGAGGTTATGGCTCTGGAGCCCGTTGTTGGTGCCGCTATTGCGGCACCTGTGGCGGGCCAACAAAACGTAATTGACCCCTGGATTAGAAACAATTTTGTACAAGCCCCTGGTGGAGAGTTCACAGTGTCCCCTAGAAACGCTCCAGGTGAGATACTGTGGAGCGCGCCCTTGGGCCCTGATCTGAACCCCTATCTTTCTCATTTGTCCAGAATGTACAATGGTTATGCAGGTGG
norovirus G2 5 G2 fTP5-1 (SEQ ID NO: 117) TACAAAATTGTTTGGCGGGCCAACAAAACG
norovirus G2 5 G2 fTP5-2 (SEQ ID NO: 118) GTACAAAATTGTGGCGGGCCAACAAAACGT
norovirus G2 5 G2 fTP5-3 (SEQ ID NO: 119) TGTACAAAATTGGCGGGCCAACAAAACGTA
norovirus G2 5 G2 fTP5-4 (SEQ ID NO: 120) TTGTACAAAATTCGGGCCAACAAAACGTAA
norovirus G2 5 G2 fTP5-5 (SEQ ID NO: 121) CTTGTACAAAATGGGCCAACAAAACGTAAT
norovirus G2 5 G2 fTP5-6 (SEQ ID NO: 122) GCTTGTACAAAAGGCCAACAAAACGTAATT
norovirus G2 5 G2 fTP5-7 (SEQ ID NO: 123) GGCTTGTACAAAGCCAACAAAACGTAATTG
norovirus G2 5 G2 fTP5-8 (SEQ ID NO: 124) GGGCTTGTACAACCAACAAAACGTAATTGA
norovirus G2 5 G2 rTP5-1 (SEQ ID NO: 125) CTGGTGGAGAGTCAGTATCTCACCTGGAGC
norovirus G2 5 G2 rTP5-2 (SEQ ID NO: 126) TGGTGGAGAGTTACAGTATCTCACCTGGAG
norovirus G2 5 G2 rTP5-3 (SEQ ID NO: 127) GGTGGAGAGTTCCACAGTATCTCACCTGGA
norovirus G2 5 G2 rTP5-4 (SEQ ID NO: 128) GTGGAGAGTTCACCACAGTATCTCACCTGG
norovirus G2 5 G2 rTP5-5 (SEQ ID NO: 129) TGGAGAGTTCACTCCACAGTATCTCACCTG
norovirus G2 5 G2 rTP5-6 (SEQ ID NO: 130) GGAGAGTTCACACTCCACAGTATCTCACCT
norovirus G2 5 G2 rTP5-7 (SEQ ID NO: 131) GAGAGTTCACAGGCTCCACAGTATCTCACC
norovirus G2 5 G2 rTP5-8 (SEQ ID NO: 132) AGAGTTCACAGTCGCTCCACAGTATCTCAC

G2_template (SEQ ID NO: 52) GGCATGGATTTTTACGTGCCCAGACAAGAGCCAATGTTCAGATGGATGAGATTCTCAGATCTGAGCACGTGGGAGGGCGATCGCAATCTGGCTCCCAGCTTTGTGAATGAAGATGGCGTCGAATGACGCCAACCCATCTGATGGGTCCGCAGCCAACCTCGTCCCAGAGGTCAATAATGAGGTTATGGCTCTGGAGCCCGTTGTTGGTGCCGCTATTGCGGCACCTGTGGCGGGCCAACAAAACGTAATTGACCCCTGGATTAGAAACAATTTTGTACAAGCCCCTGGTGGAGAGTTCACAGTGTCCCCTAGAAACGCTCCAGGTGAGATACTGTGGAGCGCGCCCTTGGGCCCTGATCTGAACCCCTATCTTTCTCATTTGTCCAGAATGTACAATGGTTATGCAGGTGG
norovirus G2 6 G2 fTP6-1 (SEQ ID NO: 133) AGGGCGCGCTCCCACAGTGTCCCCTAGAAA
norovirus G2 6 G2 fTP6-2 (SEQ ID NO: 134) AAGGGCGCGCTCACAGTGTCCCCTAGAAAC
norovirus G2 6 G2 fTP6-3 (SEQ ID NO: 135) CAAGGGCGCGCTCAGTGTCCCCTAGAAACG
norovirus G2 6 G2 fTP6-4 (SEQ ID NO: 136) CCAAGGGCGCGCAGTGTCCCCTAGAAACGC
norovirus G2 6 G2 fTP6-5 (SEQ ID NO: 137) CCCAAGGGCGCGGTGTCCCCTAGAAACGCT
norovirus G2 6 G2 fTP6-6 (SEQ ID NO: 138) GCCCAAGGGCGCTGTCCCCTAGAAACGCTC
norovirus G2 6 G2 fTP6-7 (SEQ ID NO: 139) GGCCCAAGGGCGGTCCCCTAGAAACGCTCC
norovirus G2 6 G2 fTP6-8 (SEQ ID NO: 140) GGGCCCAAGGGCTCCCCTAGAAACGCTCCA
norovirus G2 6 G2 rTP6-1 (SEQ ID NO: 141) TGATCTGAACCCCATAACCATTGTACATTC
norovirus G2 6 G2 rTP6-2 (SEQ ID NO: 142) GATCTGAACCCCGCATAACCATTGTACATT
norovirus G2 6 G2 rTP6-3 (SEQ ID NO: 143) ATCTGAACCCCTTGCATAACCATTGTACAT
norovirus G2 6 G2 rTP6-4 (SEQ ID NO: 144) TCTGAACCCCTACTGCATAACCATTGTACA
norovirus G2 6 G2 rTP6-5 (SEQ ID NO: 145) CTGAACCCCTATCCTGCATAACCATTGTAC
norovirus G2 6 G2 rTP6-6 (SEQ ID NO: 146) TGAACCCCTATCACCTGCATAACCATTGTA
norovirus G2 6 G2 rTP6-7 (SEQ ID NO: 147) GAACCCCTATCTCACCTGCATAACCATTGT
norovirus G2 6 G2 rTP6-8 (SEQ ID NO: 148) AACCCCTATCTTCCACCTGCATAACCATTG

ABCB1 (SEQ ID NO: 149) ACCTGGGCATCGTGTCCCAGGAGCCCATCCTGTTTGACTGCAGCATTGCTGAGAACATTGCCTATGGAGACAACAGCCGGGTGGTGTCACAGGAAGAGATCGTGAGGGCAGCAAAGGAGGCCAACATACATGCCTTCATCGAGTCACTGCCTAATGTACCTCCCTCTTCAGG
ABCB1 ABCB1 TP_F1 (SEQ ID NO: 150) CGATCTCTTCGCATTGCTGAGAACATTGC
ABCB1 ABCB1 TP_F2 (SEQ ID NO: 151) CGATCTCTTCAGCATTGCTGAGAACATTG
ABCB1 ABCB1 TP_F3 (SEQ ID NO: 152) CGATCTCTTCCAGCATTGCTGAGAACATT
ABCB1 ABCB1 TP_F4 (SEQ ID NO: 153) CGATCTCTTCGCAGCATTGCTGAGAACAT
ABCB1 ABCB1 TP_F5 (SEQ ID NO: 154) CGATCTCTTCTGCAGCATTGCTGAGAACA
ABCB1 ABCB1 TP_F6 (SEQ ID NO: 155) CGATCTCTTCCTGCAGCATTGCTGAGAAC
ABCB1 ABCB1 TP_F7 (SEQ ID NO: 156) CGATCTCTTCACTGCAGCATTGCTGAGAA
ABCB1 ABCB1 TP_F8 (SEQ ID NO: 157) CGATCTCTTCGACTGCAGCATTGCTGAGA
ABCB1 ABCB1 TP_R1 (SEQ ID NO: 158) TCGTGAGGGCCAGTGACTCGATGAAGGCA
ABCB1 ABCB1 TP_R2 (SEQ ID NO: 159) TCGTGAGGGCGCAGTGACTCGATGAAGGC
ABCB1 ABCB1 TP_R3 (SEQ ID NO: 160) TCGTGAGGGCGGCAGTGACTCGATGAAGG
ABCB1 ABCB1 TP_R4 (SEQ ID NO: 161) TCGTGAGGGCAGGCAGTGACTCGATGAAG
ABCB1 ABCB1 TP_R5 (SEQ ID NO: 162) TCGTGAGGGCTAGGCAGTGACTCGATGAA
ABCB1 ABCB1 TP_R6 (SEQ ID NO: 163) TCGTGAGGGCTTAGGCAGTGACTCGATGA
ABCB1 ABCB1 TP_R7 (SEQ ID NO: 164) TCGTGAGGGCATTAGGCAGTGACTCGATG
ABCB1 ABCB1 TP_R8 (SEQ ID NO: 165) TCGTGAGGGCCATTAGGCAGTGACTCGAT

ABCG2 (SEQ ID NO: 166) ATGGTCTTAGAAAAGACTCATTATCATTATGTCTCATTAAAATGCTATTTGCCTTAAGGATGATGTTGTGATGGGCACTCTGACGGTGAGAGAAAACTTACAGTTCTCAGCAGCTCTTCGGCTTGCAACAACTATGACGAATCATGAAAAAAACGAACGGATTATAAGGGGTCATTCCAGAGT
ABCG2 ABCG2 TP_F1 (SEQ ID NO: 167) TGTAAGTTTTTAAGGATGATGTTGTGATG
ABCG2 ABCG2 TP_F2 (SEQ ID NO: 168) TGTAAGTTTTTTAAGGATGATGTTGTGAT
ABCG2 ABCG2 TP_F3 (SEQ ID NO: 169) TGTAAGTTTTCTTAAGGATGATGTTGTGA
ABCG2 ABCG2 TP_F4 (SEQ ID NO: 170) TGTAAGTTTTCCTTAAGGATGATGTTGTG
ABCG2 ABCG2 TP_F5 (SEQ ID NO: 171) TGTAAGTTTTGCCTTAAGGATGATGTTGT
ABCG2 ABCG2 TP_F6 (SEQ ID NO: 172) TGTAAGTTTTTGCCTTAAGGATGATGTTG
ABCG2 ABCG2 TP_F7 (SEQ ID NO: 173) TGTAAGTTTTTTGCCTTAAGGATGATGTT
ABCG2 ABCG2 TP_F8 (SEQ ID NO: 174) TGTAAGTTTTTTTGCCTTAAGGATGATGT
ABCG2 ABCG2 TP_R1 (SEQ ID NO: 175) ACAGTTCTCAATGATTCGTCATAGTTGTT
ABCG2 ABCG2 TP_R2 (SEQ ID NO: 176) ACAGTTCTCACATGATTCGTCATAGTTGT
ABCG2 ABCG2 TP_R3 (SEQ ID NO: 177) ACAGTTCTCATCATGATTCGTCATAGTTG
ABCG2 ABCG2 TP_R4 (SEQ ID NO: 178) ACAGTTCTCATTCATGATTCGTCATAGTT
ABCG2 ABCG2 TP_R5 (SEQ ID NO: 179) ACAGTTCTCATTTCATGATTCGTCATAGT
ABCG2 ABCG2 TP_R6 (SEQ ID NO: 180) ACAGTTCTCATTTTCATGATTCGTCATAG
ABCG2 ABCG2 TP_R7 (SEQ ID NO: 181) ACAGTTCTCATTTTTCATGATTCGTCATA
ABCG2 ABCG2 TP_R8 (SEQ ID NO: 182) ACAGTTCTCATTTTTTCATGATTCGTCAT

EGFR20 (SEQ ID NO: 183) GACGTGCCTCTCCCTCCCTCCAGGAAGCCTACGTGATGGCCAGCGTGGACAACCCCCACGTGTGCCGCCTGCTGGGCATCTGCCTCACCTCCACCGTGCAgCTCATCACGCAGCTCATGCCCTTCGGCTGCCTCCTGGACTATGTCCGGGAACACAAAGACATGTGTGCA
EGFR20 EGFR20 TP_F1 (SEQ ID NO: 184) CGTGATGAGGCCGCCTGCTGGGCATCTG
EGFR20 EGFR20 TP_F2 (SEQ ID NO: 185) CGTGATGAGTGCCGCCTGCTGGGCATCT
EGFR20 EGFR20 TP_F3 (SEQ ID NO: 186) CGTGATGAGGTGCCGCCTGCTGGGCATC
EGFR20 EGFR20 TP_F4 (SEQ ID NO: 187) CGTGATGAGTGTGCCGCCTGCTGGGCAT
EGFR20 EGFR20 TP_F5 (SEQ ID NO: 188) CGTGATGAGGTGTGCCGCCTGCTGGGCA
EGFR20 EGFR20 TP_F6 (SEQ ID NO: 189) CGTGATGAGCGTGTGCCGCCTGCTGGGC
EGFR20 EGFR20 TP_F7 (SEQ ID NO: 190) CGTGATGAGACGTGTGCCGCCTGCTGGG
EGFR20 EGFR20 TP_F8 (SEQ ID NO: 191) CGTGATGAGCACGTGTGCCGCCTGCTGG
EGFR20 EGFR20 TP_R1 (SEQ ID NO: 192) ACGCAGCTCTGTTCCCGGACATAGTCCA
EGFR20 EGFR20 TP_R2 (SEQ ID NO: 193) ACGCAGCTCGTGTTCCCGGACATAGTCC
EGFR20 EGFR20 TP_R3 (SEQ ID NO: 194) ACGCAGCTCTGTGTTCCCGGACATAGTC
EGFR20 EGFR20 TP_R4 (SEQ ID NO: 195) ACGCAGCTCTTGTGTTCCCGGACATAGT
EGFR20 EGFR20 TP_R5 (SEQ ID NO: 196) ACGCAGCTCTTTGTGTTCCCGGACATAG
EGFR20 EGFR20 TP_R6 (SEQ ID NO: 197) ACGCAGCTCCTTTGTGTTCCCGGACATA
EGFR20 EGFR20 TP_R7 (SEQ ID NO: 198) ACGCAGCTCTCTTTGTGTTCCCGGACAT
EGFR20 EGFR20 TP_R8 (SEQ ID NO: 199) ACGCAGCTCGTCTTTGTGTTCCCGGACA

EGFR20 mutant (SEQ ID NO: 200) GACGTGCCTCTCCCTCCCTCCAGGAAGCCTACGTGATGGCCAGCGTGGACAACCCCCACGTGTGCCGCCTGCTGGGCATCTGCCTCACCTCCACCGTGCAgCTCATCATGCAGCTCATGCCCTTCGGCTGCCTCCTGGACTATGTCCGGGAACACAAAGACATGTG
EGFR20 EGFR20m TP_F1 (SEQ ID NO: 201) CATGATGAGGCCGCCTGCTGGGCATCTG
EGFR20 EGFR20m TP_F2 (SEQ ID NO: 202) CATGATGAGTGCCGCCTGCTGGGCATCT
EGFR20 EGFR20m TP_F3 (SEQ ID NO: 203) CATGATGAGGTGCCGCCTGCTGGGCATC
EGFR20 EGFR20m TP_F4 (SEQ ID NO: 204) CATGATGAGTGTGCCGCCTGCTGGGCAT
EGFR20 EGFR20m TP_F5 (SEQ ID NO: 205) CATGATGAGGTGTGCCGCCTGCTGGGCA
EGFR20 EGFR20m TP_F6 (SEQ ID NO: 206) CATGATGAGCGTGTGCCGCCTGCTGGGC
EGFR20 EGFR20m TP_F7 (SEQ ID NO: 207) CATGATGAGACGTGTGCCGCCTGCTGGG
EGFR20 EGFR20m TP_F8 (SEQ ID NO: 208) CATGATGAGCACGTGTGCCGCCTGCTGG
EGFR20 EGFR20m TP_R1 (SEQ ID NO: 209) CATGCAGCTTGTTCCCGGACATAGTCCA
EGFR20 EGFR20m TP_R2 (SEQ ID NO: 210) CATGCAGCTGTGTTCCCGGACATAGTCC
EGFR20 EGFR20m TP_R3 (SEQ ID NO: 211) CATGCAGCTTGTGTTCCCGGACATAGTC
EGFR20 EGFR20m TP_R4 (SEQ ID NO: 212) CATGCAGCTTTGTGTTCCCGGACATAGT
EGFR20 EGFR20m TP_R5 (SEQ ID NO: 213) CATGCAGCTTTTGTGTTCCCGGACATAG
EGFR20 EGFR20m TP_R6 (SEQ ID NO: 214) CATGCAGCTCTTTGTGTTCCCGGACATA
EGFR20 EGFR20m TP_R7 (SEQ ID NO: 215) CATGCAGCTTCTTTGTGTTCCCGGACAT
EGFR20 EGFR20m TP_R8 (SEQ ID NO: 216) CATGCAGCTGTCTTTGTGTTCCCGGACA

FTO (SEQ ID NO: 217) TTATGAGATAATGTCCTTTTTAAAAATAAACACTAACATCAGTTATGCATTTAGAATGTCTGAATTATTATTCTAGGTTCCTTGCGACTGCTGTGAATTTAGTGATGCACTTGGATAGTCTCTGTTACTCTAAAGTTTTAATAGGTAACAGTCAGAAATGGAGTGGGAGAGCTGAAAACACAATCGATG
FTO FTO TP_F1 (SEQ ID NO: 218) CTAAATTCACTAGAATGTCTGAATTATTATTC
FTO FTO TP_F2 (SEQ ID NO: 219) CTAAATTCACTTAGAATGTCTGAATTATTATT
FTO FTO TP_F3 (SEQ ID NO: 220) CTAAATTCACTTTAGAATGTCTGAATTATTAT
FTO FTO TP_F4 (SEQ ID NO: 221) CTAAATTCACATTTAGAATGTCTGAATTATTA
FTO FTO TP_F5 (SEQ ID NO: 222) CTAAATTCACCATTTAGAATGTCTGAATTATT
FTO FTO TP_F6 (SEQ ID NO: 223) CTAAATTCACGCATTTAGAATGTCTGAATTAT
FTO FTO TP_F7 (SEQ ID NO: 224) CTAAATTCACTGCATTTAGAATGTCTGAATTA
FTO FTO TP_F8 (SEQ ID NO: 225) CTAAATTCACATGCATTTAGAATGTCTGAATT
FTO FTO TP_R1 (SEQ ID NO: 226) AGTGATGCACCTGTTACCTATTAAAACTTTAG
FTO FTO TP_R2 (SEQ ID NO: 227) AGTGATGCACACTGTTACCTATTAAAACTTTA
FTO FTO TP_R3 (SEQ ID NO: 228) AGTGATGCACGACTGTTACCTATTAAAACTTT
FTO FTO TP_R4 (SEQ ID NO: 229) AGTGATGCACTGACTGTTACCTATTAAAACTT
FTO FTO TP_R5 (SEQ ID NO: 230) AGTGATGCACCTGACTGTTACCTATTAAAACT
FTO FTO TP_R6 (SEQ ID NO: 231) AGTGATGCACTCTGACTGTTACCTATTAAAAC
FTO FTO TP_R7 (SEQ ID NO: 232) AGTGATGCACTTCTGACTGTTACCTATTAAAA
FTO FTO TP_R8 (SEQ ID NO: 233) AGTGATGCACTTTCTGACTGTTACCTATTAAA

FTO TTATGAGATAATGTCCTTTTTAAAAATAAACACTAACATCAGTTATGCATTTAGAATGTCTGAATTATTATTCTAGGTTCCTTGCGACTGCTGTGAATTTAGTGATGCACTTGGATAGTCTCTGTTACTCTAAAGTTTTAATAGGTAACAGTCAGAAATGGAGTGGGAGAGCATAAAAGCAAACTGAAATGCAAATAGCTG
FTO FTO_2 TP_F1 (SEQ ID NO: 234) CTAAATTCACAGCAGTTATGCATTTAGAATGTCTGA
FTO FTO_2 TP_F2 (SEQ ID NO: 235) CTAAATTCACAGTCAGTTATGCATTTAGAATGTCTG
FTO FTO_2 TP_F3 (SEQ ID NO: 236) CTAAATTCACAGATCAGTTATGCATTTAGAATGTCT
FTO FTO_2 TP_F4 (SEQ ID NO: 237) CTAAATTCACAGCATCAGTTATGCATTTAGAATGTC
FTO FTO_2 TP_F5 (SEQ ID NO: 238) CTAAATTCACAGACATCAGTTATGCATTTAGAATGT
FTO FTO_2 TP_F6 (SEQ ID NO: 239) CTAAATTCACAGAACATCAGTTATGCATTTAGAATG
FTO FTO_2 TP_F7 (SEQ ID NO: 240) CTAAATTCACAGTAACATCAGTTATGCATTTAGAAT
FTO FTO_2 TP_F8 (SEQ ID NO: 241) CTAAATTCACAGCTAACATCAGTTATGCATTTAGAA
FTO FTO_2 TP_R1 (SEQ ID NO: 242) AGTGATGCACTTCCATTTCTGACTGTTACCTATTAA
FTO FTO_2 TP_R2 (SEQ ID NO: 243) AGTGATGCACTTTCCATTTCTGACTGTTACCTATTA
FTO FTO_2 TP_R3 (SEQ ID NO: 244) AGTGATGCACTTCTCCATTTCTGACTGTTACCTATT
FTO FTO_2 TP_R4 (SEQ ID NO: 245) AGTGATGCACTTACTCCATTTCTGACTGTTACCTAT
FTO FTO_2 TP_R5 (SEQ ID NO: 246) AGTGATGCACTTCACTCCATTTCTGACTGTTACCTA
FTO FTO_2 TP_R6 (SEQ ID NO: 247) AGTGATGCACTTCCACTCCATTTCTGACTGTTACCT
FTO FTO_2 TP_R7 (SEQ ID NO: 248) AGTGATGCACTTCCCACTCCATTTCTGACTGTTACC
FTO FTO_2 TP_R8 (SEQ ID NO: 249) AGTGATGCACTTTCCCACTCCATTTCTGACTGTTAC

GGCX (SEQ ID NO: 250) CTCCCTGCTTTCCTAGGTATGTTCTCCTACGTCATGCTGGCCAGCAGCCCTCTCTTCTGCTCCCCTGAGTGGCCTCGGAAGCTGGTGTCCTACTGCCCCCGAAGGTTGCAACAACTGTTGCCCCTCAAGGCAGCCCCTCAGCCCAGGATTTCCTGTGTGTGAAGAGGGGGCCGCGGCAAA
GGCX GGCX TP_F1 (SEQ ID NO: 251) TCGGGGGCAGTGCTCCCCTGAGTGGC
GGCX GGCX TP_F2 (SEQ ID NO: 252) TCGGGGGCAGCTGCTCCCCTGAGTGG
GGCX GGCX TP_F3 (SEQ ID NO: 253) TCGGGGGCAGTCTGCTCCCCTGAGTG
GGCX GGCX TP_F4 (SEQ ID NO: 254) TCGGGGGCAGTTCTGCTCCCCTGAGT
GGCX GGCX TP_F5 (SEQ ID NO: 255) TCGGGGGCAGCTTCTGCTCCCCTGAG
GGCX GGCX TP_F6 (SEQ ID NO: 256) TCGGGGGCAGTCTTCTGCTCCCCTGA
GGCX GGCX TP_F7 (SEQ ID NO: 257) TCGGGGGCAGCTCTTCTGCTCCCCTG
GGCX GGCX TP_F8 (SEQ ID NO: 258) TCGGGGGCAGTCTCTTCTGCTCCCCT
GGCX GGCX TP_R1 (SEQ ID NO: 259) CGAAGGTTGCGGGCTGAGGGGCTGCC
GGCX GGCX TP_R2 (SEQ ID NO: 260) CGAAGGTTGCTGGGCTGAGGGGCTGC
GGCX GGCX TP_R3 (SEQ ID NO: 261) CGAAGGTTGCCTGGGCTGAGGGGCTG
GGCX GGCX TP_R4 (SEQ ID NO: 262) CGAAGGTTGCACTGGGCTGAGGGGCT
GGCX GGCX TP_R5 (SEQ ID NO: 263) CGAAGGTTGCCACTGGGCTGAGGGGC
GGCX GGCX TP_R6 (SEQ ID NO: 264) CGAAGGTTGCACACTGGGCTGAGGGG
GGCX GGCX TP_R7 (SEQ ID NO: 265) CGAAGGTTGCAACACTGGGCTGAGGG
GGCX GGCX TP_R8 (SEQ ID NO: 266) CGAAGGTTGCAAACACTGGGCTGAGG

MTHFR (SEQ ID NO: 267) TGACTGTCATCCCTATTGGCAGGTTACCCCAAAGGCCACCCCGAAGCAGGGAGCTTTGAGGCTGACCTGAAGCACTTGAAGGAGAAGGTGTCTGCGGGAGCCGATTTCATCATCACGCAGCTTTTCTTTGAGGCTGACACATTCTTCCGCTTTGTGAAGGCATGCACTCGCATGGGGCATCACTTG
MTHFR MTHFR TP_F1 (SEQ ID NO: 268) GGCTCCCGCATTTGAGGCTGACCTGAAGC
MTHFR MTHFR TP_F2 (SEQ ID NO: 269) GGCTCCCGCACTTTGAGGCTGACCTGAAG
MTHFR MTHFR TP_F3 (SEQ ID NO: 270) GGCTCCCGCAGCTTTGAGGCTGACCTGAA
MTHFR MTHFR TP_F4 (SEQ ID NO: 271) GGCTCCCGCAAGCTTTGAGGCTGACCTGA
MTHFR MTHFR TP_F5 (SEQ ID NO: 272) GGCTCCCGCAGAGCTTTGAGGCTGACCTG
MTHFR MTHFR TP_F6 (SEQ ID NO: 273) GGCTCCCGCAGGAGCTTTGAGGCTGACCT
MTHFR MTHFR TP_F7 (SEQ ID NO: 274) GGCTCCCGCAGGGAGCTTTGAGGCTGACC
MTHFR MTHFR TP_F8 (SEQ ID NO: 275) GGCTCCCGCAAGGGAGCTTTGAGGCTGAC
MTHFR MTHFR TP_R1 (SEQ ID NO: 276) GCCGATTTCAGAAGAATGTGTCAGCCTCA
MTHFR MTHFR TP_R2 (SEQ ID NO: 277) GCCGATTTCAGGAAGAATGTGTCAGCCTC
MTHFR MTHFR TP_R3 (SEQ ID NO: 278) GCCGATTTCACGGAAGAATGTGTCAGCCT
MTHFR MTHFR TP_R4 (SEQ ID NO: 279) GCCGATTTCAGCGGAAGAATGTGTCAGCC
MTHFR MTHFR TP_R5 (SEQ ID NO: 280) GCCGATTTCAAGCGGAAGAATGTGTCAGC
MTHFR MTHFR TP_R6 (SEQ ID NO: 281) GCCGATTTCAAAGCGGAAGAATGTGTCAG
MTHFR MTHFR TP_R7 (SEQ ID NO: 282) GCCGATTTCAAAAGCGGAAGAATGTGTCA
MTHFR MTHFR TP_R8 (SEQ ID NO: 283) GCCGATTTCACAAAGCGGAAGAATGTGTC

MTHFR TGACTGTCATCCCTATTGGCAGGTTACCCCAAAGGCCACCCCGAAGCAGGGAGCTTTGAGGCTGACCTGAAGCACTTGAAGGAGAAGGTGTCTGCGGGAGCCGATTTCATCATCACGCAGCTTTTCTTTGAGGCTGACACATTCTTCCGCTTTGTGAAGGCATGCACCGACATGGGCATCACTTGCCCCATCGTCCG
MTHFR MTHFR_2 TP_F1 (SEQ ID NO: 284) GGCTCCCTTTGAGGCTGACCTGAAGC
MTHFR MTHFR_2 TP_F2 (SEQ ID NO: 285) GGCTCCCCTTTGAGGCTGACCTGAAG
MTHFR MTHFR_2 TP_F3 (SEQ ID NO: 286) GGCTCCCGCTTTGAGGCTGACCTGAA
MTHFR MTHFR_2 TP_F4 (SEQ ID NO: 287) GGCTCCCAGCTTTGAGGCTGACCTGA
MTHFR MTHFR_2 TP_F5 (SEQ ID NO: 288) GGCTCCCGAGCTTTGAGGCTGACCTG
MTHFR MTHFR_2 TP_F6 (SEQ ID NO: 289) GGCTCCCGGAGCTTTGAGGCTGACCT
MTHFR MTHFR_2 TP_F7 (SEQ ID NO: 290) GGCTCCCGGGAGCTTTGAGGCTGACC
MTHFR MTHFR_2 TP_F8 (SEQ ID NO: 291) GGCTCCCAGGGAGCTTTGAGGCTGAC
MTHFR MTHFR_2 TP_R1 (SEQ ID NO: 292) GCCGATTGAAGAATGTGTCAGCCTCA
MTHFR MTHFR_2 TP_R2 (SEQ ID NO: 293) GCCGATTGGAAGAATGTGTCAGCCTC
MTHFR MTHFR_2 TP_R3 (SEQ ID NO: 294) GCCGATTCGGAAGAATGTGTCAGCCT
MTHFR MTHFR_2 TP_R4 (SEQ ID NO: 295) GCCGATTGCGGAAGAATGTGTCAGCC
MTHFR MTHFR_2 TP_R5 (SEQ ID NO: 296) GCCGATTAGCGGAAGAATGTGTCAGC
MTHFR MTHFR_2 TP_R6 (SEQ ID NO: 297) GCCGATTAAGCGGAAGAATGTGTCAG
MTHFR MTHFR_2 TP_R7 (SEQ ID NO: 298) GCCGATTAAAGCGGAAGAATGTGTCA
MTHFR MTHFR_2 TP_R8 (SEQ ID NO: 299) GCCGATTCAAAGCGGAAGAATGTGTC

NAT2-341 (SEQ ID NO: 300) ACAATCGGTTTTCAGACCACAATGTTAGGAGGGTATTTTTACATCCCTCCAGTTAACAAATACAGCACTGGCATGGTTCACCTTCTCCTGCAGGTGACCATTGACGGCAGGAATTACATTGTCGATGCTGGGTCTGGAAGCTCCTCCCAGATGTGGCAGCCTCTAGAGCTGC
NAT2-341 NAT2-341 TP_F1 (SEQ ID NO: 301) CAATGGTCACCAGTTAACAAATACAGCACT
NAT2-341 NAT2-341 TP_F2 (sequence number 302) CAATGGTCACCCAGTTAACAAATACAGCAC
NAT2-341 NAT2-341 TP_F3 (sequence number 303) CAATGGTCACTCCAGTTAACAAATACAGCA
NAT2-341 NAT2-341 TP_F4 (sequence number 304) CAATGGTCACCTCCAGTTAACAAATACAGC
NAT2-341 NAT2-341 TP_F5 (sequence number 305) CAATGGTCACCCTCCAGTTAACAAATACAG
NAT2-341 NAT2-341 TP_F6 (sequence number 306) CAATGGTCACCCCTCCAGTTAACAAATACA
NAT2-341 NAT2-341 TP_F7 (sequence number 307) CAATGGTCACTCCCTCCAGTTAACAAATAC
NAT2-341 NAT2-341 TP_F8 (sequence number 308) CAATGGTCACATCCCTCCAGTTAACAAATA
NAT2-341 NAT2-341 TP_R1 (SEQ ID NO: 309) CATTGACGGTCTGGGAGGAGCTTCCAGAC
NAT2-341 NAT2-341 TP_R2 (SEQ ID NO: 310) CATTGACGGATCTGGGAGGAGCTTCCAGA
NAT2-341 NAT2-341 TP_R3 (SEQ ID NO: 311) CATTGACGGCATCTGGGAGGAGCTTCCAG
NAT2-341 NAT2-341 TP_R4 (SEQ ID NO: 312) CATTGACGGCTGGGAGGAGCTTCCAGAC
NAT2-341 NAT2-341 TP_R5 (SEQ ID NO: 313) CATTGACGGTCTGGGAGGAGCTTCCAGA
NAT2-341 NAT2-341 TP_R6 (SEQ ID NO: 314) CATTGACGGATCTGGGAGGAGCTTCCAG
NAT2-341 NAT2-341 TP_R7 (SEQ ID NO: 315) CATTGACGGGGCTGCCACATCTGGGAG
NAT2-341 NAT2-341 TP_R8 (SEQ ID NO: 316) CATTGACGGAGGCTGCCACATCTGGGA

NAT2-590 (SEQ ID NO: 317) ATCAGGAGAGAGCAGTATATTACAAACAAAGAATTTCTTAATTCTCATCTCCTGCCAAAGAAGAAACACCAAAAAATATACTTATTTACGCTTGAACCTCGAACAATTGAAGATTTTGAGTCTATGAATACATACCTGCAGACGTCTCCAACATCTTCATTTATAACCAGCATCATTTTGTTCCTTGCAG
NAT2-590 NAT2-590 TP_F1 (SEQ ID NO: 318) TCGAGGTTCACTGCCAAAGAAGAAACACCAAA
NAT2-590 NAT2-590 TP_F2 (SEQ ID NO: 319) TCGAGGTTCACCTGCCAAAGAAGAAACACCAA
NAT2-590 NAT2-590 TP_F3 (sequence number 320) TCGAGGTTCATCCTGCCAAAGAAGAAACACCA
NAT2-590 NAT2-590 TP_F4 (SEQ ID NO: 321) TCGAGGTTCACTCCTGCCAAAGAAGAAACACC
NAT2-590 NAT2-590 TP_F5 (SEQ ID NO: 322) TCGAGGTTCATCTCCTGCCAAAGAAGAAACAC
NAT2-590 NAT2-590 TP_F6 (SEQ ID NO: 323) TCGAGGTTCAATCTCCTGCCAAAGAAGAAACA
NAT2-590 NAT2-590 TP_F7 (SEQ ID NO: 324) TCGAGGTTCACATCTCCTGCCAAAGAAGAAAC
NAT2-590 NAT2-590 TP_F8 (SEQ ID NO: 325) TCGAGGTTCATCATCTCCTGCCAAAGAAGAAA
NAT2-590 NAT2-590 TP_R1 (SEQ ID NO: 326) CGAACAATTGTGGAGACGTCTGCAGGTATGTA
NAT2-590 NAT2-590 TP_R2 (SEQ ID NO: 327) CGAACAATTGTTGGAGACGTCTGCAGGTATGT
NAT2-590 NAT2-590 TP_R3 (SEQ ID NO: 328) CGAACAATTGGTTGGAGACGTCTGCAGGTATG
NAT2-590 NAT2-590 TP_R4 (SEQ ID NO: 329) CGAACAATTGTGTTGGAGACGTCTGCAGGTAT
NAT2-590 NAT2-590 TP_R5 (SEQ ID NO: 330) CGAACAATTGATGTTGGAGACGTCTGCAGGTA
NAT2-590 NAT2-590 TP_R6 (SEQ ID NO: 331) CGAACAATTGGATGTTGGAGACGTCTGCAGGT
NAT2-590 NAT2-590 TP_R7 (SEQ ID NO: 332) CGAACAATTGAGATGTTGGAGACGTCTGCAGG
NAT2-590 NAT2-590 TP_R8 (SEQ ID NO: 333) CGAACAATTGAAGATGTTGGAGACGTCTGCAG

NAT2-857 (SEQ ID NO: 334) GTCGAGTTTAAAACTCTCACTGAGGAAGAGGTTGAAGAAGTGCTGAAAAATATATTTAAGATTTCCTTGGGGAGAAATCTCGTGCCCAAACCTGGTGATGGATCCCTTACTATTTAGAATAAGGAACAAAATAAACCCTTGTGTATGTATCACCCAACTCACTAATTATCAACTTATGTGCTCTAGACTATATGTCCT
NAT2-857 NAT2-857 TP_F1 (SEQ ID NO: 335) TCCATCACCAATTTAAGATTTCCTTGGGGAGA
NAT2-857 NAT2-857 TP_F2 (SEQ ID NO: 336) TCCATCACCATATTTAAGATTTCCTTGGGGAG
NAT2-857 NAT2-857 TP_F3 (SEQ ID NO: 337) TCCATCACCAATATTTAAGATTTCCTTGGGGA
NAT2-857 NAT2-857 TP_F4 (SEQ ID NO: 338) TCCATCACCATATATTTAAGATTTCCTTGGGG
NAT2-857 NAT2-857 TP_F5 (SEQ ID NO: 339) TCCATCACCAATATATTTAAGATTTCCTTGGG
NAT2-857 NAT2-857 TP_F6 (SEQ ID NO: 340) TCCATCACCAAATATATTTAAGATTTCCTTGG
NAT2-857 NAT2-857 TP_F7 (Sequence No. 341) TCCATCACCAAAATATATTTAAGATTTCCTTG
NAT2-857 NAT2-857 TP_F8 (SEQ ID NO: 342) TCCATCACCAAAAATATATTTAAGATTTCCTT
NAT2-857 NAT2-857 TP_R1 (SEQ ID NO: 343) GATCCCTTACTGGGTGATACATACACAAGGGT
NAT2-857 NAT2-857 TP_R2 (SEQ ID NO: 344) GATCCCTTACTTGGGTGATACATACACAAGGG
NAT2-857 NAT2-857 TP_R3 (SEQ ID NO: 345) GATCCCTTACGTTGGGTGATACATACACAAGG
NAT2-857 NAT2-857 TP_R4 (SEQ ID NO: 346) GATCCCTTACAGTTGGGTGATACATACACAAG
NAT2-857 NAT2-857 TP_R5 (SEQ ID NO: 347) GATCCCTTACGAGTTGGGTGATACATACACAA
NAT2-857 NAT2-857 TP_R6 (SEQ ID NO: 348) GATCCCTTACTGAGTTGGGTGATACATACACA
NAT2-857 NAT2-857 TP_R7 (SEQ ID NO: 349) GATCCCTTACGTGAGTTGGGTGATACATACAC
NAT2-857 NAT2-857 TP_R8 (SEQ ID NO: 350) GATCCCTTACAGTGAGTTGGGTGATACATACA

NAT2-857 GTCGAGTTTAAAACTCTCACTGAGGAAGAGGTTGAAGAAGTGCTGAAAAATATATTTAAGATTTCCTTGGGGAGAAATCTCGTGCCCAAACCTGGTGATGGATCCCTTACTATTTAGAATAAGGAACAAAATAAACCCTTGTGTATGTATCACCCAACTCACTAATTATCAACTTATGTGCTTATCAGATATCCTCT
NAT2-857 NAT2-857_2 TP_F1 (SEQ ID NO: 351) TCCATCACCAATTTAAGATTTCCTTGGGGAGAAAT
NAT2-857 NAT2-857_2 TP_F2 (SEQ ID NO: 352) TCCATCACCATATTTAAGATTTCCTTGGGGAGAAA
NAT2-857 NAT2-857_2 TP_F3 (SEQ ID NO: 353) TCCATCACCAATATTTAAGATTTCCTTGGGGAGAA
NAT2-857 NAT2-857_2 TP_F4 (SEQ ID NO: 354) TCCATCACCATATATTTAAGATTTCCTTGGGGAGA
NAT2-857 NAT2-857_2 TP_F5 (SEQ ID NO: 355) TCCATCACCAATATATTTAAGATTTCCTTGGGGAG
NAT2-857 NAT2-857_2 TP_F6 (sequence number 356) TCCATCACCAAATATATTTAAGATTTCCTTGGGGA
NAT2-857 NAT2-857_2 TP_F7 (SEQ ID NO: 357) TCCATCACCAAAATATATTTAAGATTTCCTTGGGG
NAT2-857 NAT2-857_2 TP_F8 (SEQ ID NO: 358) TCCATCACCAAAAATATATTTAAGATTTCCTTGGG
NAT2-857 NAT2-857_2 TP_R1 (SEQ ID NO: 359) GATCCCTTACATACATACACAAGGGTTTATTTTGT
NAT2-857 NAT2-857_2 TP_R2 (SEQ ID NO: 360) GATCCCTTACGATACATACACAAGGGTTTATTTTG
NAT2-857 NAT2-857_2 TP_R3 (SEQ ID NO: 361) GATCCCTTACTGATACATACACAAGGGTTTATTTT
NAT2-857 NAT2-857_2 TP_R4 (sequence number 362) GATCCCTTACGTGATACATACACAAGGGTTTATTT
NAT2-857 NAT2-857_2 TP_R5 (SEQ ID NO: 363) GATCCCTTACGGTGATACATACACAAGGGTTTATT
NAT2-857 NAT2-857_2 TP_R6 (SEQ ID NO: 364) GATCCCTTACGGGTGATACATACACAAGGGTTTAT
NAT2-857 NAT2-857_2 TP_R7 (SEQ ID NO: 365) GATCCCTTACTGGGTGATACATACACAAGGGTTTA
NAT2-857 NAT2-857_2 TP_R8 (SEQ ID NO: 366) GATCCCTTACTTGGGTGATACATACACAAGGGTTT

rs7566605 (SEQ ID NO: 367) AGTTGATCTAATGTTCTCTCTCCTACCTCCCTCCAATACCCCATCGGAATTGAAATCATTGCAATAGCCACTGCCAAGTACTTAACAATGGATATTTGATCGTGGTCCTTTAGGTCTGTACCAGGGTGGTTTTCAGTTTTTTACTTCATCAGCACAGGAAGGCAAGGACATATTCTCCATCTCTCAT
rs7566605 rs7566605 TP_F1 (SEQ ID NO: 368) CGATCAAATAATCATTGCAATAGCCACTG
rs7566605 rs7566605 TP_F2 (SEQ ID NO: 369) CGATCAAATAAATCATTGCAATAGCCACT
rs7566605 rs7566605 TP_F3 (SEQ ID NO: 370) CGATCAAATAAAATCATTGCAATAGCCAC
rs7566605 rs7566605 TP_F4 (SEQ ID NO: 371) CGATCAAATAGAAATCATTGCAATAGCCA
rs7566605 rs7566605 TP_F5 (SEQ ID NO: 372) CGATCAAATATGAAATCATTGCAATAGCC
rs7566605 rs7566605 TP_F6 (SEQ ID NO: 373) CGATCAAATATTGAAATCATTGCAATAGC
rs7566605 rs7566605 TP_F7 (SEQ ID NO: 374) CGATCAAATAATTGAAATCATTGCAATAG
rs7566605 rs7566605 TP_F8 (SEQ ID NO: 375) CGATCAAATAAATTGAAATCATTGCAATA
rs7566605 rs7566605 TP_R1 (SEQ ID NO: 376) TCGTGGTCCTGAAGTAAAAAACTGAAAAC
rs7566605 rs7566605 TP_R2 (SEQ ID NO: 377) TCGTGGTCCTTGAAGTAAAAAACTGAAAA
rs7566605 rs7566605 TP_R3 (SEQ ID NO: 378) TCGTGGTCCTATGAAGTAAAAAACTGAAA
rs7566605 rs7566605 TP_R4 (SEQ ID NO: 379) TCGTGGTCCTGATGAAGTAAAAAACTGAA
rs7566605 rs7566605 TP_R5 (SEQ ID NO: 380) TCGTGGTCCTTGATGAAGTAAAAAACTGA
rs7566605 rs7566605 TP_R6 (SEQ ID NO: 381) TCGTGGTCCTCTGATGAAGTAAAAAACTG
rs7566605 rs7566605 TP_R7 (SEQ ID NO: 382) TCGTGGTCCTGCTGATGAAGTAAAAAACT
rs7566605 rs7566605 TP_R8 (SEQ ID NO: 383) TCGTGGTCCTTGCTGATGAAGTAAAAAAC

KCNQ1_rs2283228 (SEQ ID NO: 384) CCCAAGAAGAAAACTCTGAGAAGTCAGGACAGGAGATGGTGTCGTCAGGGGGCTGCTGGAAGGTTTGGTGACCGGAGCAGCAGGTGCCAGAGCTGAAAGCACTGGTTAAACGTGATGGGTAAACACATGTCTAATCCATCTGGTTCAAATGATCACTGG
KCNQ1_rs2283228 KCNQ1_rs2283228 TP-F1 (SEQ ID NO: 385) gTGCTTTCAGCTGGCTGCTGGAAGGTTTGGTG
KCNQ1_rs2283228 KCNQ1_rs2283228 TP-F2 (SEQ ID NO: 386) gTGCTTTCAGCTGCTGCTGGAAGGTTTGGTGA
KCNQ1_rs2283228 KCNQ1_rs2283228 TP-F3 (SEQ ID NO: 387) gTGCTTTCAGCTCTGCTGGAAGGTTTGGTGAC
KCNQ1_rs2283228 KCNQ1_rs2283228 TP-F4 (SEQ ID NO: 388) gTGCTTTCAGCTTGCTGGAAGGTTTGGTGACC
KCNQ1_rs2283228 KCNQ1_rs2283228 TP-F5 (SEQ ID NO: 389) gTGCTTTCAGCTGCTGGAAGGTTTGGTGACCG
KCNQ1_rs2283228 KCNQ1_rs2283228 TP-F6 (SEQ ID NO: 390) gTGCTTTCAGCTCTGGAAGGTTTGGTGACCGG
KCNQ1_rs2283228 KCNQ1_rs2283228 TP-F7 (SEQ ID NO: 391) gTGCTTTCAGCTTGGAAGGTTTGGTGACCGGA
KCNQ1_rs2283228 KCNQ1_rs2283228 TP-F8 (SEQ ID NO: 392) gTGCTTTCAGCTGGAAGGTTTGGTGACCGGAG
KCNQ1_rs2283228 KCNQ1_rs2283228 TP-R1 (SEQ ID NO: 393) CActggttaaacttcaaagaagctgaaataga
KCNQ1_rs2283228 KCNQ1_rs2283228 TP-R2 (SEQ ID NO: 394) CActggttaaacattcaaagaagctgaaatag
KCNQ1_rs2283228 KCNQ1_rs2283228 TP-R3 (SEQ ID NO: 395) CActggttaaacgattcaaagaagctgaaata
KCNQ1_rs2283228 KCNQ1_rs2283228 TP-R4 (SEQ ID NO: 396) CActggttaaacggattcaaagaagctgaaat
KCNQ1_rs2283228 KCNQ1_rs2283228 TP-R5 (SEQ ID NO: 397) CActggttaaacgggattcaaagaagctgaaa
KCNQ1_rs2283228 KCNQ1_rs2283228 TP-R6 (SEQ ID NO: 398) CActggttaaactgggattcaaagaagctgaa
KCNQ1_rs2283228 KCNQ1_rs2283228 TP-R7 (SEQ ID NO: 399) CActggttaaacgtgggattcaaagaagctga
KCNQ1_rs2283228 KCNQ1_rs2283228 TP-R8 (SEQ ID NO: 400) CActggttaaacagtgggattcaaagaagctg

KCNQ1_rs2237897 (SEQ ID NO: 401) AGGCCTGGGTGGAGACAGAACACAGCAGGCTGTGGGCGTGGCCAATGTTGAGGGACGGAGGTGGCCAGAGGCCTCTCAGTGGTGCCCGGGGGGCCTCATCCTTCCCCTGAGCCCCGCCCCTCAGGCCCAGCGCCTGTGCGCGCCCGCGCGT
KCNQ1_rs2237897 KCNQ1_rs2237897 TP-F1 (SEQ ID NO: 402) CGTCCCCAGCTCCCTAGGGACGGAGGTGGCCAGAG
KCNQ1_rs2237897 KCNQ1_rs2237897 TP-F2 (SEQ ID NO: 403) CGTCCCCAGCTCCCTGGGACGGAGGTGGCCAGAGG
KCNQ1_rs2237897 KCNQ1_rs2237897 TP-F3 (SEQ ID NO: 404) CGTCCCCAGCTCCCTGGACGGAGGTGGCCAGAGGC
KCNQ1_rs2237897 KCNQ1_rs2237897 TP-F4 (SEQ ID NO: 405) CGTCCCCAGCTCCCTGACGGAGGTGGCCAGAGGCC
KCNQ1_rs2237897 KCNQ1_rs2237897 TP-F5 (SEQ ID NO: 406) CGTCCCCAGCTCCCTACGGAGGTGGCCAGAGGCCT
KCNQ1_rs2237897 KCNQ1_rs2237897 TP-F6 (SEQ ID NO: 407) CGTCCCCAGCTCCCTCGGAGGTGGCCAGAGGCCTC
KCNQ1_rs2237897 KCNQ1_rs2237897 TP-F7 (SEQ ID NO: 408) CGTCCCCAGCTCCCTGGAGGTGGCCAGAGGCCTCT
KCNQ1_rs2237897 KCNQ1_rs2237897 TP-F8 (SEQ ID NO: 409) CGTCCCCAGCTCCCTGAGGTGGCCAGAGGCCTCTC
KCNQ1_rs2237897 KCNQ1_rs2237897 TP-R1 (SEQ ID NO: 410) ACGAGGGGCCTCACCCTGGGCCTGAAGGGAGGG
KCNQ1_rs2237897 KCNQ1_rs2237897 TP-R2 (SEQ ID NO: 411) ACGAGGGGCCTCATCCCTGGGCCTGAAGGGAGG
KCNQ1_rs2237897 KCNQ1_rs2237897 TP-R3 (SEQ ID NO: 412) ACGAGGGGCCTCACTCCCTGGGCCTGAAGGGAG
KCNQ1_rs2237897 KCNQ1_rs2237897 TP-R4 (SEQ ID NO: 413) ACGAGGGGCCTCAACTCCCTGGGCCTGAAGGGA
KCNQ1_rs2237897 KCNQ1_rs2237897 TP-R5 (SEQ ID NO: 414) ACGAGGGGCCTCACACTCCCTGGGCCTGAAGGG
KCNQ1_rs2237897 KCNQ1_rs2237897 TP-R6 (SEQ ID NO: 415) ACGAGGGGCCTCACCACTCCCTGGGCCTGAAGG
KCNQ1_rs2237897 KCNQ1_rs2237897 TP-R7 (SEQ ID NO: 416) ACGAGGGGCCTCAGCCACTCCCTGGGCCTGAAG
KCNQ1_rs2237897 KCNQ1_rs2237897 TP-R8 (SEQ ID NO: 417) ACGAGGGGCCTCAGGCCACTCCCTGGGCCTGAA

Preparation of template plasmid A plasmid prepared by the following method was used as a template nucleic acid for nucleic acid amplification so that genomic fragments other than the region to be amplified would not affect the result of nucleic acid amplification. First, target nucleic acid sequences of various target genes were amplified by PCR. The amplification product was inserted into a pGEM-T plasmid and cloned to obtain plasmids containing target nucleic acid sequences of various target genes. The plasmid was used as a template nucleic acid for nucleic acid amplification. The plasmid was stored at −20 ° C. until used as a template nucleic acid.

Nucleic acid amplification reaction The nucleic acid amplification reaction was performed on all target sequences using all combinations (64 ways) of F1 TP to F8 TP and R1 TP to R8 TP with the following method. The template nucleic acid plasmid was diluted to 2000 copies / μL, then heated to 98 ° C. and denatured. The composition of the nucleic acid amplification reaction solution is as shown below. A reaction solution containing no template nucleic acid was used as a negative control. The reaction solution was prepared on ice to make 25 μL. Nucleic acid was amplified by incubating the reaction solution at 60 ° C. for 90 minutes. The generation of amplification products was monitored every minute using a real-time fluorescence detection apparatus (trade name Mx3000P, manufactured by Stratagene, or trade name LightCycler R480, manufactured by Roche). The time until the fluorescence intensity monitored at each time point by the real-time fluorescence detection apparatus reaches half of the maximum fluorescence intensity was determined as the reaction time of the amplification reaction.

(Nucleic acid amplification reaction solution)
20 mmol / L Tris-HCl buffer (pH 8.0)
10 mmol / L Potassium chloride 10 mmol / L Ammonium sulfate 8 mmol / L Magnesium sulfate 5% Dimethyl sulfoxide (DMSO)
0.1% Tween20
1.4mmol / L dNTP
2 × 3.3 μmol / L 2 × TP
0.001% original SYBRGreen I
(Molecular Probe)
6units AacDNA polymerase
(Danafoam)
1μL template nucleic acid

Nucleic acid amplification reaction evaluation method When all of the following conditions (a) to (c) are satisfied, the evaluation of the combination of the TP is set to 1, and when any of the conditions is not satisfied, By setting the evaluation to 0, the speed and specificity of the nucleic acid amplification reaction were evaluated.
(A) The difference between the reaction time of the sample containing the template nucleic acid and the reaction time of the sample not containing the template nucleic acid (negative control) is 25 minutes or more.
(B) The reaction time of the sample containing the template nucleic acid is within 40 minutes.
(C) The maximum fluorescence intensity is 1 or more.
Since the number of combinations of TPs including a certain Forward / Reverse TP is 8 which is the number of corresponding Reverse / Forward TPs (R1 TP to R8 TP), the evaluation score is 0 to 8 points.

[Example 1]
In this example, the primer (0) was designed and produced based on the primer design method (0) and the primer production method (0), and nucleic acid amplification was performed using the primer (0). That is, as shown in FIG. 7A, the bases adjacent to the 5 ′ end of the TP sequence (Ac ′) are a and b in the order of 5 ′ to 3 ′, and 5 of the sequence (Bc) of the TP extension chain The bases adjacent to the terminal are c and d in the order of 5 'to 3'. If the bases a and d or the bases b and c are complementary, the TP has the sequence (W). The relationship between the bases a and d and the bases b and c being complementary to the speed and specificity of the nucleic acid amplification reaction by TP was analyzed as follows. First, various combinations of the above-mentioned TP and target nucleic acid sequence were classified according to the presence or absence of the complementary relationship between the bases a and d and the bases b and c. Next, the speed and specificity of the nucleic acid amplification reaction in each classification was analyzed. The result is shown in FIG. The horizontal axis of the graph of FIG. 5B shows the evaluation score of the nucleic acid amplification reaction of the TP combinations of each classification, and the vertical axis of the graph shows the number of TP combinations giving each evaluation score. First, Comparative Example 1 is a case where none of the bases a and d and the bases b and c have a complementary relationship. The average evaluation score at this time was 0.257, and 2.4% of the evaluation score was 3 or more. On the other hand, in Examples 1-1 and 1-2, the a and d or the bases b and c have a complementary relationship, respectively, and TP has a sequence (W) having 1 base. The average of the evaluation scores at this time is 0.286 points and 0.465 points, respectively, and those with an evaluation score of 3 points or more are 2.9% and 7.0% of the whole, respectively. It was superior to the case of 1. Furthermore, in Example 1-3, both the bases a and d and the bases b and c have a complementary relationship, and TP has a sequence (W) having 2 bases. The average evaluation score at this time was 0.875, and those with an evaluation score of 3 or more were 16.7% of the total, indicating a further excellent amplification rate and specificity. From these results, the TP having the sequence (W) exhibits an excellent amplification rate / specificity, and when the length of the sequence (W) is 2 bases, the amplification rate / excellence superior to that of 1 base It became clear to show specificity.

[Example 2]
Same as Example 1 except that the combinations of TP and target nucleic acid sequence are classified according to the complementary relationship of bases a and f, bases b and e, and bases c and d shown in FIG. The evaluation score of the nucleic acid amplification reaction was analyzed. The result is shown in FIG. The vertical axis and horizontal axis of the graph are the same as in the first embodiment. First, Comparative Example 2 is a case where none of the bases a and f, the bases b and e, and the bases c and d have a complementary relationship. The average evaluation score at this time was 0.316, and the evaluation score of 3% or more was 2.6% of the total. On the other hand, in Examples 2-1 and 2-2, the bases a and f or the bases b and e have a complementary relationship, and TP has a sequence (W) having 1 base. The average of the evaluation scores at this time is 0.34 points and 0.435 points, respectively, and those with an evaluation score of 3 points or more are 3.8% and 8.7% of the total, respectively. It was superior to the case of 2. In Examples 2-3 and 2-4, the bases a and f, the bases b and e, the bases b and e, and the bases c and d have a complementary relationship, and TP is It has a two-base sequence (W). The average of the evaluation scores at this time is 0.929 points and 0.5 points, respectively, and those with an evaluation score of 3 points or more are 14.3% and 5.0% of the whole, respectively. It was superior to the case of 2. Furthermore, in Example 2-5, the bases a and f, the bases b and e, and the bases c and d all have a complementary relationship, and TP has a sequence (W) with 3 bases. . At this time, the average of the evaluation scores was 0.8, and the evaluation scores of 3 or more were 20.0% of the total, respectively, indicating a further excellent amplification rate and specificity. From these results, the TP having the sequence (W) exhibits an excellent amplification rate / specificity, and when the length of the sequence (W) is 3 bases, the amplification rate / excellence superior to the case of 2 bases / It became clear to show specificity.

[Example 3]
In this example, based on the primer design method (1) and the primer production method (1), the primer (1) is designed and produced, and further, nucleic acid amplification is performed using the primer (1). The amplification efficiency was confirmed. That is, in this example, in the action mechanism of the nucleic acid amplification reaction (FIG. 3), when the complementary sequence of TP formed a stem-loop structure (FIGS. 3E and 3F) by folding, The effect of the probability that the site on the loop where the new TP hybridizes is a single strand on the amplification reaction was examined. The probability of being single-stranded is first determined by determining the sequence of interest in the TP hybridizing sequence on the loop, and then evaluating how many complementary sequences are present on the loop. When the number of complementary sequences is larger, the probability of being a single strand is smaller. A part of FIG. 3G is shown below.

Preparation of template plasmid A plasmid prepared by the following method was used as a template nucleic acid for nucleic acid amplification so that genomic fragments other than the region to be amplified would not affect the result of nucleic acid amplification. First, target nucleic acid sequences of various target genes were amplified by PCR. The amplification product was inserted into a pGEM-T plasmid and cloned to obtain plasmids containing target nucleic acid sequences of various target genes. The plasmid was used as a template nucleic acid for nucleic acid amplification. The plasmid was stored at −20 ° C. until used as a template nucleic acid.

Nucleic acid amplification reaction Target sequence for amplification reaction: (See below)
TP pair used (Forward TP and Reverse TP): (See below)
Nucleic acid amplification reaction was performed on the target sequence using various TP pairs. The template nucleic acid plasmid was diluted to 2000 copies / μL, then heated to 98 ° C. and denatured. The composition of the nucleic acid amplification reaction solution is as shown below. A reaction solution containing no template nucleic acid was used as a negative control. The reaction solution was prepared on ice to make 25 μL. Nucleic acid was amplified by incubating the reaction solution at 60 ° C. for 90 minutes. The generation of amplification products was monitored every minute using a real-time fluorescence detection apparatus (trade name Mx3000P, manufactured by Stratagene, or trade name LightCycler R480, manufactured by Roche). The time until the fluorescence intensity monitored at each time point by the real-time fluorescence detection apparatus reaches half of the maximum fluorescence intensity was determined as the reaction time of the amplification reaction.

(Nucleic acid amplification reaction solution)
20 mmol / L Tris-HCl buffer (pH 8.0)
10 mmol / L Potassium chloride 10 mmol / L Ammonium sulfate 8 mmol / L Magnesium sulfate 5% Dimethyl sulfoxide (DMSO)
0.1% Tween20
1.4mmol / L dNTP
2 × 3.3 μmol / L 2 × TP
0.001% original SYBRGreen I
(Molecular Probe)
6units AacDNA polymerase
(Danafoam)
1μL template nucleic acid

Nucleic acid amplification reaction evaluation method The sequence (A) on the loop corresponding to the sequence (Ac ′) on the TP that newly hybridizes on the loop (that is, a part of the sequence on the genome that is the target of nucleic acid amplification) For the same sequence, the 3 base sequence (complementary sequence) complementary to the 3 base sequence from the 5 ′ end of the sequence (A) (complementary sequence) is the entire loop (sequence (B) and sequence) In the region sandwiched between (Bc), it was calculated how many were present in the region including the intervening sequence between (A) and (B). However, the region of 9 bases in total, including the target sequence and its 3 upstream bases and 3 downstream bases, was excluded from the count calculation. The same calculation was performed for a sequence in which the target sequence was further shifted by one base to the 3 ′ side (target sequence 2-4, target sequence 3-5, target sequence 4-6... In order).

  Next, the reaction time of the sample containing the template nucleic acid for each TP used was within 45 minutes, and the difference between the reaction time of the sample containing the template nucleic acid and the reaction time of the sample not containing the template nucleic acid (negative control) Were determined to be “success” and those other than “failure”. In addition, although amplification reaction is performed by the pair of Forward TP and Reverse TP, in order to evaluate each TP, the success / failure of one TP pair was counted for each Forward TP and Reverse TP constituting the TP.

<Result>
As shown in Table 1 below, when the sequence of 3 bases from the 5 'end of the sequence (A) on the loop is the target sequence (target sequence 1-3), the success rate when the number of complementary sequences is zero 8 cases / 8 cases (100%), 8 cases / 8 cases (100%) when the number of complementary sequences is 1, whereas 0 cases / 8 when the number of complementary sequences is 2. As an example (0%), there was a marked difference between the case where the number of complementary sequences was suppressed to 1 or less and the case where the number of complementary sequences was two.
On the other hand, the data is not shown, but if a 3 base sequence (target sequence 2-4, target sequence 3-5, ...) shifted by 1 base or more downstream is selected as the target sequence, the number of complementary sequences The effect of suppressing the number to 1 or less was not noticeable.
Based on the above, when a 3 base sequence is selected from the 5 ′ end of the sequence (A) on the loop as the target sequence and the number of complementary sequences is suppressed to 1 or less, excellent amplification speed and specificity are exhibited. It became clear.

（表中「−」は９０分以内で反応が見られなかったことを示す。）

("-" In the table indicates that no reaction was observed within 90 minutes.)

The base sequence and target nucleic acid sequence of TP used in Table 1 above are as follows.

ABCG2 TP_F3 (SEQ ID NO: 169) TGTAAGTTTTCTTAAGGATGATGTTGTGA
ABCG2 TP_R6 (SEQ ID NO: 180) ACAGTTCTCATTTTCATGATTCGTCATAG
ABCG2 template 0 (SEQ ID NO: 418)
ATGGTCTTAGAAAAGACTCATTATCATTATGTCTCATTAAAATGCTATTTGCCTTAAGGATGATGTTGTGATGGGCACTCTGACGGTGAGAGAAAACTTACAGTTCTCAGCAGCTCTTCGGCTTGCAACAACTATGACGAATCATGAAAAAAACGAACGGATTAACAGGGTCATTCAAGAGTTAGGTCTGGATAAAGGCGC

ABCG2 TP_F3 (SEQ ID NO: 169) TGTAAGTTTTCTTAAGGATGATGTTGTGA
ABCG2 TP_R7 (SEQ ID NO: 181) ACAGTTCTCATTTTTCATGATTCGTCATA
ABCG2 template 0 (SEQ ID NO: 418)
ATGGTCTTAGAAAAGACTCATTATCATTATGTCTCATTAAAATGCTATTTGCCTTAAGGATGATGTTGTGATGGGCACTCTGACGGTGAGAGAAAACTTACAGTTCTCAGCAGCTCTTCGGCTTGCAACAACTATGACGAATCATGAAAAAAACGAACGGATTAACAGGGTCATTCAAGAGTTAGGTCTGGATAAAGGCGC

G2 fTP3-5 (SEQ ID NO: 89) TCCAGAGCCATATCCGCAGCCAACCTCGTC
G2 rTP3-8 (SEQ ID NO: 100) GGTGCCGCTATTGGGTCAATTACGTTTTGT
norovirus G2 3 template 0 (SEQ ID NO: 419)
CATTCTACAGCAAAATTAGCAAGTTAGTTATTGCAGAGCTAAAAGAAGGTGGCATGGATTTTTACGTGCCCAGACAAGAGCCAATGTTCAGATGGATGAGATTCTCAGATCTGAGCACGTGGGAGGGCGATCGCAATCTGGCTCCCAGCTTTGTGAATGAAGATGGCGTCGAATGACGCCAACCCATCTGATGGGTCCGCAGCCAACCTCGTCCCAGAGGTCAATAATGAGGTTATGGCTCTGGAGCCCGTTGTTGGTGCCGCTATTGCGGCACCTGTGGCGGGCCAACAAAACGTAATTGACCCCTGGATTAGAAACAATTTTGTACAAGCCCCTGGTGGAGAGTTCACAGTGTCCCCTAGAAACGCTCCAGGTGAGATACTGTGGAGCGCGCCCTTGGGCCCTGATCTGAACCCCTATCTTTCTCATTTGTCCAGAATGTACAATGGTTATGCAGGTGGTTTTGAAGTGCAGGTAATCCTTGCGGGGAACGCGTTCACCGCCGGGAAAA

G2 fTP3-6 (SEQ ID NO: 90) CTCCAGAGCCATCCGCAGCCAACCTCGTCC
G2 rTP3-8 (SEQ ID NO: 100) GGTGCCGCTATTGGGTCAATTACGTTTTGT
norovirus G2 3 template 0 (SEQ ID NO: 419)
CATTCTACAGCAAAATTAGCAAGTTAGTTATTGCAGAGCTAAAAGAAGGTGGCATGGATTTTTACGTGCCCAGACAAGAGCCAATGTTCAGATGGATGAGATTCTCAGATCTGAGCACGTGGGAGGGCGATCGCAATCTGGCTCCCAGCTTTGTGAATGAAGATGGCGTCGAATGACGCCAACCCATCTGATGGGTCCGCAGCCAACCTCGTCCCAGAGGTCAATAATGAGGTTATGGCTCTGGAGCCCGTTGTTGGTGCCGCTATTGCGGCACCTGTGGCGGGCCAACAAAACGTAATTGACCCCTGGATTAGAAACAATTTTGTACAAGCCCCTGGTGGAGAGTTCACAGTGTCCCCTAGAAACGCTCCAGGTGAGATACTGTGGAGCGCGCCCTTGGGCCCTGATCTGAACCCCTATCTTTCTCATTTGTCCAGAATGTACAATGGTTATGCAGGTGGTTTTGAAGTGCAGGTAATCCTTGCGGGGAACGCGTTCACCGCCGGGAAAA

ABCG2 TP_F4 (SEQ ID NO: 170) TGTAAGTTTTCCTTAAGGATGATGTTGTG
ABCG2 TP_R4 (SEQ ID NO: 178) ACAGTTCTCATTCATGATTCGTCATAGTT
ABCG2 template 0 (SEQ ID NO: 418)
ATGGTCTTAGAAAAGACTCATTATCATTATGTCTCATTAAAATGCTATTTGCCTTAAGGATGATGTTGTGATGGGCACTCTGACGGTGAGAGAAAACTTACAGTTCTCAGCAGCTCTTCGGCTTGCAACAACTATGACGAATCATGAAAAAAACGAACGGATTAACAGGGTCATTCAAGAGTTAGGTCTGGATAAAGGCGC

ABCG2 TP_F4 (SEQ ID NO: 170) TGTAAGTTTTCCTTAAGGATGATGTTGTG
ABCG2 TP_R5 (SEQ ID NO: 179) ACAGTTCTCATTTCATGATTCGTCATAGT
ABCG2 template 0 (SEQ ID NO: 418)
ATGGTCTTAGAAAAGACTCATTATCATTATGTCTCATTAAAATGCTATTTGCCTTAAGGATGATGTTGTGATGGGCACTCTGACGGTGAGAGAAAACTTACAGTTCTCAGCAGCTCTTCGGCTTGCAACAACTATGACGAATCATGAAAAAAACGAACGGATTAACAGGGTCATTCAAGAGTTAGGTCTGGATAAAGGCGC

G2 fTP3-2 (SEQ ID NO: 86) AGAGCCATAACCGGGTCCGCAGCCAACCTC
G2 rTP3-5 (SEQ ID NO: 97) GTTGGTGCCGCTTCAATTACGTTTTGTTGG
norovirus G2 3 template 0 (SEQ ID NO: 419)
CATTCTACAGCAAAATTAGCAAGTTAGTTATTGCAGAGCTAAAAGAAGGTGGCATGGATTTTTACGTGCCCAGACAAGAGCCAATGTTCAGATGGATGAGATTCTCAGATCTGAGCACGTGGGAGGGCGATCGCAATCTGGCTCCCAGCTTTGTGAATGAAGATGGCGTCGAATGACGCCAACCCATCTGATGGGTCCGCAGCCAACCTCGTCCCAGAGGTCAATAATGAGGTTATGGCTCTGGAGCCCGTTGTTGGTGCCGCTATTGCGGCACCTGTGGCGGGCCAACAAAACGTAATTGACCCCTGGATTAGAAACAATTTTGTACAAGCCCCTGGTGGAGAGTTCACAGTGTCCCCTAGAAACGCTCCAGGTGAGATACTGTGGAGCGCGCCCTTGGGCCCTGATCTGAACCCCTATCTTTCTCATTTGTCCAGAATGTACAATGGTTATGCAGGTGGTTTTGAAGTGCAGGTAATCCTTGCGGGGAACGCGTTCACCGCCGGGAAAA

G2 fTP3-2 (SEQ ID NO: 86) AGAGCCATAACCGGGTCCGCAGCCAACCTC
G2 rTP3-8 (SEQ ID NO: 100) GGTGCCGCTATTGGGTCAATTACGTTTTGT
norovirus G2 3 template 0 (SEQ ID NO: 419)
CATTCTACAGCAAAATTAGCAAGTTAGTTATTGCAGAGCTAAAAGAAGGTGGCATGGATTTTTACGTGCCCAGACAAGAGCCAATGTTCAGATGGATGAGATTCTCAGATCTGAGCACGTGGGAGGGCGATCGCAATCTGGCTCCCAGCTTTGTGAATGAAGATGGCGTCGAATGACGCCAACCCATCTGATGGGTCCGCAGCCAACCTCGTCCCAGAGGTCAATAATGAGGTTATGGCTCTGGAGCCCGTTGTTGGTGCCGCTATTGCGGCACCTGTGGCGGGCCAACAAAACGTAATTGACCCCTGGATTAGAAACAATTTTGTACAAGCCCCTGGTGGAGAGTTCACAGTGTCCCCTAGAAACGCTCCAGGTGAGATACTGTGGAGCGCGCCCTTGGGCCCTGATCTGAACCCCTATCTTTCTCATTTGTCCAGAATGTACAATGGTTATGCAGGTGGTTTTGAAGTGCAGGTAATCCTTGCGGGGAACGCGTTCACCGCCGGGAAAA

FTO TP_F1 (SEQ ID NO: 218) CTAAATTCACTAGAATGTCTGAATTATTATTC
FTO TP_R2 (SEQ ID NO: 227) AGTGATGCACACTGTTACCTATTAAAACTTTA
FTO template 0 (SEQ ID NO: 420)
TTATGAGATAATGTCCTTTTTAAAAATAAACACTAACATCAGTTATGCATTTAGAATGTCTGAATTATTATTCTAGGTTCCTTGCGACTGCTGTGAATTTAGTGATGCACTTGGATAGTCTCTGTTACTCTAAAGTTTTAATAGGTAACAGTCAGAAATGGAGTGGGAGAGCATAAAAGCAAACTGAAATGCAAATAGCTG

FTO TP_F1 (SEQ ID NO: 218) CTAAATTCACTAGAATGTCTGAATTATTATTC
FTO TP_R3 (SEQ ID NO: 228) AGTGATGCACGACTGTTACCTATTAAAACTTT
FTO template 0 (SEQ ID NO: 420)
TTATGAGATAATGTCCTTTTTAAAAATAAACACTAACATCAGTTATGCATTTAGAATGTCTGAATTATTATTCTAGGTTCCTTGCGACTGCTGTGAATTTAGTGATGCACTTGGATAGTCTCTGTTACTCTAAAGTTTTAATAGGTAACAGTCAGAAATGGAGTGGGAGAGCATAAAAGCAAACTGAAATGCAAATAGCTG

FTO TP_F2 (SEQ ID NO: 219) CTAAATTCACTTAGAATGTCTGAATTATTATT
FTO TP_R1 (SEQ ID NO: 226) AGTGATGCACCTGTTACCTATTAAAACTTTAG
FTO template 0 (SEQ ID NO: 420)
TTATGAGATAATGTCCTTTTTAAAAATAAACACTAACATCAGTTATGCATTTAGAATGTCTGAATTATTATTCTAGGTTCCTTGCGACTGCTGTGAATTTAGTGATGCACTTGGATAGTCTCTGTTACTCTAAAGTTTTAATAGGTAACAGTCAGAAATGGAGTGGGAGAGCATAAAAGCAAACTGAAATGCAAATAGCTG

FTO TP_F2 (SEQ ID NO: 219) CTAAATTCACTTAGAATGTCTGAATTATTATT
FTO TP_R2 (SEQ ID NO: 227) AGTGATGCACACTGTTACCTATTAAAACTTTA
FTO template 0 (SEQ ID NO: 420)
TTATGAGATAATGTCCTTTTTAAAAATAAACACTAACATCAGTTATGCATTTAGAATGTCTGAATTATTATTCTAGGTTCCTTGCGACTGCTGTGAATTTAGTGATGCACTTGGATAGTCTCTGTTACTCTAAAGTTTTAATAGGTAACAGTCAGAAATGGAGTGGGAGAGCATAAAAGCAAACTGAAATGCAAATAGCTG

[Example 4]
In this example, based on the primer design method (2) and the primer production method (2), the primer (2) is designed and produced, and further nucleic acid amplification is performed using the primer (2). The amplification efficiency was confirmed. That is, in this example, in the mechanism of action of the nucleic acid amplification reaction (FIG. 3), the site of TP hybridizes on the genomic sequence that is the target of nucleic acid amplification (FIG. 3 (a)). The influence of the probability of becoming a single strand on the influence amplification reaction was examined. The probability of being a single strand is determined by first determining the sequence of interest in the TP hybridizing sequence on the genome sequence, and then how many complementary sequences to the sequence of interest exist on the genome sequence. It was assumed that the probability of being a single strand is smaller when the number of complementary sequences is larger. A part of FIG. 3A is shown below.

Preparation of template plasmid The template plasmid was prepared in the same manner as in Example 3 (primer (1) of the present invention).

Nucleic acid amplification reaction The nucleic acid amplification reaction was performed in the same manner as in Example 3 (Primer (1) of the present invention).

Nucleic Acid Amplification Reaction Evaluation Method For the sequence (A) on the genomic sequence corresponding to the sequence (Ac ′) on the TP that hybridizes on the genomic sequence targeted for nucleic acid amplification, 3 from the 5 ′ end of the sequence (A) A base sequence (complementary sequence) of 3 bases complementary to the base sequence (target sequence 1-3) is composed of 20 bases upstream of the sequence (A), 20 sequences upstream of the sequence (A), and 20 downstream of the sequence (A). The number of bases present in the base region was calculated. However, the region of 9 bases in total, including the target sequence and its 3 upstream bases and 3 downstream bases, was excluded from the count calculation. The same calculation was performed for a sequence in which the target sequence was further shifted by one base to the 3 ′ side (target sequence 2-4, target sequence 3-5, target sequence 4-6... In order).

  Next, the reaction time of the sample containing the template nucleic acid for each TP used was within 45 minutes, and the difference between the reaction time of the sample containing the template nucleic acid and the reaction time of the sample not containing the template nucleic acid (negative control) Were determined to be “success” and those other than “failure”. In addition, although amplification reaction is performed by the pair of Forward TP and Reverse TP, in order to evaluate each TP, the success / failure of one TP pair was counted for each Forward TP and Reverse TP constituting the TP.

<Result>
As shown in Table 2 below, when the sequence of 3 bases from the 5 'end of the sequence (A) on the genome sequence is the target sequence (target sequence 1-3), the success is achieved when the number of complementary sequences is zero. The ratio of 8 cases / 8 cases (100%) was 8/8 cases (100%) when the number of complementary sequences was 1, whereas the number of complementary sequences was 2 or 3. 0 cases / 8 cases (0%), and a remarkable difference was observed between the case where the number of complementary sequences was suppressed to 1 or less and the case where 2 or more complementary sequences were suppressed.
On the other hand, the data is not shown, but if a 3 base sequence (target sequence 2-4, target sequence 3-5, ...) shifted by 1 base or more downstream is selected as the target sequence, the number of complementary sequences The effect of suppressing the number to 1 or less was not noticeable.
From the above, when a 3 base sequence is selected from the 5 'end of the sequence (A) on the genomic sequence as the target sequence and the number of complementary sequences is suppressed to 1 or less, excellent amplification speed and specificity are obtained. It became clear to show.

（表中「−」は９０分以内で反応が見られなかったことを示す。）

("-" In the table indicates that no reaction was observed within 90 minutes.)

The base sequence of TP and the target nucleic acid sequence used in Table 2 above are as follows.

EGFR20 TP_F4 (SEQ ID NO: 187) CGTGATGAGTGTGCCGCCTGCTGGGCAT
EGFR20 TP_R4 (SEQ ID NO: 195) ACGCAGCTCTTGTGTTCCCGGACATAGT
EGFR20 template 0 (SEQ ID NO: 421)
GACGTGCCTCTCCCTCCCTCCAGGAAGCCTACGTGATGGCCAGCGTGGACAACCCCCACGTGTGCCGCCTGCTGGGCATCTGCCTCACCTCCACCGTGCAGCTCATCACGCAGCTCATGCCCTTCGGCTGCCTCCTGGACTATGTCCGGGAACACAAAGACAATATTGGCTCCCAGTACCTGCTCAACTGGTGTGGA

EGFR20 TP_F4 (SEQ ID NO: 187) CGTGATGAGTGTGCCGCCTGCTGGGCAT
EGFR20 TP_R7 (SEQ ID NO: 198) ACGCAGCTCTCTTTGTGTTCCCGGACAT
EGFR20 template 0 (SEQ ID NO: 421)
GACGTGCCTCTCCCTCCCTCCAGGAAGCCTACGTGATGGCCAGCGTGGACAACCCCCACGTGTGCCGCCTGCTGGGCATCTGCCTCACCTCCACCGTGCAGCTCATCACGCAGCTCATGCCCTTCGGCTGCCTCCTGGACTATGTCCGGGAACACAAAGACAATATTGGCTCCCAGTACCTGCTCAACTGGTGTGGA

G2 fTP4-7 (SEQ ID NO: 107) CCGCCACAGGTGTATGGCTCTGGAGCCCGT
G2 rTP4-6 (SEQ ID NO: 114) AAAACGTAATTGACCAGGGGCTTGTACAAA
norovirus G2 4 template 0 (SEQ ID NO: 422)
CATTCTACAGCAAAATTAGCAAGTTAGTTATTGCAGAGCTAAAAGAAGGTGGCATGGATTTTTACGTGCCCAGACAAGAGCCAATGTTCAGATGGATGAGATTCTCAGATCTGAGCACGTGGGAGGGCGATCGCAATCTGGCTCCCAGCTTTGTGAATGAAGATGGCGTCGAATGACGCCAACCCATCTGATGGGTCCGCAGCCAACCTCGTCCCAGAGGTCAATAATGAGGTTATGGCTCTGGAGCCCGTTGTTGGTGCCGCTATTGCGGCACCTGTGGCGGGCCAACAAAACGTAATTGACCCCTGGATTAGAAACAATTTTGTACAAGCCCCTGGTGGAGAGTTCACAGTGTCCCCTAGAAACGCTCCAGGTGAGATACTGTGGAGCGCGCCCTTGGGCCCTGATCTGAACCCCTATCTTTCTCATTTGTCCAGAATGTACAATGGTTATGCAGGTGGTTTTGAAGTGCAGGTAATCCTTGCGGGGAACGCGTTCACCGCCGGGAAAA

G2 fTP4-8 (SEQ ID NO: 108) CCCGCCACAGGTATGGCTCTGGAGCCCGTT
G2 rTP4-6 (SEQ ID NO: 114) AAAACGTAATTGACCAGGGGCTTGTACAAA
norovirus G2 4 template 0 (SEQ ID NO: 422)
CATTCTACAGCAAAATTAGCAAGTTAGTTATTGCAGAGCTAAAAGAAGGTGGCATGGATTTTTACGTGCCCAGACAAGAGCCAATGTTCAGATGGATGAGATTCTCAGATCTGAGCACGTGGGAGGGCGATCGCAATCTGGCTCCCAGCTTTGTGAATGAAGATGGCGTCGAATGACGCCAACCCATCTGATGGGTCCGCAGCCAACCTCGTCCCAGAGGTCAATAATGAGGTTATGGCTCTGGAGCCCGTTGTTGGTGCCGCTATTGCGGCACCTGTGGCGGGCCAACAAAACGTAATTGACCCCTGGATTAGAAACAATTTTGTACAAGCCCCTGGTGGAGAGTTCACAGTGTCCCCTAGAAACGCTCCAGGTGAGATACTGTGGAGCGCGCCCTTGGGCCCTGATCTGAACCCCTATCTTTCTCATTTGTCCAGAATGTACAATGGTTATGCAGGTGGTTTTGAAGTGCAGGTAATCCTTGCGGGGAACGCGTTCACCGCCGGGAAAA

G2 fTP4-1 (SEQ ID NO: 101) CAGGTGCCGCAATGAGGTTATGGCTCTGGA
G2 rTP4-1 (SEQ ID NO: 109) CCAACAAAACGTGGGCTTGTACAAAATTGT
norovirus G2 4 template 0 (SEQ ID NO: 422)
CATTCTACAGCAAAATTAGCAAGTTAGTTATTGCAGAGCTAAAAGAAGGTGGCATGGATTTTTACGTGCCCAGACAAGAGCCAATGTTCAGATGGATGAGATTCTCAGATCTGAGCACGTGGGAGGGCGATCGCAATCTGGCTCCCAGCTTTGTGAATGAAGATGGCGTCGAATGACGCCAACCCATCTGATGGGTCCGCAGCCAACCTCGTCCCAGAGGTCAATAATGAGGTTATGGCTCTGGAGCCCGTTGTTGGTGCCGCTATTGCGGCACCTGTGGCGGGCCAACAAAACGTAATTGACCCCTGGATTAGAAACAATTTTGTACAAGCCCCTGGTGGAGAGTTCACAGTGTCCCCTAGAAACGCTCCAGGTGAGATACTGTGGAGCGCGCCCTTGGGCCCTGATCTGAACCCCTATCTTTCTCATTTGTCCAGAATGTACAATGGTTATGCAGGTGGTTTTGAAGTGCAGGTAATCCTTGCGGGGAACGCGTTCACCGCCGGGAAAA

G2 fTP4-5 (SEQ ID NO: 105) GCCACAGGTGCCGTTATGGCTCTGGAGCCC
G2 rTP4-1 (SEQ ID NO: 109) CCAACAAAACGTGGGCTTGTACAAAATTGT
norovirus G2 4 template 0 (SEQ ID NO: 422)
CATTCTACAGCAAAATTAGCAAGTTAGTTATTGCAGAGCTAAAAGAAGGTGGCATGGATTTTTACGTGCCCAGACAAGAGCCAATGTTCAGATGGATGAGATTCTCAGATCTGAGCACGTGGGAGGGCGATCGCAATCTGGCTCCCAGCTTTGTGAATGAAGATGGCGTCGAATGACGCCAACCCATCTGATGGGTCCGCAGCCAACCTCGTCCCAGAGGTCAATAATGAGGTTATGGCTCTGGAGCCCGTTGTTGGTGCCGCTATTGCGGCACCTGTGGCGGGCCAACAAAACGTAATTGACCCCTGGATTAGAAACAATTTTGTACAAGCCCCTGGTGGAGAGTTCACAGTGTCCCCTAGAAACGCTCCAGGTGAGATACTGTGGAGCGCGCCCTTGGGCCCTGATCTGAACCCCTATCTTTCTCATTTGTCCAGAATGTACAATGGTTATGCAGGTGGTTTTGAAGTGCAGGTAATCCTTGCGGGGAACGCGTTCACCGCCGGGAAAA

G2 fTP2-2 (SEQ ID NO: 70) GTCATTCGACGCCGATCGCAATCTGGCTCC
G2 rTP2-3 (SEQ ID NO: 79) ATCTGATGGGTCTAACCTCATTATTGACCT
norovirus G2 2 template 0 (SEQ ID NO: 423)
CATTCTACAGCAAAATTAGCAAGTTAGTTATTGCAGAGCTAAAAGAAGGTGGCATGGATTTTTACGTGCCCAGACAAGAGCCAATGTTCAGATGGATGAGATTCTCAGATCTGAGCACGTGGGAGGGCGATCGCAATCTGGCTCCCAGCTTTGTGAATGAAGATGGCGTCGAATGACGCCAACCCATCTGATGGGTCCGCAGCCAACCTCGTCCCAGAGGTCAATAATGAGGTTATGGCTCTGGAGCCCGTTGTTGGTGCCGCTATTGCGGCACCTGTGGCGGGCCAACAAAACGTAATTGACCCCTGGATTAGAAACAATTTTGTACAAGCCCCTGGTGGAGAGTTCACAGTGTCCCCTAGAAACGCTCCAGGTGAGATACTGTGGAGCGCGCCCTTGGGCCCTGATCTGAACCCCTATCTTTCTCATTTGTCCAGAATGTACAATGGTTATGCAGGTGGTTTTGAAGTGCAGGTAATCCTTGCGGGGAACGCGTTCACCGCCGGGAAAA

G2 fTP2-6 (SEQ ID NO: 74) TGGCGTCATTCGCGCAATCTGGCTCCCAGC
G2 rTP2-3 (SEQ ID NO: 79) ATCTGATGGGTCTAACCTCATTATTGACCT
norovirus G2 2 template 0 (SEQ ID NO: 423)
CATTCTACAGCAAAATTAGCAAGTTAGTTATTGCAGAGCTAAAAGAAGGTGGCATGGATTTTTACGTGCCCAGACAAGAGCCAATGTTCAGATGGATGAGATTCTCAGATCTGAGCACGTGGGAGGGCGATCGCAATCTGGCTCCCAGCTTTGTGAATGAAGATGGCGTCGAATGACGCCAACCCATCTGATGGGTCCGCAGCCAACCTCGTCCCAGAGGTCAATAATGAGGTTATGGCTCTGGAGCCCGTTGTTGGTGCCGCTATTGCGGCACCTGTGGCGGGCCAACAAAACGTAATTGACCCCTGGATTAGAAACAATTTTGTACAAGCCCCTGGTGGAGAGTTCACAGTGTCCCCTAGAAACGCTCCAGGTGAGATACTGTGGAGCGCGCCCTTGGGCCCTGATCTGAACCCCTATCTTTCTCATTTGTCCAGAATGTACAATGGTTATGCAGGTGGTTTTGAAGTGCAGGTAATCCTTGCGGGGAACGCGTTCACCGCCGGGAAAA

ABCB1 TP_F1 (SEQ ID NO: 150) CGATCTCTTCGCATTGCTGAGAACATTGC
ABCB1 TP_R3 (SEQ ID NO: 160) TCGTGAGGGCGGCAGTGACTCGATGAAGG
ABCB1 template 0 (SEQ ID NO: 424)
ACCTGGGCATCGTGTCCCAGGAGCCCATCCTGTTTGACTGCAGCATTGCTGAGAACATTGCCTATGGAGACAACAGCCGGGTGGTGTCACAGGAAGAGATCGTGAGGGCAGCAAAGGAGGCCAACATACATGCCTTCATCGAGTCACTGCCTAATGTAAGTCTCTCTTCAAATAAACAGCCTGGGAGCATGTGGCCC

ABCB1 TP_F1 (SEQ ID NO: 150) CGATCTCTTCGCATTGCTGAGAACATTGC
ABCB1 TP_R7 (SEQ ID NO: 164) TCGTGAGGGCATTAGGCAGTGACTCGATG
ABCB1 template 0 (SEQ ID NO: 424)
ACCTGGGCATCGTGTCCCAGGAGCCCATCCTGTTTGACTGCAGCATTGCTGAGAACATTGCCTATGGAGACAACAGCCGGGTGGTGTCACAGGAAGAGATCGTGAGGGCAGCAAAGGAGGCCAACATACATGCCTTCATCGAGTCACTGCCTAATGTAAGTCTCTCTTCAAATAAACAGCCTGGGAGCATGTGGCCC

EGFR20 TP_F6 (SEQ ID NO: 189) CGTGATGAGCGTGTGCCGCCTGCTGGGC
EGFR20 TP_R7 (SEQ ID NO: 198) ACGCAGCTCTCTTTGTGTTCCCGGACAT
EGFR20 template 0 (SEQ ID NO: 421)
GACGTGCCTCTCCCTCCCTCCAGGAAGCCTACGTGATGGCCAGCGTGGACAACCCCCACGTGTGCCGCCTGCTGGGCATCTGCCTCACCTCCACCGTGCAGCTCATCACGCAGCTCATGCCCTTCGGCTGCCTCCTGGACTATGTCCGGGAACACAAAGACAATATTGGCTCCCAGTACCTGCTCAACTGGTGTGGA

EGFR20 TP_F6 (SEQ ID NO: 189) CGTGATGAGCGTGTGCCGCCTGCTGGGC
EGFR20 TP_R8 (SEQ ID NO: 199) ACGCAGCTCGTCTTTGTGTTCCCGGACA
EGFR20 template 0 (SEQ ID NO: 421)
GACGTGCCTCTCCCTCCCTCCAGGAAGCCTACGTGATGGCCAGCGTGGACAACCCCCACGTGTGCCGCCTGCTGGGCATCTGCCTCACCTCCACCGTGCAGCTCATCACGCAGCTCATGCCCTTCGGCTGCCTCCTGGACTATGTCCGGGAACACAAAGACAATATTGGCTCCCAGTACCTGCTCAACTGGTGTGGA

[Example 5]
In this example, based on the primer design method (3) and the primer production method (3), the primer (3) is designed and produced, and further nucleic acid amplification is performed using the primer (3). The amplification efficiency was confirmed. That is, in this example, in the action mechanism of the nucleic acid amplification reaction (FIG. 3), when the complementary sequence of TP formed a stem-loop structure (FIGS. 3E and 3F) by folding, The influence of the binding strength of the sequence (Bc) (hereinafter referred to as “turnback sequence”) hybridizing to the sequence (B) on the amplification reaction was examined. The strength of binding is determined by first determining the sequence of interest in the turnback sequence, and then how many G (guanine) or C (cytosine) are included in the base sequence of the sequence of interest (number of G / C). When the number is larger, the bond strength is stronger. In addition, the said G / C number corresponds with the G / C number in the attention sequence seen from the 5 'end of the sequence (B') of the primer (3). A part of FIG. 3F is shown below.

Preparation of template plasmid The template plasmid was prepared in the same manner as in Example 3 (Primer (1)).

Nucleic acid amplification reaction The nucleic acid amplification reaction was performed in the same manner as in Example 3 (the primer (1)).

Nucleic acid amplification reaction evaluation method The target sequence is a sequence of 6 bases from the 3 ′ end of the turnback sequence (Bc) (target sequence 1-6), and the number of G or C contained in the base constituting the sequence is counted. Further, the sequences in which the target sequence is further shifted by one base to the 5 ′ side (the target sequence 2-7, the target sequence 3-8, the target sequence 4-9,...) Are counted in the same manner. Next, the reaction time of the sample containing the template nucleic acid for each TP used was within 45 minutes, and the difference between the reaction time of the sample containing the template nucleic acid and the reaction time of the sample not containing the template nucleic acid (negative control) Is defined as “success”, and the others are defined as “failure”. The amplification reaction is performed by a pair of Forward TP and Reverse TP. In order to evaluate each TP, success / failure of one TP pair is counted for each of Forward TP and Reverse TP constituting the TP.

<Result>
As shown in Table 3 below, when the G / C number is 4, 5, and 6 in the sequence of 6 bases from the 3 ′ end of the turnback sequence (Bc), the success rate is 7 cases / 7 cases, respectively. (100%), 5 cases / 5 cases (100%), 1 case / 1 case (100%), and the success rate when the G / C number is 3 or less is 1 case / 13 cases (7.7%) ), There was a significant difference. Note that the only successful example when the G / C number is 3 or less is an amplification reaction using a primer set (pair) combined with a Forward TP having a G / C number of 6, so the G / C of the Forward TP It is conceivable that the C number (= 6) is involved in improving the amplification rate and specificity.
On the other hand, although data is not shown, if a 6 base sequence (target sequence 2-7, target sequence 3-8,...) Shifted by one base upstream is selected as the target sequence, G / C The effect of making the number above a certain number was not noticeable.
From the above, by setting 4 or more of the 6 bases counted from the 3 ′ end of the turnback sequence (Bc) to G or C, an appropriate binding strength for hybridizing the turnback sequence is obtained. As a result, it was revealed that the amplification rate and specificity were excellent.

（表中「−」は９０分以内で反応が見られなかったことを示す。）

("-" In the table indicates that no reaction was observed within 90 minutes.)

The base sequence of TP and the target nucleic acid sequence used in Table 3 above are as follows.

G2 fTP4-8 (SEQ ID NO: 108) CCCGCCACAGGTATGGCTCTGGAGCCCGTT
G2 rTP4-1 (SEQ ID NO: 109) CCAACAAAACGTGGGCTTGTACAAAATTGT
norovirus G2 1 template 0 (SEQ ID NO: 432)
CATTCTACAGCAAAATTAGCAAGTTAGTTATTGCAGAGCTAAAAGAAGGTGGCATGGATTTTTACGTGCCCAGACAAGAGCCAATGTTCAGATGGATGAGATTCTCAGATCTGAGCACGTGGGAGGGCGATCGCAATCTGGCTCCCAGCTTTGTGAATGAAGATGGCGTCGAATGACGCCAACCCATCTGATGGGTCCGCAGCCAACCTCGTCCCAGAGGTCAATAATGAGGTTATGGCTCTGGAGCCCGTTGTTGGTGCCGCTATTGCGGCACCTGTGGCGGGCCAACAAAACGTAATTGACCCCTGGATTAGAAACAATTTTGTACAAGCCCCTGGTGGAGAGTTCACAGTGTCCCCTAGAAACGCTCCAGGTGAGATACTGTGGAGCGCGCCCTTGGGCCCTGATCTGAACCCCTATCTTTCTCATTTGTCCAGAATGTACAATGGTTATGCAGGTGGTTTTGAAGTGCAGGTAATCCTTGCGGGGAACGCGTTCACCGCCGGGAAAA

G2 fTP3-8 (SEQ ID NO: 92) GGCTCCAGAGCCGCAGCCAACCTCGTCCCA
G2 rTP3-7 (SEQ ID NO: 99) TGGTGCCGCTATGGTCAATTACGTTTTGTT
norovirus G2 3 template 0 (SEQ ID NO: 419)
CATTCTACAGCAAAATTAGCAAGTTAGTTATTGCAGAGCTAAAAGAAGGTGGCATGGATTTTTACGTGCCCAGACAAGAGCCAATGTTCAGATGGATGAGATTCTCAGATCTGAGCACGTGGGAGGGCGATCGCAATCTGGCTCCCAGCTTTGTGAATGAAGATGGCGTCGAATGACGCCAACCCATCTGATGGGTCCGCAGCCAACCTCGTCCCAGAGGTCAATAATGAGGTTATGGCTCTGGAGCCCGTTGTTGGTGCCGCTATTGCGGCACCTGTGGCGGGCCAACAAAACGTAATTGACCCCTGGATTAGAAACAATTTTGTACAAGCCCCTGGTGGAGAGTTCACAGTGTCCCCTAGAAACGCTCCAGGTGAGATACTGTGGAGCGCGCCCTTGGGCCCTGATCTGAACCCCTATCTTTCTCATTTGTCCAGAATGTACAATGGTTATGCAGGTGGTTTTGAAGTGCAGGTAATCCTTGCGGGGAACGCGTTCACCGCCGGGAAAA

G2 fTP3-8 (SEQ ID NO: 92) GGCTCCAGAGCCGCAGCCAACCTCGTCCCA
G2 rTP3-8 (SEQ ID NO: 100) GGTGCCGCTATTGGGTCAATTACGTTTTGT
norovirus G2 3 template 0 (SEQ ID NO: 419)
CATTCTACAGCAAAATTAGCAAGTTAGTTATTGCAGAGCTAAAAGAAGGTGGCATGGATTTTTACGTGCCCAGACAAGAGCCAATGTTCAGATGGATGAGATTCTCAGATCTGAGCACGTGGGAGGGCGATCGCAATCTGGCTCCCAGCTTTGTGAATGAAGATGGCGTCGAATGACGCCAACCCATCTGATGGGTCCGCAGCCAACCTCGTCCCAGAGGTCAATAATGAGGTTATGGCTCTGGAGCCCGTTGTTGGTGCCGCTATTGCGGCACCTGTGGCGGGCCAACAAAACGTAATTGACCCCTGGATTAGAAACAATTTTGTACAAGCCCCTGGTGGAGAGTTCACAGTGTCCCCTAGAAACGCTCCAGGTGAGATACTGTGGAGCGCGCCCTTGGGCCCTGATCTGAACCCCTATCTTTCTCATTTGTCCAGAATGTACAATGGTTATGCAGGTGGTTTTGAAGTGCAGGTAATCCTTGCGGGGAACGCGTTCACCGCCGGGAAAA

GGCX TP_F1 (SEQ ID NO: 251) TCGGGGGCAGTGCTCCCCTGAGTGGC
GGCX TP_R4 (SEQ ID NO: 262) CGAAGGTTGCACTGGGCTGAGGGGCT
GGCX template 0 (SEQ ID NO: 425)
CTCCCTGCTTTCCTAGGTATGTTCTCCTACGTCATGCTGGCCAGCAGCCCTCTCTTCTGCTCCCCTGAGTGGCCTCGGAAGCTGGTGTCCTACTGCCCCCGAAGGTTGCAACAACTGTTGCCCCTCAAGGCAGCCCCTCAGCCCAGTGTTTCCTGTGTGTATAAGAGGAGCCGGGGCAAAAGTGGCCAGAAGCCAGGGTGGCTG

GGCX TP_F1 (SEQ ID NO: 251) TCGGGGGCAGTGCTCCCCTGAGTGGC
GGCX TP_R8 (SEQ ID NO: 266) CGAAGGTTGCAAACACTGGGCTGAGG
GGCX template 0 (SEQ ID NO: 425)
CTCCCTGCTTTCCTAGGTATGTTCTCCTACGTCATGCTGGCCAGCAGCCCTCTCTTCTGCTCCCCTGAGTGGCCTCGGAAGCTGGTGTCCTACTGCCCCCGAAGGTTGCAACAACTGTTGCCCCTCAAGGCAGCCCCTCAGCCCAGTGTTTCCTGTGTGTATAAGAGGAGCCGGGGCAAAAGTGGCCAGAAGCCAGGGTGGCTG

G2 fTP3-2 (SEQ ID NO: 86) AGAGCCATAACCGGGTCCGCAGCCAACCTC
G2 rTP3-7 (SEQ ID NO: 99) TGGTGCCGCTATGGTCAATTACGTTTTGTT
norovirus G2 3 template 0 (SEQ ID NO: 419)
CATTCTACAGCAAAATTAGCAAGTTAGTTATTGCAGAGCTAAAAGAAGGTGGCATGGATTTTTACGTGCCCAGACAAGAGCCAATGTTCAGATGGATGAGATTCTCAGATCTGAGCACGTGGGAGGGCGATCGCAATCTGGCTCCCAGCTTTGTGAATGAAGATGGCGTCGAATGACGCCAACCCATCTGATGGGTCCGCAGCCAACCTCGTCCCAGAGGTCAATAATGAGGTTATGGCTCTGGAGCCCGTTGTTGGTGCCGCTATTGCGGCACCTGTGGCGGGCCAACAAAACGTAATTGACCCCTGGATTAGAAACAATTTTGTACAAGCCCCTGGTGGAGAGTTCACAGTGTCCCCTAGAAACGCTCCAGGTGAGATACTGTGGAGCGCGCCCTTGGGCCCTGATCTGAACCCCTATCTTTCTCATTTGTCCAGAATGTACAATGGTTATGCAGGTGGTTTTGAAGTGCAGGTAATCCTTGCGGGGAACGCGTTCACCGCCGGGAAAA

G2 fTP3-4 (SEQ ID NO: 88) CCAGAGCCATAAGTCCGCAGCCAACCTCGT
G2 rTP3-7 (SEQ ID NO: 99) TGGTGCCGCTATGGTCAATTACGTTTTGTT
norovirus G2 3 template 0 (SEQ ID NO: 419)
CATTCTACAGCAAAATTAGCAAGTTAGTTATTGCAGAGCTAAAAGAAGGTGGCATGGATTTTTACGTGCCCAGACAAGAGCCAATGTTCAGATGGATGAGATTCTCAGATCTGAGCACGTGGGAGGGCGATCGCAATCTGGCTCCCAGCTTTGTGAATGAAGATGGCGTCGAATGACGCCAACCCATCTGATGGGTCCGCAGCCAACCTCGTCCCAGAGGTCAATAATGAGGTTATGGCTCTGGAGCCCGTTGTTGGTGCCGCTATTGCGGCACCTGTGGCGGGCCAACAAAACGTAATTGACCCCTGGATTAGAAACAATTTTGTACAAGCCCCTGGTGGAGAGTTCACAGTGTCCCCTAGAAACGCTCCAGGTGAGATACTGTGGAGCGCGCCCTTGGGCCCTGATCTGAACCCCTATCTTTCTCATTTGTCCAGAATGTACAATGGTTATGCAGGTGGTTTTGAAGTGCAGGTAATCCTTGCGGGGAACGCGTTCACCGCCGGGAAAA

G2 fTP2-3 (SEQ ID NO: 71) CGTCATTCGACGGATCGCAATCTGGCTCCC
G2 rTP2-5 (SEQ ID NO: 81) CTGATGGGTCCGCATAACCTCATTATTGAC
norovirus G2 2 template 0 (SEQ ID NO: 423)
CATTCTACAGCAAAATTAGCAAGTTAGTTATTGCAGAGCTAAAAGAAGGTGGCATGGATTTTTACGTGCCCAGACAAGAGCCAATGTTCAGATGGATGAGATTCTCAGATCTGAGCACGTGGGAGGGCGATCGCAATCTGGCTCCCAGCTTTGTGAATGAAGATGGCGTCGAATGACGCCAACCCATCTGATGGGTCCGCAGCCAACCTCGTCCCAGAGGTCAATAATGAGGTTATGGCTCTGGAGCCCGTTGTTGGTGCCGCTATTGCGGCACCTGTGGCGGGCCAACAAAACGTAATTGACCCCTGGATTAGAAACAATTTTGTACAAGCCCCTGGTGGAGAGTTCACAGTGTCCCCTAGAAACGCTCCAGGTGAGATACTGTGGAGCGCGCCCTTGGGCCCTGATCTGAACCCCTATCTTTCTCATTTGTCCAGAATGTACAATGGTTATGCAGGTGGTTTTGAAGTGCAGGTAATCCTTGCGGGGAACGCGTTCACCGCCGGGAAAA

G2 fTP2-3 (SEQ ID NO: 71) CGTCATTCGACGGATCGCAATCTGGCTCCC
G2 rTP2-6 (SEQ ID NO: 82) TGATGGGTCCGCCCATAACCTCATTATTGA
norovirus G2 2 template 0 (SEQ ID NO: 423)
CATTCTACAGCAAAATTAGCAAGTTAGTTATTGCAGAGCTAAAAGAAGGTGGCATGGATTTTTACGTGCCCAGACAAGAGCCAATGTTCAGATGGATGAGATTCTCAGATCTGAGCACGTGGGAGGGCGATCGCAATCTGGCTCCCAGCTTTGTGAATGAAGATGGCGTCGAATGACGCCAACCCATCTGATGGGTCCGCAGCCAACCTCGTCCCAGAGGTCAATAATGAGGTTATGGCTCTGGAGCCCGTTGTTGGTGCCGCTATTGCGGCACCTGTGGCGGGCCAACAAAACGTAATTGACCCCTGGATTAGAAACAATTTTGTACAAGCCCCTGGTGGAGAGTTCACAGTGTCCCCTAGAAACGCTCCAGGTGAGATACTGTGGAGCGCGCCCTTGGGCCCTGATCTGAACCCCTATCTTTCTCATTTGTCCAGAATGTACAATGGTTATGCAGGTGGTTTTGAAGTGCAGGTAATCCTTGCGGGGAACGCGTTCACCGCCGGGAAAA

G2 fTP2-1 (SEQ ID NO: 69) TCATTCGACGCCGCGATCGCAATCTGGCTC
G2 rTP2-4 (SEQ ID NO: 80) TCTGATGGGTCCATAACCTCATTATTGACC
norovirus G2 2 template 0 (SEQ ID NO: 423)
CATTCTACAGCAAAATTAGCAAGTTAGTTATTGCAGAGCTAAAAGAAGGTGGCATGGATTTTTACGTGCCCAGACAAGAGCCAATGTTCAGATGGATGAGATTCTCAGATCTGAGCACGTGGGAGGGCGATCGCAATCTGGCTCCCAGCTTTGTGAATGAAGATGGCGTCGAATGACGCCAACCCATCTGATGGGTCCGCAGCCAACCTCGTCCCAGAGGTCAATAATGAGGTTATGGCTCTGGAGCCCGTTGTTGGTGCCGCTATTGCGGCACCTGTGGCGGGCCAACAAAACGTAATTGACCCCTGGATTAGAAACAATTTTGTACAAGCCCCTGGTGGAGAGTTCACAGTGTCCCCTAGAAACGCTCCAGGTGAGATACTGTGGAGCGCGCCCTTGGGCCCTGATCTGAACCCCTATCTTTCTCATTTGTCCAGAATGTACAATGGTTATGCAGGTGGTTTTGAAGTGCAGGTAATCCTTGCGGGGAACGCGTTCACCGCCGGGAAAA

G2 fTP2-2 (SEQ ID NO: 70) GTCATTCGACGCCGATCGCAATCTGGCTCC
G2 rTP2-4 (SEQ ID NO: 80) TCTGATGGGTCCATAACCTCATTATTGACC
norovirus G2 2 template 0 (SEQ ID NO: 423)
CATTCTACAGCAAAATTAGCAAGTTAGTTATTGCAGAGCTAAAAGAAGGTGGCATGGATTTTTACGTGCCCAGACAAGAGCCAATGTTCAGATGGATGAGATTCTCAGATCTGAGCACGTGGGAGGGCGATCGCAATCTGGCTCCCAGCTTTGTGAATGAAGATGGCGTCGAATGACGCCAACCCATCTGATGGGTCCGCAGCCAACCTCGTCCCAGAGGTCAATAATGAGGTTATGGCTCTGGAGCCCGTTGTTGGTGCCGCTATTGCGGCACCTGTGGCGGGCCAACAAAACGTAATTGACCCCTGGATTAGAAACAATTTTGTACAAGCCCCTGGTGGAGAGTTCACAGTGTCCCCTAGAAACGCTCCAGGTGAGATACTGTGGAGCGCGCCCTTGGGCCCTGATCTGAACCCCTATCTTTCTCATTTGTCCAGAATGTACAATGGTTATGCAGGTGGTTTTGAAGTGCAGGTAATCCTTGCGGGGAACGCGTTCACCGCCGGGAAAA

UCP1_fTP_3 (SEQ ID NO: 426) TTGAATAGTGCCAGTGGTGGCTAATGAGAG
UCP1_rTP (0.5) (SEQ ID NO: 427) GTTAATGTGTTATGTAACAAATTCTCCTTTCCTTT
UCP1 template 0 (SEQ ID NO: 428)
TTGTATTTCTTCTTTTAGAAATTGATTCTTCTGTCATTTGCACATTTATCTGTATAATTATAACAGGGTATTTCCCAGTGGTGGCTAATGAGAGAATTATGGGAAAGTATAGAACACTATTCAAATGCAAAGCACTGTATGATTTTTATTTAATAGGAAGACATTTTGTGCAGCGATTTCTGATTGACCACAGTTTGATCAAGTGCATTTGTTAATGTGTTCTACATTTTCAAAAAGGAAAGGAGAATTTGTTACATTCAGAACTTGCTGCCACTCCTTTGCTACGTCATAAAGGGTCAGTTGCCCTTGCTCATACTGACCTATTCTTTACCTCTCTGCTTCTTCTTTGTGCCAGAAGAGTAGAAATCTGACCCTTTGGGGATACCACCCTCTCCCCTACT

UCP1_fTP_3 (SEQ ID NO: 426) TTGAATAGTGCCAGTGGTGGCTAATGAGAG
UCP1_rTP (1) (SEQ ID NO: 429) TACATTTTCAAATGTAACAAATTCTCCTTTCCTTT
UCP1 template 0 (SEQ ID NO: 428)
TTGTATTTCTTCTTTTAGAAATTGATTCTTCTGTCATTTGCACATTTATCTGTATAATTATAACAGGGTATTTCCCAGTGGTGGCTAATGAGAGAATTATGGGAAAGTATAGAACACTATTCAAATGCAAAGCACTGTATGATTTTTATTTAATAGGAAGACATTTTGTGCAGCGATTTCTGATTGACCACAGTTTGATCAAGTGCATTTGTTAATGTGTTCTACATTTTCAAAAAGGAAAGGAGAATTTGTTACATTCAGAACTTGCTGCCACTCCTTTGCTACGTCATAAAGGGTCAGTTGCCCTTGCTCATACTGACCTATTCTTTACCTCTCTGCTTCTTCTTTGTGCCAGAAGAGTAGAAATCTGACCCTTTGGGGATACCACCCTCTCCCCTACT

[Example 6]
In this example, based on the primer design method (4) and the primer production method (4), the primer (4) is designed and produced, and further nucleic acid amplification is performed using the primer (4). The amplification efficiency was confirmed. That is, in this example, in the action mechanism of the nucleic acid amplification reaction (FIG. 3), when the complementary sequence of TP formed a stem-loop structure (FIGS. 3E and 3F) by folding, The effect of the probability that the site on the loop where the new TP hybridizes is a single strand on the amplification reaction was examined. The probability of being single-stranded is first determined by determining the sequence of interest in the TP hybridizing sequence on the loop, and then evaluating how many complementary sequences are present on the loop. When the number of complementary sequences is larger, the probability of being a single strand is smaller. A part of FIG. 3G is shown below.

As described above, the scientific reason why the effect can be seen when the probability that a specific part on the loop becomes a single strand is increased is considered as follows. However, these are merely examples of theoretical considerations and do not limit the present invention.
1. Since the end of the loop is fixed by the stem structure, it is necessary for the new TP to hybridize helically.
2. Since the 5 ′ end of the TP has a turnback region and is long, it is advantageous that the 5 ′ side first forms a nucleus and the 3 ′ side hybridizes in a spiral shape.
Since the 3.5 'end has steric hindrance of the stem structure, it is advantageous that the initial nucleation is some distance away from the 5' end.

Preparation of template plasmid The template plasmid was prepared in the same manner as in Example 3 (Primer (1)).

Nucleic acid amplification reaction The nucleic acid amplification reaction was performed in the same manner as in Example 3 (the primer (1)).

Nucleic acid amplification reaction evaluation method The sequence (A) on the loop corresponding to the sequence (Ac ′) on the TP that newly hybridizes on the loop (that is, a part of the sequence on the genome that is the target of nucleic acid amplification) For the same sequence, a sequence of 3 bases complementary to the sequence of 3 bases from the 5 ′ end of sequence (A) (1-3 of the target sequence 5 ′ side) (complementary sequence) is the “target sequence 5 ′ A maximum of 20 bases from the 4th base to the 23rd base of the sequence counted upstream from the 5 ′ end of “Side 1-3” (not including the sequence (B) and its upstream sequence), and attention In the total of 20 bases (not including the sequence (Bc) and its downstream sequence) from the 4th base to the 23rd base of the sequence counted downstream from the 3 ′ end of the sequence 1-3, The total number was calculated. Then, the sequence of interest is further shifted by one base to the 3 ′ side (in order of “target sequence 5 ′ side 2-4”, “target sequence 5 ′ side 3-5”, “target sequence 5 ′ side 4-6”). The same calculation was performed for. Similarly, a sequence of 3 bases from the 3 ′ end of sequence (A) (target sequence 3 ′ side 1-3) and a sequence shifted by one base to the 5 ′ side (in order of “target sequence 3 ′ side 2-4 ”,“ Target sequence 3 ′ side 3-5 ”,“ target sequence 3 ′ side 4-6 ”...)).
Next, the reaction time of the sample containing the template nucleic acid for each TP used was within 45 minutes, and the difference between the reaction time of the sample containing the template nucleic acid and the reaction time of the sample not containing the template nucleic acid (negative control) Were determined to be “success” and those other than “failure”.

<Result>
When the sequence of interest is determined by counting from the 5 ′ end of the sequence (A) on the loop, as shown in Table 4 below, 3 from the 9th base to the 11th base from the 5 ′ end of the sequence (A) on the loop When the base sequence is the target sequence (target sequence 5 ′ side 9-11), the success rate when the number of complementary sequences is 0 is 4/4 cases (100%), the number of complementary sequences is 1 In the case of 4 cases / 4 cases (100%), the number of complementary sequences is 2 cases, 0 cases / 2 cases (0%), and the number of complementary sequences is suppressed to 1 or less. A marked difference was seen between the two cases.

  When the sequence of 3 bases from the 10th base to the 12th base from the 5 ′ end of the sequence (A) on the loop is the target sequence (target sequence 5 ′ side 10-12), the number of complementary sequences is 0. The success rate was 5 cases / 5 cases (100%). When the number of complementary sequences was 1, it was 3/3 cases (100%), whereas the number of complementary sequences was 2 or more. In all cases, there were 0 cases / 4 cases (0%), and a remarkable difference was observed between the case where the number of complementary sequences was suppressed to 1 or less and the case where 2 or more complementary sequences were suppressed.

  Furthermore, when the sequence of 3 bases from the 11th base to the 13th base from the 5 ′ end of the sequence (A) on the loop is the target sequence (target sequence 5 ′ side 11-13), the number of complementary sequences is 0. The success rate was 4 cases / 4 cases (100%), and 4 cases / 4 cases (100%) when the number of complementary sequences was 1, whereas the number of complementary sequences was 2 or more. In all cases, there were 0 cases / 4 cases (0%), and a remarkable difference was observed between the case where the number of complementary sequences was suppressed to 1 or less and the case where 2 or more complementary sequences were suppressed.

  On the other hand, although data is not shown, in the case of the target sequence 5 ′ side 7-9, the target sequence 5 ′ side 8-10, and the target sequence 5 ′ side 12-14, the effect of reducing the number of complementary sequences is remarkable. I couldn't see it.

  From the above, among the 5 bases from the 9th base to the 13th base counted from the 5 ′ end of the sequence (A) on the loop as the target sequence, 3 bases from the 9th base to the 11th base, 10 bases It has been clarified that excellent amplification rate and specificity are exhibited when the number of complementary sequences of 3 bases from the 12th base to the 3rd base or 3 bases from the 11th base to the 13th base is suppressed to 1 or less. It was.

When the sequence of interest is determined by counting from the 3 ′ end of the sequence (A) on the loop, as shown in Table 5 below, the 3rd from the 6th base to the 8th base from the 3 ′ end of the sequence (A) on the loop When the base sequence is the target sequence (target sequence 3 ′ side 6-8), the success rate when the number of complementary sequences is 0 is 5/5 cases (100%), the number of complementary sequences is 1 In some cases, 3 cases / 3 cases (100%), whereas in the case of 2 complementary sequences, 0 cases / 4 cases (0%), and when the number of complementary sequences is suppressed to 1 or less, A marked difference was seen between the two cases.
Further, when the sequence of 3 bases from the 7th base to the 9th base from the 3 ′ end of the sequence (A) on the loop is the target sequence (target sequence 3 ′ side 7-9), the number of complementary sequences is 0. In the case of 4 cases / 4 cases (100%), the success rate was 4 cases / 4 cases (100%) when the number of complementary sequences was 1, whereas the number of complementary sequences was 2 cases. 0 cases / 4 cases (0%), and a remarkable difference was observed between the case where the number of complementary sequences was suppressed to 1 or less and the case where two complementary sequences were suppressed.

  Furthermore, when the sequence of 3 bases from the 8th base to the 10th base from the 3 ′ end of the sequence (A) on the loop is the target sequence (target sequence 3 ′ side 8-10), the number of complementary sequences is 0. In the case of 5 cases / 5 cases (100%), the number of complementary sequences was 3 cases / 3 cases (100%), whereas the number of complementary sequences was 2 cases. 0 cases / 4 cases (0%), and a remarkable difference was observed between the case where the number of complementary sequences was suppressed to 1 or less and the case where two complementary sequences were suppressed.

  On the other hand, although data is not shown, in the case of the target sequence 3 ′ side 4-6, the target sequence 3 ′ side 5-7, the target sequence 3 ′ side 9-11, and the target sequence 3 ′ side 10-12, the complementary sequence The effect of reducing the number was not noticeable.

  From the above, among the 5 bases from the 6th base to the 10th base from the 3 ′ end of the sequence (A) on the loop as the target sequence, the 6th base to the 8th base, 3 bases, the 7th base It was revealed that excellent amplification rate and specificity were exhibited when the number of complementary sequences of the 9 base 3 bases or the 8 base to 10 base 3 base sequences was suppressed to 1 or less.

（表４〜表５中「−」は９０分以内で反応が見られなかったことを示す。）

("-" In Table 4 to Table 5 indicates that no reaction was observed within 90 minutes.)

The base sequences of TP and target nucleic acid sequences used in Tables 4 and 5 above are as follows.

5'end 9-11 base

ABCG2 TP_F4 (SEQ ID NO: 170) TGTAAGTTTTCCTTAAGGATGATGTTGTG
ABCG2 TP_R6 (SEQ ID NO: 180) ACAGTTCTCATTTTCATGATTCGTCATAG
ABCG2 template 0 (SEQ ID NO: 418)
ATGGTCTTAGAAAAGACTCATTATCATTATGTCTCATTAAAATGCTATTTGCCTTAAGGATGATGTTGTGATGGGCACTCTGACGGTGAGAGAAAACTTACAGTTCTCAGCAGCTCTTCGGCTTGCAACAACTATGACGAATCATGAAAAAAACGAACGGATTAACAGGGTCATTCAAGAGTTAGGTCTGGATAAAGGCGC

ABCG2 TP_F4 (SEQ ID NO: 170) TGTAAGTTTTCCTTAAGGATGATGTTGTG
ABCG2 TP_R7 (SEQ ID NO: 181) ACAGTTCTCATTTTTCATGATTCGTCATA
ABCG2 template 0 (SEQ ID NO: 418)
ATGGTCTTAGAAAAGACTCATTATCATTATGTCTCATTAAAATGCTATTTGCCTTAAGGATGATGTTGTGATGGGCACTCTGACGGTGAGAGAAAACTTACAGTTCTCAGCAGCTCTTCGGCTTGCAACAACTATGACGAATCATGAAAAAAACGAACGGATTAACAGGGTCATTCAAGAGTTAGGTCTGGATAAAGGCGC

NAT2-590 TP_F6 (SEQ ID NO: 323) TCGAGGTTCAATCTCCTGCCAAAGAAGAAACA
NAT2-590 TP_R4 (SEQ ID NO: 329) CGAACAATTGTGTTGGAGACGTCTGCAGGTAT
NAT2-590 template 0 (sequence number 430)
ATCAGGAGAGAGCAGTATATTACAAACAAAGAATTTCTTAATTCTCATCTCCTGCCAAAGAAGAAACACCAAAAAATATACTTATTTACGCTTGAACCTCGAACAATTGAAGATTTTGAGTCTATGAATACATACCTGCAGACGTCTCCAACATCTTCATTTATAACCACATCATTTTGTTCCTTGCAGACCCCAGAAGGG

NAT2-590 TP_F6 (SEQ ID NO: 323) TCGAGGTTCAATCTCCTGCCAAAGAAGAAACA
NAT2-590 TP_R5 (SEQ ID NO: 330) CGAACAATTGATGTTGGAGACGTCTGCAGGTA
NAT2-590 template 0 (sequence number 430)
ATCAGGAGAGAGCAGTATATTACAAACAAAGAATTTCTTAATTCTCATCTCCTGCCAAAGAAGAAACACCAAAAAATATACTTATTTACGCTTGAACCTCGAACAATTGAAGATTTTGAGTCTATGAATACATACCTGCAGACGTCTCCAACATCTTCATTTATAACCACATCATTTTGTTCCTTGCAGACCCCAGAAGGG

FTO TP_F2 (SEQ ID NO: 219) CTAAATTCACTTAGAATGTCTGAATTATTATT
FTO TP_R1 (SEQ ID NO: 226) AGTGATGCACCTGTTACCTATTAAAACTTTAG
FTO template 0 (SEQ ID NO: 420)
TTATGAGATAATGTCCTTTTTAAAAATAAACACTAACATCAGTTATGCATTTAGAATGTCTGAATTATTATTCTAGGTTCCTTGCGACTGCTGTGAATTTAGTGATGCACTTGGATAGTCTCTGTTACTCTAAAGTTTTAATAGGTAACAGTCAGAAATGGAGTGGGAGAGCATAAAAGCAAACTGAAATGCAAATAGCTG

5'end 10-12 base

G2 fTP3-7 (SEQ ID NO: 91) GCTCCAGAGCCACGCAGCCAACCTCGTCCC
G2 rTP3-5 (SEQ ID NO: 97) GTTGGTGCCGCTTCAATTACGTTTTGTTGG
norovirus G2 3 template 0 (SEQ ID NO: 419)
CATTCTACAGCAAAATTAGCAAGTTAGTTATTGCAGAGCTAAAAGAAGGTGGCATGGATTTTTACGTGCCCAGACAAGAGCCAATGTTCAGATGGATGAGATTCTCAGATCTGAGCACGTGGGAGGGCGATCGCAATCTGGCTCCCAGCTTTGTGAATGAAGATGGCGTCGAATGACGCCAACCCATCTGATGGGTCCGCAGCCAACCTCGTCCCAGAGGTCAATAATGAGGTTATGGCTCTGGAGCCCGTTGTTGGTGCCGCTATTGCGGCACCTGTGGCGGGCCAACAAAACGTAATTGACCCCTGGATTAGAAACAATTTTGTACAAGCCCCTGGTGGAGAGTTCACAGTGTCCCCTAGAAACGCTCCAGGTGAGATACTGTGGAGCGCGCCCTTGGGCCCTGATCTGAACCCCTATCTTTCTCATTTGTCCAGAATGTACAATGGTTATGCAGGTGGTTTTGAAGTGCAGGTAATCCTTGCGGGGAACGCGTTCACCGCCGGGAAAA

G2 fTP3-7 (SEQ ID NO: 91) GCTCCAGAGCCACGCAGCCAACCTCGTCCC
G2 rTP3-8 (SEQ ID NO: 100) GGTGCCGCTATTGGGTCAATTACGTTTTGT
norovirus G2 3 template 0 (SEQ ID NO: 419)
CATTCTACAGCAAAATTAGCAAGTTAGTTATTGCAGAGCTAAAAGAAGGTGGCATGGATTTTTACGTGCCCAGACAAGAGCCAATGTTCAGATGGATGAGATTCTCAGATCTGAGCACGTGGGAGGGCGATCGCAATCTGGCTCCCAGCTTTGTGAATGAAGATGGCGTCGAATGACGCCAACCCATCTGATGGGTCCGCAGCCAACCTCGTCCCAGAGGTCAATAATGAGGTTATGGCTCTGGAGCCCGTTGTTGGTGCCGCTATTGCGGCACCTGTGGCGGGCCAACAAAACGTAATTGACCCCTGGATTAGAAACAATTTTGTACAAGCCCCTGGTGGAGAGTTCACAGTGTCCCCTAGAAACGCTCCAGGTGAGATACTGTGGAGCGCGCCCTTGGGCCCTGATCTGAACCCCTATCTTTCTCATTTGTCCAGAATGTACAATGGTTATGCAGGTGGTTTTGAAGTGCAGGTAATCCTTGCGGGGAACGCGTTCACCGCCGGGAAAA

GGCX TP_F3 (SEQ ID NO: 253) TCGGGGGCAGTCTGCTCCCCTGAGTG
GGCX TP_R4 (SEQ ID NO: 262) CGAAGGTTGCACTGGGCTGAGGGGCT
GGCX template 0 (SEQ ID NO: 425)
CTCCCTGCTTTCCTAGGTATGTTCTCCTACGTCATGCTGGCCAGCAGCCCTCTCTTCTGCTCCCCTGAGTGGCCTCGGAAGCTGGTGTCCTACTGCCCCCGAAGGTTGCAACAACTGTTGCCCCTCAAGGCAGCCCCTCAGCCCAGTGTTTCCTGTGTGTATAAGAGGAGCCGGGGCAAAAGTGGCCAGAAGCCAGGGTGGCTG

G2 fTP4-5 (SEQ ID NO: 105) GCCACAGGTGCCGTTATGGCTCTGGAGCCC
G2 rTP4-2 (SEQ ID NO: 110) CAACAAAACGTAGGGGCTTGTACAAAATTG
norovirus G2 4 template 0 (SEQ ID NO: 422)
CATTCTACAGCAAAATTAGCAAGTTAGTTATTGCAGAGCTAAAAGAAGGTGGCATGGATTTTTACGTGCCCAGACAAGAGCCAATGTTCAGATGGATGAGATTCTCAGATCTGAGCACGTGGGAGGGCGATCGCAATCTGGCTCCCAGCTTTGTGAATGAAGATGGCGTCGAATGACGCCAACCCATCTGATGGGTCCGCAGCCAACCTCGTCCCAGAGGTCAATAATGAGGTTATGGCTCTGGAGCCCGTTGTTGGTGCCGCTATTGCGGCACCTGTGGCGGGCCAACAAAACGTAATTGACCCCTGGATTAGAAACAATTTTGTACAAGCCCCTGGTGGAGAGTTCACAGTGTCCCCTAGAAACGCTCCAGGTGAGATACTGTGGAGCGCGCCCTTGGGCCCTGATCTGAACCCCTATCTTTCTCATTTGTCCAGAATGTACAATGGTTATGCAGGTGGTTTTGAAGTGCAGGTAATCCTTGCGGGGAACGCGTTCACCGCCGGGAAAA

FTO TP_F8 (SEQ ID NO: 225) CTAAATTCACATGCATTTAGAATGTCTGAATT
FTO TP_R8 (SEQ ID NO: 233) AGTGATGCACTTTCTGACTGTTACCTATTAAA
FTO template 0 (SEQ ID NO: 420)
TTATGAGATAATGTCCTTTTTAAAAATAAACACTAACATCAGTTATGCATTTAGAATGTCTGAATTATTATTCTAGGTTCCTTGCGACTGCTGTGAATTTAGTGATGCACTTGGATAGTCTCTGTTACTCTAAAGTTTTAATAGGTAACAGTCAGAAATGGAGTGGGAGAGCATAAAAGCAAACTGAAATGCAAATAGCTG

FTO TP_F1 (SEQ ID NO: 218) CTAAATTCACTAGAATGTCTGAATTATTATTC
FTO TP_R8 (SEQ ID NO: 233) AGTGATGCACTTTCTGACTGTTACCTATTAAA
FTO template 0 (SEQ ID NO: 420)
TTATGAGATAATGTCCTTTTTAAAAATAAACACTAACATCAGTTATGCATTTAGAATGTCTGAATTATTATTCTAGGTTCCTTGCGACTGCTGTGAATTTAGTGATGCACTTGGATAGTCTCTGTTACTCTAAAGTTTTAATAGGTAACAGTCAGAAATGGAGTGGGAGAGCATAAAAGCAAACTGAAATGCAAATAGCTG

5'end 11-13 base

G2 fTP6-5 (SEQ ID NO: 137) CCCAAGGGCGCGGTGTCCCCTAGAAACGCT
G2 rTP6-5 (SEQ ID NO: 145) CTGAACCCCTATCCTGCATAACCATTGTAC
norovirus G2 6 template 0 (SEQ ID NO: 431)
CATTCTACAGCAAAATTAGCAAGTTAGTTATTGCAGAGCTAAAAGAAGGTGGCATGGATTTTTACGTGCCCAGACAAGAGCCAATGTTCAGATGGATGAGATTCTCAGATCTGAGCACGTGGGAGGGCGATCGCAATCTGGCTCCCAGCTTTGTGAATGAAGATGGCGTCGAATGACGCCAACCCATCTGATGGGTCCGCAGCCAACCTCGTCCCAGAGGTCAATAATGAGGTTATGGCTCTGGAGCCCGTTGTTGGTGCCGCTATTGCGGCACCTGTGGCGGGCCAACAAAACGTAATTGACCCCTGGATTAGAAACAATTTTGTACAAGCCCCTGGTGGAGAGTTCACAGTGTCCCCTAGAAACGCTCCAGGTGAGATACTGTGGAGCGCGCCCTTGGGCCCTGATCTGAACCCCTATCTTTCTCATTTGTCCAGAATGTACAATGGTTATGCAGGTGGTTTTGAAGTGCAGGTAATCCTTGCGGGGAACGCGTTCACCGCCGGGAAAA

G2 fTP6-5 (SEQ ID NO: 137) CCCAAGGGCGCGGTGTCCCCTAGAAACGCT
G2 rTP6-7 (SEQ ID NO: 147) GAACCCCTATCTCACCTGCATAACCATTGT
norovirus G2 6 template 0 (SEQ ID NO: 431)
CATTCTACAGCAAAATTAGCAAGTTAGTTATTGCAGAGCTAAAAGAAGGTGGCATGGATTTTTACGTGCCCAGACAAGAGCCAATGTTCAGATGGATGAGATTCTCAGATCTGAGCACGTGGGAGGGCGATCGCAATCTGGCTCCCAGCTTTGTGAATGAAGATGGCGTCGAATGACGCCAACCCATCTGATGGGTCCGCAGCCAACCTCGTCCCAGAGGTCAATAATGAGGTTATGGCTCTGGAGCCCGTTGTTGGTGCCGCTATTGCGGCACCTGTGGCGGGCCAACAAAACGTAATTGACCCCTGGATTAGAAACAATTTTGTACAAGCCCCTGGTGGAGAGTTCACAGTGTCCCCTAGAAACGCTCCAGGTGAGATACTGTGGAGCGCGCCCTTGGGCCCTGATCTGAACCCCTATCTTTCTCATTTGTCCAGAATGTACAATGGTTATGCAGGTGGTTTTGAAGTGCAGGTAATCCTTGCGGGGAACGCGTTCACCGCCGGGAAAA

EGFR20 TP_F4 (SEQ ID NO: 187) CGTGATGAGTGTGCCGCCTGCTGGGCAT
EGFR20 TP_R4 (SEQ ID NO: 195) ACGCAGCTCTTGTGTTCCCGGACATAGT
EGFR20 template 0 (SEQ ID NO: 421)
GACGTGCCTCTCCCTCCCTCCAGGAAGCCTACGTGATGGCCAGCGTGGACAACCCCCACGTGTGCCGCCTGCTGGGCATCTGCCTCACCTCCACCGTGCAGCTCATCACGCAGCTCATGCCCTTCGGCTGCCTCCTGGACTATGTCCGGGAACACAAAGACAATATTGGCTCCCAGTACCTGCTCAACTGGTGTGGA

EGFR20 TP_F4 (SEQ ID NO: 187) CGTGATGAGTGTGCCGCCTGCTGGGCAT
EGFR20 TP_R7 (SEQ ID NO: 198) ACGCAGCTCTCTTTGTGTTCCCGGACAT
EGFR20 template 0 (SEQ ID NO: 421)
GACGTGCCTCTCCCTCCCTCCAGGAAGCCTACGTGATGGCCAGCGTGGACAACCCCCACGTGTGCCGCCTGCTGGGCATCTGCCTCACCTCCACCGTGCAGCTCATCACGCAGCTCATGCCCTTCGGCTGCCTCCTGGACTATGTCCGGGAACACAAAGACAATATTGGCTCCCAGTACCTGCTCAACTGGTGTGGA

FTO TP_F7 (SEQ ID NO: 224) CTAAATTCACTGCATTTAGAATGTCTGAATTA
FTO TP_R7 (SEQ ID NO: 232) AGTGATGCACTTCTGACTGTTACCTATTAAAA
FTO template 0 (SEQ ID NO: 420)
TTATGAGATAATGTCCTTTTTAAAAATAAACACTAACATCAGTTATGCATTTAGAATGTCTGAATTATTATTCTAGGTTCCTTGCGACTGCTGTGAATTTAGTGATGCACTTGGATAGTCTCTGTTACTCTAAAGTTTTAATAGGTAACAGTCAGAAATGGAGTGGGAGAGCATAAAAGCAAACTGAAATGCAAATAGCTG

FTO TP_F7 (SEQ ID NO: 224) CTAAATTCACTGCATTTAGAATGTCTGAATTA
FTO TP_R8 (SEQ ID NO: 233) AGTGATGCACTTTCTGACTGTTACCTATTAAA
FTO template 0 (SEQ ID NO: 420)
TTATGAGATAATGTCCTTTTTAAAAATAAACACTAACATCAGTTATGCATTTAGAATGTCTGAATTATTATTCTAGGTTCCTTGCGACTGCTGTGAATTTAGTGATGCACTTGGATAGTCTCTGTTACTCTAAAGTTTTAATAGGTAACAGTCAGAAATGGAGTGGGAGAGCATAAAAGCAAACTGAAATGCAAATAGCTG

3'end 6-8 base

G2 fTP3-3 (SEQ ID NO: 87) CAGAGCCATAACGGTCCGCAGCCAACCTCG
G2 rTP3-3 (SEQ ID NO: 95) TTGTTGGTGCCGAATTACGTTTTGTTGGCC
norovirus G2 3 template 0 (SEQ ID NO: 419)
CATTCTACAGCAAAATTAGCAAGTTAGTTATTGCAGAGCTAAAAGAAGGTGGCATGGATTTTTACGTGCCCAGACAAGAGCCAATGTTCAGATGGATGAGATTCTCAGATCTGAGCACGTGGGAGGGCGATCGCAATCTGGCTCCCAGCTTTGTGAATGAAGATGGCGTCGAATGACGCCAACCCATCTGATGGGTCCGCAGCCAACCTCGTCCCAGAGGTCAATAATGAGGTTATGGCTCTGGAGCCCGTTGTTGGTGCCGCTATTGCGGCACCTGTGGCGGGCCAACAAAACGTAATTGACCCCTGGATTAGAAACAATTTTGTACAAGCCCCTGGTGGAGAGTTCACAGTGTCCCCTAGAAACGCTCCAGGTGAGATACTGTGGAGCGCGCCCTTGGGCCCTGATCTGAACCCCTATCTTTCTCATTTGTCCAGAATGTACAATGGTTATGCAGGTGGTTTTGAAGTGCAGGTAATCCTTGCGGGGAACGCGTTCACCGCCGGGAAAA

G2 fTP3-3 (SEQ ID NO: 87) CAGAGCCATAACGGTCCGCAGCCAACCTCG
G2 rTP3-8 (SEQ ID NO: 100) GGTGCCGCTATTGGGTCAATTACGTTTTGT
norovirus G2 3 template 0 (SEQ ID NO: 419)
CATTCTACAGCAAAATTAGCAAGTTAGTTATTGCAGAGCTAAAAGAAGGTGGCATGGATTTTTACGTGCCCAGACAAGAGCCAATGTTCAGATGGATGAGATTCTCAGATCTGAGCACGTGGGAGGGCGATCGCAATCTGGCTCCCAGCTTTGTGAATGAAGATGGCGTCGAATGACGCCAACCCATCTGATGGGTCCGCAGCCAACCTCGTCCCAGAGGTCAATAATGAGGTTATGGCTCTGGAGCCCGTTGTTGGTGCCGCTATTGCGGCACCTGTGGCGGGCCAACAAAACGTAATTGACCCCTGGATTAGAAACAATTTTGTACAAGCCCCTGGTGGAGAGTTCACAGTGTCCCCTAGAAACGCTCCAGGTGAGATACTGTGGAGCGCGCCCTTGGGCCCTGATCTGAACCCCTATCTTTCTCATTTGTCCAGAATGTACAATGGTTATGCAGGTGGTTTTGAAGTGCAGGTAATC
CTTGCGGGGAACGCGTTCACCGCCGGGAAAA

G2 fTP2-2 (SEQ ID NO: 70) GTCATTCGACGCCGATCGCAATCTGGCTCC
G2 rTP2-3 (SEQ ID NO: 79) ATCTGATGGGTCTAACCTCATTATTGACCT
norovirus G2 2 template 0 (SEQ ID NO: 423)
CATTCTACAGCAAAATTAGCAAGTTAGTTATTGCAGAGCTAAAAGAAGGTGGCATGGATTTTTACGTGCCCAGACAAGAGCCAATGTTCAGATGGATGAGATTCTCAGATCTGAGCACGTGGGAGGGCGATCGCAATCTGGCTCCCAGCTTTGTGAATGAAGATGGCGTCGAATGACGCCAACCCATCTGATGGGTCCGCAGCCAACCTCGTCCCAGAGGTCAATAATGAGGTTATGGCTCTGGAGCCCGTTGTTGGTGCCGCTATTGCGGCACCTGTGGCGGGCCAACAAAACGTAATTGACCCCTGGATTAGAAACAATTTTGTACAAGCCCCTGGTGGAGAGTTCACAGTGTCCCCTAGAAACGCTCCAGGTGAGATACTGTGGAGCGCGCCCTTGGGCCCTGATCTGAACCCCTATCTTTCTCATTTGTCCAGAATGTACAATGGTTATGCAGGTGGTTTTGAAGTGCAGGTAATCCTTGCGGGGAACGCGTTCACCGCCGGGAAAA

G2 fTP2-2 (SEQ ID NO: 70) GTCATTCGACGCCGATCGCAATCTGGCTCC
G2 rTP2-3 (SEQ ID NO: 79) ATCTGATGGGTCTAACCTCATTATTGACCT
norovirus G2 2 template 0 (SEQ ID NO: 423)
CATTCTACAGCAAAATTAGCAAGTTAGTTATTGCAGAGCTAAAAGAAGGTGGCATGGATTTTTACGTGCCCAGACAAGAGCCAATGTTCAGATGGATGAGATTCTCAGATCTGAGCACGTGGGAGGGCGATCGCAATCTGGCTCCCAGCTTTGTGAATGAAGATGGCGTCGAATGACGCCAACCCATCTGATGGGTCCGCAGCCAACCTCGTCCCAGAGGTCAATAATGAGGTTATGGCTCTGGAGCCCGTTGTTGGTGCCGCTATTGCGGCACCTGTGGCGGGCCAACAAAACGTAATTGACCCCTGGATTAGAAACAATTTTGTACAAGCCCCTGGTGGAGAGTTCACAGTGTCCCCTAGAAACGCTCCAGGTGAGATACTGTGGAGCGCGCCCTTGGGCCCTGATCTGAACCCCTATCTTTCTCATTTGTCCAGAATGTACAATGGTTATGCAGGTGGTTTTGAAGTGCAGGTAATCCTTGCGGGGAACGCGTTCACCGCCGGGAAAA

FTO TP_F5 (SEQ ID NO: 222) CTAAATTCACCATTTAGAATGTCTGAATTATT
FTO TP_R3 (SEQ ID NO: 228) AGTGATGCACGACTGTTACCTATTAAAACTTT
FTO template 0 (SEQ ID NO: 420)
TTATGAGATAATGTCCTTTTTAAAAATAAACACTAACATCAGTTATGCATTTAGAATGTCTGAATTATTATTCTAGGTTCCTTGCGACTGCTGTGAATTTAGTGATGCACTTGGATAGTCTCTGTTACTCTAAAGTTTTAATAGGTAACAGTCAGAAATGGAGTGGGAGAGCATAAAAGCAAACTGAAATGCAAATAGCTG

FTO TP_F5 (SEQ ID NO: 222) CTAAATTCACCATTTAGAATGTCTGAATTATT
FTO TP_R4 (SEQ ID NO: 229) AGTGATGCACTGACTGTTACCTATTAAAACTT
FTO template 0 (SEQ ID NO: 420)
TTATGAGATAATGTCCTTTTTAAAAATAAACACTAACATCAGTTATGCATTTAGAATGTCTGAATTATTATTCTAGGTTCCTTGCGACTGCTGTGAATTTAGTGATGCACTTGGATAGTCTCTGTTACTCTAAAGTTTTAATAGGTAACAGTCAGAAATGGAGTGGGAGAGCATAAAAGCAAACTGAAATGCAAATAGCTG

3'end 7-9 base

ABCG2 TP_F3 (SEQ ID NO: 169) TGTAAGTTTTCTTAAGGATGATGTTGTGA
ABCG2 TP_R4 (SEQ ID NO: 178) ACAGTTCTCATTCATGATTCGTCATAGTT
ABCG2 template 0 (SEQ ID NO: 418)
ATGGTCTTAGAAAAGACTCATTATCATTATGTCTCATTAAAATGCTATTTGCCTTAAGGATGATGTTGTGATGGGCACTCTGACGGTGAGAGAAAACTTACAGTTCTCAGCAGCTCTTCGGCTTGCAACAACTATGACGAATCATGAAAAAAACGAACGGATTAACAGGGTCATTCAAGAGTTAGGTCTGGATAAAGGCGC

ABCG2 TP_F3 (SEQ ID NO: 169) TGTAAGTTTTCTTAAGGATGATGTTGTGA
ABCG2 TP_R5 (SEQ ID NO: 179) ACAGTTCTCATTTCATGATTCGTCATAGT
ABCG2 template 0 (SEQ ID NO: 418)
ATGGTCTTAGAAAAGACTCATTATCATTATGTCTCATTAAAATGCTATTTGCCTTAAGGATGATGTTGTGATGGGCACTCTGACGGTGAGAGAAAACTTACAGTTCTCAGCAGCTCTTCGGCTTGCAACAACTATGACGAATCATGAAAAAAACGAACGGATTAACAGGGTCATTCAAGAGTTAGGTCTGGATAAAGGCGC

EGFR20 TP_F1 (SEQ ID NO: 184) CGTGATGAGGCCGCCTGCTGGGCATCTG
EGFR20 TP_R4 (SEQ ID NO: 195) ACGCAGCTCTTGTGTTCCCGGACATAGT
EGFR20 template 0 (SEQ ID NO: 421)
GACGTGCCTCTCCCTCCCTCCAGGAAGCCTACGTGATGGCCAGCGTGGACAACCCCCACGTGTGCCGCCTGCTGGGCATCTGCCTCACCTCCACCGTGCAGCTCATCACGCAGCTCATGCCCTTCGGCTGCCTCCTGGACTATGTCCGGGAACACAAAGACAATATTGGCTCCCAGTACCTGCTCAACTGGTGTGGA

EGFR20 TP_F1 (SEQ ID NO: 184) CGTGATGAGGCCGCCTGCTGGGCATCTG
EGFR20 TP_R7 (SEQ ID NO: 198) ACGCAGCTCTCTTTGTGTTCCCGGACAT
EGFR20 template 0 (SEQ ID NO: 421)
GACGTGCCTCTCCCTCCCTCCAGGAAGCCTACGTGATGGCCAGCGTGGACAACCCCCACGTGTGCCGCCTGCTGGGCATCTGCCTCACCTCCACCGTGCAGCTCATCACGCAGCTCATGCCCTTCGGCTGCCTCCTGGACTATGTCCGGGAACACAAAGACAATATTGGCTCCCAGTACCTGCTCAACTGGTGTGGA

FTO TP_F6 (SEQ ID NO: 223) CTAAATTCACGCATTTAGAATGTCTGAATTAT
FTO TP_R6 (SEQ ID NO: 231) AGTGATGCACTCTGACTGTTACCTATTAAAAC
FTO template 0 (SEQ ID NO: 420)
TTATGAGATAATGTCCTTTTTAAAAATAAACACTAACATCAGTTATGCATTTAGAATGTCTGAATTATTATTCTAGGTTCCTTGCGACTGCTGTGAATTTAGTGATGCACTTGGATAGTCTCTGTTACTCTAAAGTTTTAATAGGTAACAGTCAGAAATGGAGTGGGAGAGCATAAAAGCAAACTGAAATGCAAATAGCTG

FTO TP_F6 (SEQ ID NO: 223) CTAAATTCACGCATTTAGAATGTCTGAATTAT
FTO TP_R7 (SEQ ID NO: 232) AGTGATGCACTTCTGACTGTTACCTATTAAAA
FTO template 0 (SEQ ID NO: 420)
TTATGAGATAATGTCCTTTTTAAAAATAAACACTAACATCAGTTATGCATTTAGAATGTCTGAATTATTATTCTAGGTTCCTTGCGACTGCTGTGAATTTAGTGATGCACTTGGATAGTCTCTGTTACTCTAAAGTTTTAATAGGTAACAGTCAGAAATGGAGTGGGAGAGCATAAAAGCAAACTGAAATGCAAATAGCTG

3'end 8-10 base

G2 fTP1-7 (SEQ ID NO: 59) GAGAATCTCATCGATTTTTACGTGCCCAGA
G2 rTP1-6 (SEQ ID NO: 66) GAGCACGTGGGACTTCATTCACAAAGCTGG
norovirus G2 1 template 0 (SEQ ID NO: 432)
CATTCTACAGCAAAATTAGCAAGTTAGTTATTGCAGAGCTAAAAGAAGGTGGCATGGATTTTTACGTGCCCAGACAAGAGCCAATGTTCAGATGGATGAGATTCTCAGATCTGAGCACGTGGGAGGGCGATCGCAATCTGGCTCCCAGCTTTGTGAATGAAGATGGCGTCGAATGACGCCAACCCATCTGATGGGTCCGCAGCCAACCTCGTCCCAGAGGTCAATAATGAGGTTATGGCTCTGGAGCCCGTTGTTGGTGCCGCTATTGCGGCACCTGTGGCGGGCCAACAAAACGTAATTGACCCCTGGATTAGAAACAATTTTGTACAAGCCCCTGGTGGAGAGTTCACAGTGTCCCCTAGAAACGCTCCAGGTGAGATACTGTGGAGCGCGCCCTTGGGCCCTGATCTGAACCCCTATCTTTCTCATTTGTCCAGAATGTACAATGGTTATGCAGGTGGTTTTGAAGTGCAGGTAATCCTTGCGGGGAACGCGTTCACCGCCGGGAAAA

G2 fTP1-7 (SEQ ID NO: 59) GAGAATCTCATCGATTTTTACGTGCCCAGA
G2 rTP1-7 (SEQ ID NO: 67) AGCACGTGGGAGTCTTCATTCACAAAGCTG
norovirus G2 1 template 0 (SEQ ID NO: 432)
CATTCTACAGCAAAATTAGCAAGTTAGTTATTGCAGAGCTAAAAGAAGGTGGCATGGATTTTTACGTGCCCAGACAAGAGCCAATGTTCAGATGGATGAGATTCTCAGATCTGAGCACGTGGGAGGGCGATCGCAATCTGGCTCCCAGCTTTGTGAATGAAGATGGCGTCGAATGACGCCAACCCATCTGATGGGTCCGCAGCCAACCTCGTCCCAGAGGTCAATAATGAGGTTATGGCTCTGGAGCCCGTTGTTGGTGCCGCTATTGCGGCACCTGTGGCGGGCCAACAAAACGTAATTGACCCCTGGATTAGAAACAATTTTGTACAAGCCCCTGGTGGAGAGTTCACAGTGTCCCCTAGAAACGCTCCAGGTGAGATACTGTGGAGCGCGCCCTTGGGCCCTGATCTGAACCCCTATCTTTCTCATTTGTCCAGAATGTACAATGGTTATGCAGGTGGTTTTGAAGTGCAGGTAATCCTTGCGGGGAACGCGTTCACCGCCGGGAAAA

EGFR20m TP_F2 (SEQ ID NO: 202) CATGATGAGTGCCGCCTGCTGGGCATCT
EGFR20m TP_R6 (SEQ ID NO: 214) CATGCAGCTCTTTGTGTTCCCGGACATA
EGFR20 template 1 (SEQ ID NO: 433)
GACGTGCCTCTCCCTCCCTCCAGGAAGCCTACGTGATGGCCAGCGTGGACAACCCCCACGTGTGCCGCCTGCTGGGCATCTGCCTCACCTCCACCGTGCAGCTCATCACGCAGCTCATGCCCTTCGGCTGCCTCCTGGACTATGTCCGGGAACACAAAGACAATATTGGCTCCCAGTACCTGCTCAACTGGTGTGGA

G2 fTP3-2 (SEQ ID NO: 86) AGAGCCATAACCGGGTCCGCAGCCAACCTC
G2 rTP3-1 (SEQ ID NO: 93) CGTTGTTGGTGCTTACGTTTTGTTGGCCCG
norovirus G2 3 template 0 (SEQ ID NO: 419)
CATTCTACAGCAAAATTAGCAAGTTAGTTATTGCAGAGCTAAAAGAAGGTGGCATGGATTTTTACGTGCCCAGACAAGAGCCAATGTTCAGATGGATGAGATTCTCAGATCTGAGCACGTGGGAGGGCGATCGCAATCTGGCTCCCAGCTTTGTGAATGAAGATGGCGTCGAATGACGCCAACCCATCTGATGGGTCCGCAGCCAACCTCGTCCCAGAGGTCAATAATGAGGTTATGGCTCTGGAGCCCGTTGTTGGTGCCGCTATTGCGGCACCTGTGGCGGGCCAACAAAACGTAATTGACCCCTGGATTAGAAACAATTTTGTACAAGCCCCTGGTGGAGAGTTCACAGTGTCCCCTAGAAACGCTCCAGGTGAGATACTGTGGAGCGCGCCCTTGGGCCCTGATCTGAACCCCTATCTTTCTCATTTGTCCAGAATGTACAATGGTTATGCAGGTGGTTTTGAAGTGCAGGTAATCCTTGCGGGGAACGCGTTCACCGCCGGGAAAA

FTO TP_F7 (SEQ ID NO: 224) CTAAATTCACTGCATTTAGAATGTCTGAATTA
FTO TP_R5 (SEQ ID NO: 230) AGTGATGCACCTGACTGTTACCTATTAAAACT
FTO template 0 (SEQ ID NO: 420)
TTATGAGATAATGTCCTTTTTAAAAATAAACACTAACATCAGTTATGCATTTAGAATGTCTGAATTATTATTCTAGGTTCCTTGCGACTGCTGTGAATTTAGTGATGCACTTGGATAGTCTCTGTTACTCTAAAGTTTTAATAGGTAACAGTCAGAAATGGAGTGGGAGAGCATAAAAGCAAACTGAAATGCAAATAGCTG

FTO TP_F7 (SEQ ID NO: 224) CTAAATTCACTGCATTTAGAATGTCTGAATTA
FTO TP_R6 (SEQ ID NO: 231) AGTGATGCACTCTGACTGTTACCTATTAAAAC
FTO template 0 (SEQ ID NO: 420)
TTATGAGATAATGTCCTTTTTAAAAATAAACACTAACATCAGTTATGCATTTAGAATGTCTGAATTATTATTCTAGGTTCCTTGCGACTGCTGTGAATTTAGTGATGCACTTGGATAGTCTCTGTTACTCTAAAGTTTTAATAGGTAACAGTCAGAAATGGAGTGGGAGAGCATAAAAGCAAACTGAAATGCAAATAGCTG

[Example 7]
In this example, based on the primer design method (5) and the primer production method (5), the primer (5) is designed and produced, and further nucleic acid amplification is performed using the primer (5). The amplification efficiency was confirmed. That is, in this example, the influence of the number of TPs hybridizing to a place other than the originally designed place on the amplification reaction was examined. In addition, paying attention to the base sequence at the 3 ′ end of TP, it was decided to evaluate how many sequences complementary to the sequence appear in the sequence of the region sandwiched by TP (sequence to be calculated). Here, the sequence of the region sandwiched between the TPs was defined in this example as the sequence of the region sandwiched between the TP of the sense strand and the TP of the antisense strand as shown in the figure below.

Preparation of template plasmid The template plasmid was prepared in the same manner as in Example 3 (Primer (1)).

Nucleic acid amplification reaction The nucleic acid amplification reaction was performed in the same manner as in Example 3 (the primer (1)).

Nucleic acid amplification reaction evaluation method First, the length of the 3 ′ end sequence of the TP of interest (both the TP of the sense strand and the TP of the antisense strand) is set to 4 to 8 bases from the 3 ′ side. The number of complementary sequences in the sequence was calculated in the sequence of the calculation target sequence (both sense strand and antisense strand). That is, for example, for a 6-base sequence from the 3 ′ side of the TP of a certain sense strand, a sequence complementary to that sequence (a sequence that may hybridize) is the sense strand and antisense strand of the sequence to be calculated. I counted how many in total.
Next, for each TP pair used, the reaction time of the sample containing the template nucleic acid was within 45 minutes, and the reaction time of the sample containing the template nucleic acid and the reaction time of the sample not containing the template nucleic acid (negative control) Those with a difference of 30 minutes or more were judged as “success”, and those other than that were judged as “failure”. In addition, although amplification reaction is performed by the pair of Forward TP and Reverse TP, in order to evaluate each TP, the success / failure of one TP pair was counted for each Forward TP and Reverse TP constituting the TP. However, when investigating the relationship between the total number of complementary sequences of each TP pair and success / failure, only one success / failure of one TP pair was counted.

<Result>
As shown in Table 6 below, when 6 bases from the 3 ′ end of TP are used as the target sequence, the success rate when the total number of complementary sequences counted for each TP of the TP pair is 0 is 4 4 cases (100%) compared to 0 cases / 4 cases (0%) in the case of 1 case, which is remarkable when the total number of complementary sequences is 0 and 1 or more. The difference was seen.
On the other hand, although data are not shown, when the number of sequences of interest was 5 bases or less, the effect of reducing the total number of complementary sequences to 0 was not noticeable. As a matter of course, when the number of target sequences is 7 bases or more, the same effect as in the case of 6 bases was observed.
Based on the above, when 6 or more bases from the 3 ′ end of Forward TP or Reverse TP are selected as the target sequence, and no complementary sequence of the target sequence exists on the sequence between the TPs. In addition, it was revealed that the amplification rate and specificity were excellent.

（表中「−」は９０分以内で反応が見られなかったことを示す。）

("-" In the table indicates that no reaction was observed within 90 minutes.)

The base sequence and target nucleic acid sequence of TP used in Table 6 above are as follows.

G2 fTP4-7 (SEQ ID NO: 107) CCGCCACAGGTGTATGGCTCTGGAGCCCGT
G2 rTP4-6 (SEQ ID NO: 114) AAAACGTAATTGACCAGGGGCTTGTACAAA
norovirus G2 4 template 0 (SEQ ID NO: 422)
CATTCTACAGCAAAATTAGCAAGTTAGTTATTGCAGAGCTAAAAGAAGGTGGCATGGATTTTTACGTGCCCAGACAAGAGCCAATGTTCAGATGGATGAGATTCTCAGATCTGAGCACGTGGGAGGGCGATCGCAATCTGGCTCCCAGCTTTGTGAATGAAGATGGCGTCGAATGACGCCAACCCATCTGATGGGTCCGCAGCCAACCTCGTCCCAGAGGTCAATAATGAGGTTATGGCTCTGGAGCCCGTTGTTGGTGCCGCTATTGCGGCACCTGTGGCGGGCCAACAAAACGTAATTGACCCCTGGATTAGAAACAATTTTGTACAAGCCCCTGGTGGAGAGTTCACAGTGTCCCCTAGAAACGCTCCAGGTGAGATACTGTGGAGCGCGCCCTTGGGCCCTGATCTGAACCCCTATCTTTCTCATTTGTCCAGAATGTACAATGGTTATGCAGGTGGTTTTGAAGTGCAGGTAATCCTTGCGGGGAACGCGTTCACCGCCGGGAAAA

G2 fTP4-7 (SEQ ID NO: 107) CCGCCACAGGTGTATGGCTCTGGAGCCCGT
G2 rTP4-8 (SEQ ID NO: 116) AACGTAATTGACCCACCAGGGGCTTGTACA
norovirus G2 4 template 0 (SEQ ID NO: 422)
CATTCTACAGCAAAATTAGCAAGTTAGTTATTGCAGAGCTAAAAGAAGGTGGCATGGATTTTTACGTGCCCAGACAAGAGCCAATGTTCAGATGGATGAGATTCTCAGATCTGAGCACGTGGGAGGGCGATCGCAATCTGGCTCCCAGCTTTGTGAATGAAGATGGCGTCGAATGACGCCAACCCATCTGATGGGTCCGCAGCCAACCTCGTCCCAGAGGTCAATAATGAGGTTATGGCTCTGGAGCCCGTTGTTGGTGCCGCTATTGCGGCACCTGTGGCGGGCCAACAAAACGTAATTGACCCCTGGATTAGAAACAATTTTGTACAAGCCCCTGGTGGAGAGTTCACAGTGTCCCCTAGAAACGCTCCAGGTGAGATACTGTGGAGCGCGCCCTTGGGCCCTGATCTGAACCCCTATCTTTCTCATTTGTCCAGAATGTACAATGGTTATGCAGGTGGTTTTGAAGTGCAGGTAATCCTTGCGGGGAACGCGTTCACCGCCGGGAAAA

G2 fTP4-8 (SEQ ID NO: 108) CCCGCCACAGGTATGGCTCTGGAGCCCGTT
G2 rTP4-6 (SEQ ID NO: 114) AAAACGTAATTGACCAGGGGCTTGTACAAA
norovirus G2 4 template 0 (SEQ ID NO: 422)
CATTCTACAGCAAAATTAGCAAGTTAGTTATTGCAGAGCTAAAAGAAGGTGGCATGGATTTTTACGTGCCCAGACAAGAGCCAATGTTCAGATGGATGAGATTCTCAGATCTGAGCACGTGGGAGGGCGATCGCAATCTGGCTCCCAGCTTTGTGAATGAAGATGGCGTCGAATGACGCCAACCCATCTGATGGGTCCGCAGCCAACCTCGTCCCAGAGGTCAATAATGAGGTTATGGCTCTGGAGCCCGTTGTTGGTGCCGCTATTGCGGCACCTGTGGCGGGCCAACAAAACGTAATTGACCCCTGGATTAGAAACAATTTTGTACAAGCCCCTGGTGGAGAGTTCACAGTGTCCCCTAGAAACGCTCCAGGTGAGATACTGTGGAGCGCGCCCTTGGGCCCTGATCTGAACCCCTATCTTTCTCATTTGTCCAGAATGTACAATGGTTATGCAGGTGGTTTTGAAGTGCAGGTAATCCTTGCGGGGAACGCGTTCACCGCCGGGAAAA

G2 fTP4-8 (SEQ ID NO: 108) CCCGCCACAGGTATGGCTCTGGAGCCCGTT
G2 rTP4-8 (SEQ ID NO: 116) AACGTAATTGACCCACCAGGGGCTTGTACA
norovirus G2 4 template 0 (SEQ ID NO: 422)
CATTCTACAGCAAAATTAGCAAGTTAGTTATTGCAGAGCTAAAAGAAGGTGGCATGGATTTTTACGTGCCCAGACAAGAGCCAATGTTCAGATGGATGAGATTCTCAGATCTGAGCACGTGGGAGGGCGATCGCAATCTGGCTCCCAGCTTTGTGAATGAAGATGGCGTCGAATGACGCCAACCCATCTGATGGGTCCGCAGCCAACCTCGTCCCAGAGGTCAATAATGAGGTTATGGCTCTGGAGCCCGTTGTTGGTGCCGCTATTGCGGCACCTGTGGCGGGCCAACAAAACGTAATTGACCCCTGGATTAGAAACAATTTTGTACAAGCCCCTGGTGGAGAGTTCACAGTGTCCCCTAGAAACGCTCCAGGTGAGATACTGTGGAGCGCGCCCTTGGGCCCTGATCTGAACCCCTATCTTTCTCATTTGTCCAGAATGTACAATGGTTATGCAGGTGGTTTTGAAGTGCAGGTAATCCTTGCGGGGAACGCGTTCACCGCCGGGAAAA

G2 fTP6-8 (SEQ ID NO: 140) GGGCCCAAGGGCTCCCCTAGAAACGCTCCA
G2 rTP6-3 (SEQ ID NO: 143) ATCTGAACCCCTTGCATAACCATTGTACAT
norovirus G2 6 template 0 (SEQ ID NO: 431)
CATTCTACAGCAAAATTAGCAAGTTAGTTATTGCAGAGCTAAAAGAAGGTGGCATGGATTTTTACGTGCCCAGACAAGAGCCAATGTTCAGATGGATGAGATTCTCAGATCTGAGCACGTGGGAGGGCGATCGCAATCTGGCTCCCAGCTTTGTGAATGAAGATGGCGTCGAATGACGCCAACCCATCTGATGGGTCCGCAGCCAACCTCGTCCCAGAGGTCAATAATGAGGTTATGGCTCTGGAGCCCGTTGTTGGTGCCGCTATTGCGGCACCTGTGGCGGGCCAACAAAACGTAATTGACCCCTGGATTAGAAACAATTTTGTACAAGCCCCTGGTGGAGAGTTCACAGTGTCCCCTAGAAACGCTCCAGGTGAGATACTGTGGAGCGCGCCCTTGGGCCCTGATCTGAACCCCTATCTTTCTCATTTGTCCAGAATGTACAATGGTTATGCAGGTGGTTTTGAAGTGCAGGTAATCCTTGCGGGGAACGCGTTCACCGCCGGGAAAA

G2 fTP6-8 (SEQ ID NO: 140) GGGCCCAAGGGCTCCCCTAGAAACGCTCCA
G2 rTP6-4 (SEQ ID NO: 144) TCTGAACCCCTACTGCATAACCATTGTACA
norovirus G2 6 template 0 (SEQ ID NO: 431)
CATTCTACAGCAAAATTAGCAAGTTAGTTATTGCAGAGCTAAAAGAAGGTGGCATGGATTTTTACGTGCCCAGACAAGAGCCAATGTTCAGATGGATGAGATTCTCAGATCTGAGCACGTGGGAGGGCGATCGCAATCTGGCTCCCAGCTTTGTGAATGAAGATGGCGTCGAATGACGCCAACCCATCTGATGGGTCCGCAGCCAACCTCGTCCCAGAGGTCAATAATGAGGTTATGGCTCTGGAGCCCGTTGTTGGTGCCGCTATTGCGGCACCTGTGGCGGGCCAACAAAACGTAATTGACCCCTGGATTAGAAACAATTTTGTACAAGCCCCTGGTGGAGAGTTCACAGTGTCCCCTAGAAACGCTCCAGGTGAGATACTGTGGAGCGCGCCCTTGGGCCCTGATCTGAACCCCTATCTTTCTCATTTGTCCAGAATGTACAATGGTTATGCAGGTGGTTTTGAAGTGCAGGTAATCCTTGCGGGGAACGCGTTCACCGCCGGGAAAA

G2 fTP6-7 (SEQ ID NO: 139) GGCCCAAGGGCGGTCCCCTAGAAACGCTCC
G2 rTP6-2 (SEQ ID NO: 142) GATCTGAACCCCGCATAACCATTGTACATT
norovirus G2 6 template 0 (SEQ ID NO: 431)
CATTCTACAGCAAAATTAGCAAGTTAGTTATTGCAGAGCTAAAAGAAGGTGGCATGGATTTTTACGTGCCCAGACAAGAGCCAATGTTCAGATGGATGAGATTCTCAGATCTGAGCACGTGGGAGGGCGATCGCAATCTGGCTCCCAGCTTTGTGAATGAAGATGGCGTCGAATGACGCCAACCCATCTGATGGGTCCGCAGCCAACCTCGTCCCAGAGGTCAATAATGAGGTTATGGCTCTGGAGCCCGTTGTTGGTGCCGCTATTGCGGCACCTGTGGCGGGCCAACAAAACGTAATTGACCCCTGGATTAGAAACAATTTTGTACAAGCCCCTGGTGGAGAGTTCACAGTGTCCCCTAGAAACGCTCCAGGTGAGATACTGTGGAGCGCGCCCTTGGGCCCTGATCTGAACCCCTATCTTTCTCATTTGTCCAGAATGTACAATGGTTATGCAGGTGGTTTTGAAGTGCAGGTAATCCTTGCGGGGAACGCGTTCACCGCCGGGAAAA

G2 fTP5-8 (SEQ ID NO: 124) GGGCTTGTACAACCAACAAAACGTAATTGA
G2 rTP5-3 (SEQ ID NO: 127) GGTGGAGAGTTCCACAGTATCTCACCTGGA
norovirus G2 5 template 0 (SEQ ID NO: 434)
CATTCTACAGCAAAATTAGCAAGTTAGTTATTGCAGAGCTAAAAGAAGGTGGCATGGATTTTTACGTGCCCAGACAAGAGCCAATGTTCAGATGGATGAGATTCTCAGATCTGAGCACGTGGGAGGGCGATCGCAATCTGGCTCCCAGCTTTGTGAATGAAGATGGCGTCGAATGACGCCAACCCATCTGATGGGTCCGCAGCCAACCTCGTCCCAGAGGTCAATAATGAGGTTATGGCTCTGGAGCCCGTTGTTGGTGCCGCTATTGCGGCACCTGTGGCGGGCCAACAAAACGTAATTGACCCCTGGATTAGAAACAATTTTGTACAAGCCCCTGGTGGAGAGTTCACAGTGTCCCCTAGAAACGCTCCAGGTGAGATACTGTGGAGCGCGCCCTTGGGCCCTGATCTGAACCCCTATCTTTCTCATTTGTCCAGAATGTACAATGGTTATGCAGGTGGTTTTGAAGTGCAGGTAATCCTTGCGGGGAACGCGTTCACCGCCGGGAAAA

[Example 8]
In this example, based on the primer design method (6) and the primer production method (6), the primer (6) is designed and produced, and further nucleic acid amplification is performed using the primer (6). The amplification efficiency of the target nucleic acid sequence was confirmed.

[1] Dimer (homodimer) between one type of TP
The influence of the number of 3 ′ ends of TP hybridized to the same TP on the amplification reaction was examined. Therefore, attention was paid to the nucleotide sequence at the 3 ′ end of TP, and the number of sequences complementary to it was evaluated in the same TP sequence.

[2] Dimer (heterodimer) between two types of TPs (Forward, Reverse)
The influence of the number of hybridizing the 3 ′ end of one TP to the opposite TP on the amplification reaction was examined. Therefore, attention was paid to the nucleotide sequence at the 3 ′ end of TP, and the number of sequences complementary to the nucleotide sequence appeared in the opposite TP sequence.

Preparation of template plasmid The template plasmid was prepared in the same manner as in Example 3 (Primer (1)).

Nucleic acid amplification reaction The nucleic acid amplification reaction was performed in the same manner as in Example 3 (the primer (1)).

Nucleic acid amplification reaction evaluation method First, the length of the 3 ′ end sequence of the target TP is set to 2 to 6 bases from the 3 ′ side, and the complementary sequence of each target sequence is the same TP sequence or the opposite TP. We counted how many in the array.
Next, the reaction time of the sample containing the template nucleic acid for each TP used was within 45 minutes, and the difference between the reaction time of the sample containing the template nucleic acid and the reaction time of the sample not containing the template nucleic acid (negative control) Were determined to be “success” and those other than “failure”. In addition, although amplification reaction is performed by the pair of Forward TP and Reverse TP, in order to evaluate each TP, the success / failure of one TP pair was counted for each Forward TP and Reverse TP constituting the TP. However, when investigating the relationship between the total number of complementary sequences of each TP pair and success / failure, only one success / failure of one TP pair was counted.

<Result>
[1] Dimer (homodimer) between one type of TP
As shown in Table 7 below, when 3 bases from the 3 ′ end of TP were used as the target sequence, the success rate when the number of complementary sequences was 0 was 8 cases / 8 cases (100%). On the other hand, when the number of complementary sequences was one, there were 0 cases / 8 cases (0%), and a remarkable difference was observed between the case where no complementary sequences existed and the case where they existed.
On the other hand, although not shown in the data, when 2 bases from the 3 ′ end of TP were used as the target sequence, the effect of preventing the presence of complementary sequences was not noticeable. Of course, when 4 or more bases from the 3 ′ end of TP were used as the target sequence, the same effect as in the case of 3 bases was observed.
From the above, when 3 or more bases from the 3 ′ end of TP are selected as a target sequence, and no complementary sequence of the target sequence exists in the same TP sequence, excellent amplification speed and specificity can be obtained. It became clear to show.

（表中「−」は９０分以内で反応が見られなかったことを示す。）

("-" In the table indicates that no reaction was observed within 90 minutes.)

In addition, the base sequence and target nucleic acid sequence of TP used in Table 7 are as follows.

NAT2-590 TP_F6 (SEQ ID NO: 323) TCGAGGTTCAATCTCCTGCCAAAGAAGAAACA
NAT2-590 TP_R1 (SEQ ID NO: 326) CGAACAATTGTGGAGACGTCTGCAGGTATGTA
NAT2-590 template 0 (sequence number 430)
ATCAGGAGAGAGCAGTATATTACAAACAAAGAATTTCTTAATTCTCATCTCCTGCCAAAGAAGAAACACCAAAAAATATACTTATTTACGCTTGAACCTCGAACAATTGAAGATTTTGAGTCTATGAATACATACCTGCAGACGTCTCCAACATCTTCATTTATAACCACATCATTTTGTTCCTTGCAGACCCCAGAAGGG

NAT2-590 TP_F6 (SEQ ID NO: 323) TCGAGGTTCAATCTCCTGCCAAAGAAGAAACA
NAT2-590 TP_R3 (SEQ ID NO: 328) CGAACAATTGGTTGGAGACGTCTGCAGGTATG
NAT2-590 template 0 (sequence number 430)
ATCAGGAGAGAGCAGTATATTACAAACAAAGAATTTCTTAATTCTCATCTCCTGCCAAAGAAGAAACACCAAAAAATATACTTATTTACGCTTGAACCTCGAACAATTGAAGATTTTGAGTCTATGAATACATACCTGCAGACGTCTCCAACATCTTCATTTATAACCACATCATTTTGTTCCTTGCAGACCCCAGAAGGG

NAT2-590 TP_F1 (SEQ ID NO: 318) TCGAGGTTCACTGCCAAAGAAGAAACACCAAA
NAT2-590 TP_R5 (SEQ ID NO: 330) CGAACAATTGATGTTGGAGACGTCTGCAGGTA
NAT2-590 template 0 (sequence number 430)
ATCAGGAGAGAGCAGTATATTACAAACAAAGAATTTCTTAATTCTCATCTCCTGCCAAAGAAGAAACACCAAAAAATATACTTATTTACGCTTGAACCTCGAACAATTGAAGATTTTGAGTCTATGAATACATACCTGCAGACGTCTCCAACATCTTCATTTATAACCACATCATTTTGTTCCTTGCAGACCCCAGAAGGG

NAT2-590 TP_F3 (SEQ ID NO: 320) TCGAGGTTCATCCTGCCAAAGAAGAAACACCA
NAT2-590 TP_R4 (SEQ ID NO: 329) CGAACAATTGTGTTGGAGACGTCTGCAGGTAT
NAT2-590 template 0 (sequence number 430)
ATCAGGAGAGAGCAGTATATTACAAACAAAGAATTTCTTAATTCTCATCTCCTGCCAAAGAAGAAACACCAAAAAATATACTTATTTACGCTTGAACCTCGAACAATTGAAGATTTTGAGTCTATGAATACATACCTGCAGACGTCTCCAACATCTTCATTTATAACCACATCATTTTGTTCCTTGCAGACCCCAGAAGGG

NAT2-590 TP_F4 (SEQ ID NO: 321) TCGAGGTTCACTCCTGCCAAAGAAGAAACACC
NAT2-590 TP_R2 (SEQ ID NO: 327) CGAACAATTGTTGGAGACGTCTGCAGGTATGT
NAT2-590 template 0 (sequence number 430)
ATCAGGAGAGAGCAGTATATTACAAACAAAGAATTTCTTAATTCTCATCTCCTGCCAAAGAAGAAACACCAAAAAATATACTTATTTACGCTTGAACCTCGAACAATTGAAGATTTTGAGTCTATGAATACATACCTGCAGACGTCTCCAACATCTTCATTTATAACCACATCATTTTGTTCCTTGCAGACCCCAGAAGGG

NAT2-590 TP_F4 (SEQ ID NO: 321) TCGAGGTTCACTCCTGCCAAAGAAGAAACACC
NAT2-590 TP_R8 (SEQ ID NO: 333) CGAACAATTGAAGATGTTGGAGACGTCTGCAG
NAT2-590 template 0 (sequence number 430)
ATCAGGAGAGAGCAGTATATTACAAACAAAGAATTTCTTAATTCTCATCTCCTGCCAAAGAAGAAACACCAAAAAATATACTTATTTACGCTTGAACCTCGAACAATTGAAGATTTTGAGTCTATGAATACATACCTGCAGACGTCTCCAACATCTTCATTTATAACCACATCATTTTGTTCCTTGCAGACCCCAGAAGGG

NAT2-590 TP_F7 (SEQ ID NO: 324) TCGAGGTTCACATCTCCTGCCAAAGAAGAAAC
NAT2-590 TP_R2 (SEQ ID NO: 327) CGAACAATTGTTGGAGACGTCTGCAGGTATGT
NAT2-590 template 0 (sequence number 430)
ATCAGGAGAGAGCAGTATATTACAAACAAAGAATTTCTTAATTCTCATCTCCTGCCAAAGAAGAAACACCAAAAAATATACTTATTTACGCTTGAACCTCGAACAATTGAAGATTTTGAGTCTATGAATACATACCTGCAGACGTCTCCAACATCTTCATTTATAACCACATCATTTTGTTCCTTGCAGACCCCAGAAGGG

NAT2-590 TP_F7 (SEQ ID NO: 324) TCGAGGTTCACATCTCCTGCCAAAGAAGAAAC
NAT2-590 TP_R8 (SEQ ID NO: 333) CGAACAATTGAAGATGTTGGAGACGTCTGCAG
NAT2-590 template 0 (sequence number 430)
ATCAGGAGAGAGCAGTATATTACAAACAAAGAATTTCTTAATTCTCATCTCCTGCCAAAGAAGAAACACCAAAAAATATACTTATTTACGCTTGAACCTCGAACAATTGAAGATTTTGAGTCTATGAATACATACCTGCAGACGTCTCCAACATCTTCATTTATAACCACATCATTTTGTTCCTTGCAGACCCCAGAAGGG

[2] Dimer (heterodimer) between two types of TPs (Forward, Reverse)
Table 8 below shows 5 bases from the 3 ′ end of each TP in each TP pair as the target sequence, and for each TP pair, the number of complementary sequences in which the target sequence of one TP exists in the opposite TP sequence The relationship between the total number of the target sequence of the opposite TP and the number of complementary sequences present in the one TP sequence and the success / failure is shown. When the total number of complementary sequences is 0, the success rate was 4 cases / 4 cases (100%), whereas when the number of complementary sequences is 1, 0 cases / 4 cases (0%) There was a marked difference between the absence and presence of the complementary sequence.
On the other hand, although not shown in the data, when 4 or less bases from the 3 ′ end of TP were used as the target sequence, the effect of preventing the complementary sequence from being present in the reverse TP sequence was not noticeable. . As a matter of course, when 6 or more bases from the 3 ′ end of TP were used as the target sequence, the same effect as in the case of using 5 bases was observed.
From the above, when 5 or more bases from the 3 ′ end of Forward TP or Reverse TP were selected as the target sequence, and the complementary sequence of the target sequence was not present in the reverse TP sequence, it was excellent. It became clear that the amplification rate and specificity were exhibited.

（表中「−」は９０分以内で反応が見られなかったことを示す。）

("-" In the table indicates that no reaction was observed within 90 minutes.)

The base sequence of TP and the target nucleic acid sequence used in Table 8 are as follows.

G2 fTP1-7 (SEQ ID NO: 59) GAGAATCTCATCGATTTTTACGTGCCCAGA
G2 rTP1-6 (SEQ ID NO: 66) GAGCACGTGGGACTTCATTCACAAAGCTGG
norovirus G2 1 template 0 (SEQ ID NO: 432)
CATTCTACAGCAAAATTAGCAAGTTAGTTATTGCAGAGCTAAAAGAAGGTGGCATGGATTTTTACGTGCCCAGACAAGAGCCAATGTTCAGATGGATGAGATTCTCAGATCTGAGCACGTGGGAGGGCGATCGCAATCTGGCTCCCAGCTTTGTGAATGAAGATGGCGTCGAATGACGCCAACCCATCTGATGGGTCCGCAGCCAACCTCGTCCCAGAGGTCAATAATGAGGTTATGGCTCTGGAGCCCGTTGTTGGTGCCGCTATTGCGGCACCTGTGGCGGGCCAACAAAACGTAATTGACCCCTGGATTAGAAACAATTTTGTACAAGCCCCTGGTGGAGAGTTCACAGTGTCCCCTAGAAACGCTCCAGGTGAGATACTGTGGAGCGCGCCCTTGGGCCCTGATCTGAACCCCTATCTTTCTCATTTGTCCAGAATGTACAATGGTTATGCAGGTGGTTTTGAAGTGCAGGTAATCCTTGCGGGGAACGCGTTCACCGCCGGGAAAA

G2 fTP1-7 (SEQ ID NO: 59) GAGAATCTCATCGATTTTTACGTGCCCAGA
G2 rTP1-7 (SEQ ID NO: 67) AGCACGTGGGAGTCTTCATTCACAAAGCTG
norovirus G2 1 template 0 (SEQ ID NO: 432)
CATTCTACAGCAAAATTAGCAAGTTAGTTATTGCAGAGCTAAAAGAAGGTGGCATGGATTTTTACGTGCCCAGACAAGAGCCAATGTTCAGATGGATGAGATTCTCAGATCTGAGCACGTGGGAGGGCGATCGCAATCTGGCTCCCAGCTTTGTGAATGAAGATGGCGTCGAATGACGCCAACCCATCTGATGGGTCCGCAGCCAACCTCGTCCCAGAGGTCAATAATGAGGTTATGGCTCTGGAGCCCGTTGTTGGTGCCGCTATTGCGGCACCTGTGGCGGGCCAACAAAACGTAATTGACCCCTGGATTAGAAACAATTTTGTACAAGCCCCTGGTGGAGAGTTCACAGTGTCCCCTAGAAACGCTCCAGGTGAGATACTGTGGAGCGCGCCCTTGGGCCCTGATCTGAACCCCTATCTTTCTCATTTGTCCAGAATGTACAATGGTTATGCAGGTGGTTTTGAAGTGCAGGTAATCCTTGCGGGGAACGCGTTCACCGCCGGGAAAA

G2 fTP1-7 (SEQ ID NO: 59) GAGAATCTCATCGATTTTTACGTGCCCAGA
G2 rTP1-8 (SEQ ID NO: 68) GCACGTGGGAGGATCTTCATTCACAAAGCT
norovirus G2 1 template 0 (SEQ ID NO: 432)
CATTCTACAGCAAAATTAGCAAGTTAGTTATTGCAGAGCTAAAAGAAGGTGGCATGGATTTTTACGTGCCCAGACAAGAGCCAATGTTCAGATGGATGAGATTCTCAGATCTGAGCACGTGGGAGGGCGATCGCAATCTGGCTCCCAGCTTTGTGAATGAAGATGGCGTCGAATGACGCCAACCCATCTGATGGGTCCGCAGCCAACCTCGTCCCAGAGGTCAATAATGAGGTTATGGCTCTGGAGCCCGTTGTTGGTGCCGCTATTGCGGCACCTGTGGCGGGCCAACAAAACGTAATTGACCCCTGGATTAGAAACAATTTTGTACAAGCCCCTGGTGGAGAGTTCACAGTGTCCCCTAGAAACGCTCCAGGTGAGATACTGTGGAGCGCGCCCTTGGGCCCTGATCTGAACCCCTATCTTTCTCATTTGTCCAGAATGTACAATGGTTATGCAGGTGGTTTTGAAGTGCAGGTAATCCTTGCGGGGAACGCGTTCACCGCCGGGAAAA

G2 fTP1-4 (SEQ ID NO: 56) AATCTCATCCATATGGATTTTTACGTGCCC
G2 rTP1-7 (SEQ ID NO: 67) AGCACGTGGGAGTCTTCATTCACAAAGCTG
norovirus G2 1 template 0 (SEQ ID NO: 432)
CATTCTACAGCAAAATTAGCAAGTTAGTTATTGCAGAGCTAAAAGAAGGTGGCATGGATTTTTACGTGCCCAGACAAGAGCCAATGTTCAGATGGATGAGATTCTCAGATCTGAGCACGTGGGAGGGCGATCGCAATCTGGCTCCCAGCTTTGTGAATGAAGATGGCGTCGAATGACGCCAACCCATCTGATGGGTCCGCAGCCAACCTCGTCCCAGAGGTCAATAATGAGGTTATGGCTCTGGAGCCCGTTGTTGGTGCCGCTATTGCGGCACCTGTGGCGGGCCAACAAAACGTAATTGACCCCTGGATTAGAAACAATTTTGTACAAGCCCCTGGTGGAGAGTTCACAGTGTCCCCTAGAAACGCTCCAGGTGAGATACTGTGGAGCGCGCCCTTGGGCCCTGATCTGAACCCCTATCTTTCTCATTTGTCCAGAATGTACAATGGTTATGCAGGTGGTTTTGAAGTGCAGGTAATCCTTGCGGGGAACGCGTTCACCGCCGGGAAAA

G2 fTP1-1 (SEQ ID NO: 53) CTCATCCATCTGGGCATGGATTTTTACGTG
G2 rTP1-1 (SEQ ID NO: 61) GATCTGAGCACGTTCACAAAGCTGGGAGCC
norovirus G2 1 template 0 (SEQ ID NO: 432)
CATTCTACAGCAAAATTAGCAAGTTAGTTATTGCAGAGCTAAAAGAAGGTGGCATGGATTTTTACGTGCCCAGACAAGAGCCAATGTTCAGATGGATGAGATTCTCAGATCTGAGCACGTGGGAGGGCGATCGCAATCTGGCTCCCAGCTTTGTGAATGAAGATGGCGTCGAATGACGCCAACCCATCTGATGGGTCCGCAGCCAACCTCGTCCCAGAGGTCAATAATGAGGTTATGGCTCTGGAGCCCGTTGTTGGTGCCGCTATTGCGGCACCTGTGGCGGGCCAACAAAACGTAATTGACCCCTGGATTAGAAACAATTTTGTACAAGCCCCTGGTGGAGAGTTCACAGTGTCCCCTAGAAACGCTCCAGGTGAGATACTGTGGAGCGCGCCCTTGGGCCCTGATCTGAACCCCTATCTTTCTCATTTGTCCAGAATGTACAATGGTTATGCAGGTGGTTTTGAAGTGCAGGTAATCCTTGCGGGGAACGCGTTCACCGCCGGGAAAA

G2 fTP1-1 (SEQ ID NO: 53) CTCATCCATCTGGGCATGGATTTTTACGTG
G2 rTP1-2 (SEQ ID NO: 62) ATCTGAGCACGTATTCACAAAGCTGGGAGC
norovirus G2 1 template 0 (SEQ ID NO: 432)
CATTCTACAGCAAAATTAGCAAGTTAGTTATTGCAGAGCTAAAAGAAGGTGGCATGGATTTTTACGTGCCCAGACAAGAGCCAATGTTCAGATGGATGAGATTCTCAGATCTGAGCACGTGGGAGGGCGATCGCAATCTGGCTCCCAGCTTTGTGAATGAAGATGGCGTCGAATGACGCCAACCCATCTGATGGGTCCGCAGCCAACCTCGTCCCAGAGGTCAATAATGAGGTTATGGCTCTGGAGCCCGTTGTTGGTGCCGCTATTGCGGCACCTGTGGCGGGCCAACAAAACGTAATTGACCCCTGGATTAGAAACAATTTTGTACAAGCCCCTGGTGGAGAGTTCACAGTGTCCCCTAGAAACGCTCCAGGTGAGATACTGTGGAGCGCGCCCTTGGGCCCTGATCTGAACCCCTATCTTTCTCATTTGTCCAGAATGTACAATGGTTATGCAGGTGGTTTTGAAGTGCAGGTAATCCTTGCGGGGAACGCGTTCACCGCCGGGAAAA

G2 fTP1-2 (SEQ ID NO: 54) TCTCATCCATCTGCATGGATTTTTACGTGC
G2 rTP1-7 (SEQ ID NO: 67) AGCACGTGGGAGTCTTCATTCACAAAGCTG
norovirus G2 1 template 0 (SEQ ID NO: 432)
CATTCTACAGCAAAATTAGCAAGTTAGTTATTGCAGAGCTAAAAGAAGGTGGCATGGATTTTTACGTGCCCAGACAAGAGCCAATGTTCAGATGGATGAGATTCTCAGATCTGAGCACGTGGGAGGGCGATCGCAATCTGGCTCCCAGCTTTGTGAATGAAGATGGCGTCGAATGACGCCAACCCATCTGATGGGTCCGCAGCCAACCTCGTCCCAGAGGTCAATAATGAGGTTATGGCTCTGGAGCCCGTTGTTGGTGCCGCTATTGCGGCACCTGTGGCGGGCCAACAAAACGTAATTGACCCCTGGATTAGAAACAATTTTGTACAAGCCCCTGGTGGAGAGTTCACAGTGTCCCCTAGAAACGCTCCAGGTGAGATACTGTGGAGCGCGCCCTTGGGCCCTGATCTGAACCCCTATCTTTCTCATTTGTCCAGAATGTACAATGGTTATGCAGGTGGTTTTGAAGTGCAGGTAATCCTTGCGGGGAACGCGTTCACCGCCGGGAAAA

G2 fTP1-2 (SEQ ID NO: 54) TCTCATCCATCTGCATGGATTTTTACGTGC
G2 rTP1-8 (SEQ ID NO: 68) GCACGTGGGAGGATCTTCATTCACAAAGCT
norovirus G2 1 template 0 (SEQ ID NO: 432)
CATTCTACAGCAAAATTAGCAAGTTAGTTATTGCAGAGCTAAAAGAAGGTGGCATGGATTTTTACGTGCCCAGACAAGAGCCAATGTTCAGATGGATGAGATTCTCAGATCTGAGCACGTGGGAGGGCGATCGCAATCTGGCTCCCAGCTTTGTGAATGAAGATGGCGTCGAATGACGCCAACCCATCTGATGGGTCCGCAGCCAACCTCGTCCCAGAGGTCAATAATGAGGTTATGGCTCTGGAGCCCGTTGTTGGTGCCGCTATTGCGGCACCTGTGGCGGGCCAACAAAACGTAATTGACCCCTGGATTAGAAACAATTTTGTACAAGCCCCTGGTGGAGAGTTCACAGTGTCCCCTAGAAACGCTCCAGGTGAGATACTGTGGAGCGCGCCCTTGGGCCCTGATCTGAACCCCTATCTTTCTCATTTGTCCAGAATGTACAATGGTTATGCAGGTGGTTTTGAAGTGCAGGTAATCCTTGCGGGGAACGCGTTCACCGCCGGGAAAA

[Example 9]
In this example, based on the primer design method (7) and the primer production method (7), the primer (7) is designed and produced, and further, nucleic acid amplification is performed using the primer (7). The amplification efficiency was confirmed.

  The influence of the number of hybridizing the 5 ′ end of TP to the same TP on the amplification reaction was examined. Therefore, the base sequence at the 5 ′ end of TP was focused on, and it was decided to evaluate how many complementary sequences appeared in the same TP sequence.

Preparation of template plasmid The template plasmid was prepared in the same manner as in Example 3 (Primer (1)).

Nucleic acid amplification reaction The nucleic acid amplification reaction was performed in the same manner as in Example 3 (the primer (1)).

Nucleic acid amplification reaction evaluation method First, the length of the 5 ′ end sequence of the target TP is set to 2 to 6 bases from the 5 ′ side, and how many complementary sequences of each target sequence exist in the same TP sequence I counted.
Next, the reaction time of the sample containing the template nucleic acid for each TP used was within 45 minutes, and the difference between the reaction time of the sample containing the template nucleic acid and the reaction time of the sample not containing the template nucleic acid (negative control) Were determined to be “success” and those other than “failure”. In addition, although amplification reaction is performed by the pair of Forward TP and Reverse TP, in order to evaluate each TP, the success / failure of one TP pair was counted for each Forward TP and Reverse TP constituting the TP.

<Result>
As shown in Table 9 below, when 3 bases from the 5 ′ end of TP are used as the target sequence, the success rate when the number of complementary sequences is 0 is 4 cases / 4 cases (100%). The success rate when the number is 1 was 6 cases / 9 cases (67%), while the success rate when the number of complementary sequences was 2 was 2 cases / 2 cases (100%), When the number of complementary sequences was 3, it was 0/3 cases (0%), and a remarkable difference was observed between the case where the number of complementary sequences was 2 or less and the case where 3 were present.
On the other hand, although not shown in the data, when 2 bases from the 5 ′ end of TP were used as the target sequence, the effect of reducing the number of complementary sequences to a certain number or less was not noticeable. Of course, when 4 or more bases from the 5 ′ end of TP were used as the target sequence, the same tendency as in the case of 3 bases was observed.
From the above, when 3 bases from the 5 ′ end of TP are selected as the target sequence and the number of complementary sequences of the target sequence is set to 2 or less in the same TP sequence, an excellent amplification rate / It became clear to show specificity.

（表中「−」は９０分以内で反応が見られなかったことを示す。）

("-" In the table indicates that no reaction was observed within 90 minutes.)

The base sequence and target nucleic acid sequence of TP used in Table 9 above are as follows.

FTO_2 TP_F1 (SEQ ID NO: 234) CTAAATTCACAGCAGTTATGCATTTAGAATGTCTGA
FTO_2 TP_R2 (SEQ ID NO: 243) AGTGATGCACTTTCCATTTCTGACTGTTACCTATTA
FTO_2 TP_R3 (SEQ ID NO: 244) AGTGATGCACTTCTCCATTTCTGACTGTTACCTATT
FTO_2 TP_R5 (SEQ ID NO: 246) AGTGATGCACTTCACTCCATTTCTGACTGTTACCTA
FTO_2 TP_R7 (SEQ ID NO: 248) AGTGATGCACTTCCCACTCCATTTCTGACTGTTACC
FTO_2_template (SEQ ID NO: 435)
tatcttttggcagatcagaacataatgaaaataaaataaaaaaattcaaaactggctcttgaatgaaataggattcagaagagatgatctcaaatctactTTATGAGATAATGTCCTTTTTAAAAATAAACACTAACATCAGTTATGCATTTAGAATGTCTGAATTATTATTCTAGGTTCCTTGCGACTGCTGTGAATTTAGTGATGCACTTGGATAGTCTCTGTTACTCTAAAGTTTTAATAGGTAACAGTCAGAAATGGAGTGGGAGAGCATAAAAGCAAACTGAAATGCAAATAGCTGgtaccctgaagccattaactttaagctggttattcctgacctactgtttggacataagatggtagagaggctgagtgtgacttgaacatttgttccttag

G2 fTP6-3 (SEQ ID NO: 135) CAAGGGCGCGCTCAGTGTCCCCTAGAAACG
G2 fTP6-5 (SEQ ID NO: 137) CCCAAGGGCGCGGTGTCCCCTAGAAACGCT
G2 fTP6-7 (SEQ ID NO: 139) GGCCCAAGGGCGGTCCCCTAGAAACGCTCC
G2 fTP6-8 (SEQ ID NO: 140) GGGCCCAAGGGCTCCCCTAGAAACGCTCCA
G2 rTP6-5 (SEQ ID NO: 145) CTGAACCCCTATCCTGCATAACCATTGTAC
G2 rTP6-6 (SEQ ID NO: 146) TGAACCCCTATCACCTGCATAACCATTGTA
G2 rTP6-7 (SEQ ID NO: 147) GAACCCCTATCTCACCTGCATAACCATTGT
G2_template (SEQ ID NO: 52)
GGCATGGATTTTTACGTGCCCAGACAAGAGCCAATGTTCAGATGGATGAGATTCTCAGATCTGAGCACGTGGGAGGGCGATCGCAATCTGGCTCCCAGCTTTGTGAATGAAGATGGCGTCGAATGACGCCAACCCATCTGATGGGTCCGCAGCCAACCTCGTCCCAGAGGTCAATAATGAGGTTATGGCTCTGGAGCCCGTTGTTGGTGCCGCTATTGCGGCACCTGTGGCGGGCCAACAAAACGTAATTGACCCCTGGATTAGAAACAATTTTGTACAAGCCCCTGGTGGAGAGTTCACAGTGTCCCCTAGAAACGCTCCAGGTGAGATACTGTGGAGCGCGCCCTTGGGCCCTGATCTGAACCCCTATCTTTCTCATTTGTCCAGAATGTACAATGGTTATGCAGGTGG

[Example 10]
In this example, based on the primer design method (8) and the primer production method (8), the primer (8) is designed and produced, and further, nucleic acid amplification is performed using the primer (8). The amplification efficiency was confirmed. That is, in this example, in the mechanism of action of the nucleic acid amplification reaction (FIG. 3), the site of TP hybridizes on the genomic sequence that is the target of nucleic acid amplification (FIG. 3 (a)). The influence of the probability of becoming a single strand on the amplification reaction was examined. The probability of being a single strand is determined by first determining the sequence of interest in the sequence to which TP hybridizes on the genome sequence, and then how many complementary sequences to the sequence of interest exist in the nearby genome sequence. When the number of complementary sequences is larger, the possibility of forming a stem loop structure having the target sequence portion as a stem portion increases, and as a result, there is one portion where TP hybridizes. The probability of chaining is greater. A part of FIG. 3A is shown below.

Preparation of template plasmid The template plasmid was prepared in the same manner as in Example 3 (Primer (1)).

Nucleic acid amplification reaction The nucleic acid amplification reaction was performed in the same manner as in Example 3 (the primer (1)).

Nucleic acid amplification reaction evaluation method Regarding the sequence (A) on the genomic sequence corresponding to the sequence (Ac ′) on the TP hybridized on the genomic sequence that is the target of nucleic acid amplification, from the 5 ′ end side of the sequence (A) The 4 base sequence (complementary sequence) complementary to the 4 base sequence from the 1st base to the 4th base (target sequence 1-4) is the 20 base upstream sequence of the sequence (A). It was calculated how many were present in the region of 20 bases downstream of (A) and sequence (A). However, the region of 10 bases in total, including the target sequence, 3 bases upstream thereof and 3 bases downstream thereof, was excluded from the count calculation. And the same calculation is performed for the sequences (target sequence 2-5, target sequence 3-6, target sequence 4-7, target sequence 5-8. went.

  Next, the reaction time of the sample containing the template nucleic acid for each TP used was within 45 minutes, and the difference between the reaction time of the sample containing the template nucleic acid and the reaction time of the sample not containing the template nucleic acid (negative control) Were determined to be “success” and those other than “failure”. In addition, although amplification reaction is performed by the pair of Forward TP and Reverse TP, in order to evaluate each TP, the success / failure of one TP pair was counted for each Forward TP and Reverse TP constituting the TP.

<Result>
As shown in Table 10 below, when a sequence of 4 bases from the 4th base to the 7th base counted from the 5 ′ end of the sequence (A) on the genome sequence is the target sequence (target sequence 4-7 ), The success rate when the number of complementary sequences was 1 or less was 2 cases / 6 cases (33%), whereas when the number of complementary sequences was 2 or more, 2 cases / 2 cases (100%) Thus, there was a significant difference between the case where the number of complementary sequences was 1 or less and the case where 2 or more complementary sequences were used. Similarly, in the case of the target sequence 5-8, the success rate when the number of complementary sequences is 1 or less was 2/6 cases (33%), whereas the number of complementary sequences was 2 or more. Is 2 cases / 2 cases (100%). In the case of the target sequence 6-9, the success rate when the number of complementary sequences is 1 or less was 2 cases / 6 cases (33%). When the number of complementary sequences is 2 or more, 2 cases / 2 cases (100%). In the case of the target sequence 7-10, the success rate when the number of complementary sequences is 1 or less is 2 cases / 6 cases (33 In contrast, when the number of complementary sequences is 2 or more, there are 2 cases / 2 cases (100%). In both cases, the number of complementary sequences is 1 or less and the case where 2 or more are used. Significant differences were seen.
On the other hand, the data is not shown, but as a target sequence, a 4-base sequence shifted by 1 to 5 bases before and after (target sequence 1-4, 2-5, 3-6, 8-11, 9-12) 10-13, 11-14, and 12-15), the effect of increasing the number of complementary sequences to 2 or more was not noticeable.
From the above, 4 bases from the 4th base to the 7th base out of a total of 7 bases from the 4th base to the 10th base counted from the 5 ′ end of the sequence (A) on the genome sequence as the target sequence When the number of complementary sequences of 4 bases from the 5th base to the 8th base, 4 bases from the 6th base to the 9th base, or 4 bases from the 7th base to the 10th base is 2 or more In addition, it was revealed that the amplification rate and specificity were excellent.

（表中「−」は９０分以内で反応が見られなかったことを示す。）

("-" In the table indicates that no reaction was observed within 90 minutes.)

The base sequence and target nucleic acid sequence of TP used in Table 10 above are as follows.

G2 fTP3-1 (SEQ ID NO: 85) GAGCCATAACCTTGGGTCCGCAGCCAACCT
G2 fTP3-2 (SEQ ID NO: 86) AGAGCCATAACCGGGTCCGCAGCCAACCTC
G2 fTP3-4 (SEQ ID NO: 88) CCAGAGCCATAAGTCCGCAGCCAACCTCGT
G2 fTP3-5 (SEQ ID NO: 89) TCCAGAGCCATATCCGCAGCCAACCTCGTC
G2 fTP3-6 (SEQ ID NO: 90) CTCCAGAGCCATCCGCAGCCAACCTCGTCC
G2 fTP3-7 (SEQ ID NO: 91) GCTCCAGAGCCACGCAGCCAACCTCGTCCC
G2 fTP3-8 (SEQ ID NO: 92) GGCTCCAGAGCCGCAGCCAACCTCGTCCCA
G2 rTP3-5 (SEQ ID NO: 97) GTTGGTGCCGCTTCAATTACGTTTTGTTGG
G2 rTP3-6 (SEQ ID NO: 98) TTGGTGCCGCTAGTCAATTACGTTTTGTTG
G2 rTP3-7 (SEQ ID NO: 99) TGGTGCCGCTATGGTCAATTACGTTTTGTT
G2 rTP3-8 (SEQ ID NO: 100) GGTGCCGCTATTGGGTCAATTACGTTTTGT
G2_template (SEQ ID NO: 52)
GGCATGGATTTTTACGTGCCCAGACAAGAGCCAATGTTCAGATGGATGAGATTCTCAGATCTGAGCACGTGGGAGGGCGATCGCAATCTGGCTCCCAGCTTTGTGAATGAAGATGGCGTCGAATGACGCCAACCCATCTGATGGGTCCGCAGCCAACCTCGTCCCAGAGGTCAATAATGAGGTTATGGCTCTGGAGCCCGTTGTTGGTGCCGCTATTGCGGCACCTGTGGCGGGCCAACAAAACGTAATTGACCCCTGGATTAGAAACAATTTTGTACAAGCCCCTGGTGGAGAGTTCACAGTGTCCCCTAGAAACGCTCCAGGTGAGATACTGTGGAGCGCGCCCTTGGGCCCTGATCTGAACCCCTATCTTTCTCATTTGTCCAGAATGTACAATGGTTATGCAGGTGG

KCNQ1_rs2237897 TP-F1 (SEQ ID NO: 402) CGTCCCCAGCTCCCTAGGGACGGAGGTGGCCAGAG
KCNQ1_rs2237897 TP-F2 (SEQ ID NO: 403) CGTCCCCAGCTCCCTGGGACGGAGGTGGCCAGAGG
KCNQ1_rs2237897 TP-F8 (SEQ ID NO: 409) CGTCCCCAGCTCCCTGAGGTGGCCAGAGGCCTCTC
KCNQ1_rs2237897 TP-R1 (SEQ ID NO: 410) ACGAGGGGCCTCACCCTGGGCCTGAAGGGAGGG
KCNQ1_rs2237897 TP-R2 (SEQ ID NO: 411) ACGAGGGGCCTCATCCCTGGGCCTGAAGGGAGG
KCNQ1_rs2237897 TP-R3 (SEQ ID NO: 412) ACGAGGGGCCTCACTCCCTGGGCCTGAAGGGAG
KCNQ1_rs2237897 TP-R6 (SEQ ID NO: 415) ACGAGGGGCCTCACCACTCCCTGGGCCTGAAGG
KCNQ1_rs2237897 TP-R7 (SEQ ID NO: 416) ACGAGGGGCCTCAGCCACTCCCTGGGCCTGAAG
KCNQ1_rs2237897_template (SEQ ID NO: 436)
catggtgtgcgtgtttgggagcaccctgggcagcctctcgagtgtccagagagaggacaccgtcttgagccagcagggacagccaagagcgcccggggagAGGCCTGGGTGGAGACAGAACACAGCAGGCTGTGGGCGTGGCCAATGTTGAGGGACGGAGGTGGCCAGAGGCCTCTCAGTGGTGCCCAGGGAGCTGGGGACGAGGGGCCTCATCCTTCCCCTGAGCCCCTCCCTTCAGGCCCAGGGAGTGGCCTGGGGCCCAGGAGAGCCCGCTCCGCGCTCACAGCCTCCGTTCCCAGACacgcccgggcctgagcccccaggctgcactgtgccacagaacagactccccggcccctccctgctcgagaggtgaccaaggcccagggtctcccagcgag

[Example 11]
In this example, based on the primer design method (9) and the primer production method (9), the primer (9) is designed and produced, and further nucleic acid amplification is performed using the primer (9). The amplification efficiency was confirmed. That is, in this example, in the mechanism of action of the nucleic acid amplification reaction (FIG. 3), TP hybridizes on the genomic sequence that is the target of nucleic acid amplification (FIG. 3 (a)), and then the extended chain. The effect of the probability that the region turning back on (FIG. 3B) is a single strand on the amplification reaction was examined. The probability of becoming a single strand is as follows: First, the target sequence is determined in the sequence where TP turns back on the genomic sequence, and then there are several complementary sequences to the target sequence in the neighboring genomic sequence. When the number of complementary sequences is larger, the possibility of forming a stem loop structure having the target sequence portion as a stem portion increases, and as a result, one region where the TP turns back is obtained. The probability of chaining is greater. A part of FIG. 3B is shown below.

Preparation of template plasmid The template plasmid was prepared in the same manner as in Example 3 (Primer (1)).

Nucleic acid amplification reaction The nucleic acid amplification reaction was performed in the same manner as in Example 3 (the primer (1)).

Nucleic Acid Amplification Reaction Evaluation Method When TP is hybridized on a genomic sequence targeted for nucleic acid amplification and then turned back on the extended strand, the sequence (B) on the genomic sequence corresponding to the turnback region, A 4 base reverse sequence (complementary sequence) complementary to the 4 base sequence (target sequence 1-4) from the 3 'end of sequence (B) is counted upstream from the 4 base 5' end. Number of 20 bases from the 4th base to the 23rd base of the sequence and 20 bases from the 4th base to the 23rd base of the sequence counted downstream from the 3 ′ end of the 4 bases Was calculated. In other words, the region of 10 bases in total, including the target sequence, 3 bases upstream and 3 bases downstream thereof, was excluded from the number calculation. The same calculation was performed for the sequences (target sequence 2-5, target sequence 3-6, target sequence 4-7,...) In which the target sequence was further shifted by one base to the 5 ′ side.

  Next, the reaction time of the sample containing the template nucleic acid for each TP used was within 45 minutes, and the difference between the reaction time of the sample containing the template nucleic acid and the reaction time of the sample not containing the template nucleic acid (negative control) Were determined to be “success” and those other than “failure”. In addition, although amplification reaction is performed by the pair of Forward TP and Reverse TP, in order to evaluate each TP, the success / failure of one TP pair was counted for each Forward TP and Reverse TP constituting the TP.

<Result>
As shown in Table 11 below, when the sequence of 4 bases from the 3 ′ end of the sequence (B) on the genome sequence is the target sequence (target sequence 1-4), the number of complementary sequences is 0. The success rate was 0 cases / 4 cases (0%), whereas when the number of complementary sequences was 1 or more, it was 6 cases / 8 cases (75%). There was a marked difference from the case of more than one.
On the other hand, although data is not shown, if a 4 base sequence (target sequence 2-5, 3-6, 4-7) shifted by 1 to 3 bases on the 5 'side is selected as the target sequence, it is complementary. The effect of setting the number of sequences to 1 or more was not noticeable.
From the above, as the target sequence, the sequence complementary to the 3 ′ end or 4 bases of the sequence (B) on the genome sequence is from the 4th base of the sequence counted upstream from the 5 ′ end of the 4 bases. Excellent amplification when there are 1 or more of 20 bases up to the 23rd base and 20 bases from the 4th base to the 23rd base in the sequence counted downstream from the 3 ′ end of the 4 bases It became clear to show speed and specificity.

（表中「−」は９０分以内で反応が見られなかったことを示す。）

("-" In the table indicates that no reaction was observed within 90 minutes.)

The base sequence of TP and the target nucleic acid sequence used in Table 11 are as follows.

G2 fTP2-2 (SEQ ID NO: 70) GTCATTCGACGCCGATCGCAATCTGGCTCC
G2 rTP2-1 (SEQ ID NO: 69) CCATCTGATGGGACCTCATTATTGACCTCT
norovirus G2 1 template 0 (SEQ ID NO: 432)
CATTCTACAGCAAAATTAGCAAGTTAGTTATTGCAGAGCTAAAAGAAGGTGGCATGGATTTTTACGTGCCCAGACAAGAGCCAATGTTCAGATGGATGAGATTCTCAGATCTGAGCACGTGGGAGGGCGATCGCAATCTGGCTCCCAGCTTTGTGAATGAAGATGGCGTCGAATGACGCCAACCCATCTGATGGGTCCGCAGCCAACCTCGTCCCAGAGGTCAATAATGAGGTTATGGCTCTGGAGCCCGTTGTTGGTGCCGCTATTGCGGCACCTGTGGCGGGCCAACAAAACGTAATTGACCCCTGGATTAGAAACAATTTTGTACAAGCCCCTGGTGGAGAGTTCACAGTGTCCCCTAGAAACGCTCCAGGTGAGATACTGTGGAGCGCGCCCTTGGGCCCTGATCTGAACCCCTATCTTTCTCATTTGTCCAGAATGTACAATGGTTATGCAGGTGGTTTTGAAGTGCAGGTAATCCTTGCGGGGAACGCGTTCACCGCCGGGAAAA

ABCG2 TP_F3 (SEQ ID NO: 169) TGTAAGTTTTCTTAAGGATGATGTTGTGA
ABCG2 TP_F6 (SEQ ID NO: 172) TGTAAGTTTTTGCCTTAAGGATGATGTTG
ABCG2 TP_R3 (SEQ ID NO: 177) ACAGTTCTCATCATGATTCGTCATAGTTG
ABCG2_template (SEQ ID NO: 437)
ggagttaactgtcatttgcagaactgcaggttcatcattagctagaactttaccttagttatgttatctttgtggattatgttatgtatactaaacagtcATGGTCTTAGAAAAGACTCATTATCATTATGTCTCATTAAAATGCTATTTGCCTTAAGGATGATGTTGTGATGGGCACTCTGACGGTGAGAGAAAACTTACAGTTCTCAGCAGCTCTTCGGCTTGCAACAACTATGACGAATCATGAAAAAAACGAACGGATTAACAGGGTCATTCAAGAGTTAGGTCTGGATAAAGTGGCagactccaaggtaatgtggaaaaactgaaagcatcatgatcagcataagtaggactttccctgtgtggataaaatgactctgattcagaagtttcctgta

G2 fTP4-3 (SEQ ID NO: 103) CACAGGTGCCGCAGGTTATGGCTCTGGAGC
G2 fTP4-6 (SEQ ID NO: 106) CGCCACAGGTGCTTATGGCTCTGGAGCCCG
G2 rTP4-4 (SEQ ID NO: 112) ACAAAACGTAATCAGGGGCTTGTACAAAAT
G2 rTP4-7 (SEQ ID NO: 115) AAACGTAATTGACACCAGGGGCTTGTACAA
G2_template (SEQ ID NO: 52)
GGCATGGATTTTTACGTGCCCAGACAAGAGCCAATGTTCAGATGGATGAGATTCTCAGATCTGAGCACGTGGGAGGGCGATCGCAATCTGGCTCCCAGCTTTGTGAATGAAGATGGCGTCGAATGACGCCAACCCATCTGATGGGTCCGCAGCCAACCTCGTCCCAGAGGTCAATAATGAGGTTATGGCTCTGGAGCCCGTTGTTGGTGCCGCTATTGCGGCACCTGTGGCGGGCCAACAAAACGTAATTGACCCCTGGATTAGAAACAATTTTGTACAAGCCCCTGGTGGAGAGTTCACAGTGTCCCCTAGAAACGCTCCAGGTGAGATACTGTGGAGCGCGCCCTTGGGCCCTGATCTGAACCCCTATCTTTCTCATTTGTCCAGAATGTACAATGGTTATGCAGGTGG

[Example 12]
In this example, based on the primer design method (10) and the primer production method (10), the primer (10) is designed and produced, and further, nucleic acid amplification is performed using the primer (10). The amplification efficiency was confirmed.

  The influence of the number of arbitrary 5 consecutive bases contained in the region (Ac ′) in the TP that hybridizes to the target nucleic acid sequence on the same TP on the amplification reaction was examined. Therefore, attention was paid to the base sequence shifted by 1 base at a length of 5 bases from the 3 ′ end of TP, and the number of sequences complementary thereto was evaluated in the same TP sequence.

Preparation of template plasmid The template plasmid was prepared in the same manner as in Example 3 (Primer (1)).

Nucleic acid amplification reaction The nucleic acid amplification reaction was performed in the same manner as in Example 3 (the primer (1)).

Nucleic acid amplification reaction evaluation method First, the base sequence of interest is shifted from the 3 ′ end of the TP by 5 bases in length by one base (target sequences 1-5, 2-6, 3-7, 4-8, ...), the number of complementary sequences of each target sequence was counted in the same TP sequence.
Next, the reaction time of the sample containing the template nucleic acid for each TP used was within 45 minutes, and the difference between the reaction time of the sample containing the template nucleic acid and the reaction time of the sample not containing the template nucleic acid (negative control) Were determined to be “success” and those other than “failure”. In addition, although amplification reaction is performed by the pair of Forward TP and Reverse TP, in order to evaluate each TP, the success / failure of one TP pair was counted for each Forward TP and Reverse TP constituting the TP.

<Result>
As shown in Table 12 below, when a sequence of 5 bases from the 6th base to the 10th base counted from the 3 ′ end side of TP is used as a target sequence (target sequence 6-10), the number of complementary sequences is The success rate in the case of 0 was 6 cases / 9 cases (67%), whereas in the case where the number of complementary sequences was 1 or more, it was 0 cases / 3 cases (0%). There was a marked difference between the case of zero and the case of one or more. Similarly, in the case of the target sequence 7-11, the target sequence 8-12, and the target sequence 9-13, the success rate when the number of complementary sequences is 0 is 6 cases / 9 cases (67%), respectively. 6 cases / 9 cases (67%) and 6 cases / 9 cases (67%), whereas when the number of complementary sequences is 1 or more, 0 cases / 3 cases (0%), respectively. 0 cases / 3 cases (0%), 0 cases / 3 cases (0%), and there was a significant difference between the case where the number of complementary sequences was 0 and the case where there were 1 or more complementary sequences.
On the other hand, the data is not shown, but in the case of the target sequence 1-5, 2-6, 3-7, 4-8, 5-9, 10-14, 11-15, The effect of reducing the number of complementary sequences to zero was not noticeable.
From the above, it is assumed that there is no sequence complementary to the sequence of any 5 bases consecutive among 8 bases from the 6th base to the 13th base counted from the 3 ′ end of TP in the same TP sequence. In this case, it was revealed that the amplification rate and specificity were excellent.

（表中「−」は９０分以内で反応が見られなかったことを示す。）

("-" In the table indicates that no reaction was observed within 90 minutes.)

In addition, the base sequence and target nucleic acid sequence of TP used in Table 12 are as follows.

B3 TP-F5 (SEQ ID NO: 23) CAGGCGATGCGGTGCTGGCCACCGTGGGA
B3 TP-F6 (SEQ ID NO: 24) CAGGCGATGGCGGTGCTGGCCACCGTGGG
B3 TP-R4 (SEQ ID NO: 30) CTGGACTCCGCCAGCGAAGTCACGAACAC
Beta-3 AR (SEQ ID NO: 18)
TAGCCGGGGCCCTGCTGGCGCTGGCGGTGCTGGCCACCGTGGGAGGCAACCTGCTGGTCATCGTGGCCATCGCCTGGACTCCGAGACTCCAGACCATGACCAACGTGTTCGTGACTTCGCTGGCCGCAGCCGACCTGGTGATGGGACTC

NAT2-590 TP_F6 (SEQ ID NO: 323) TCGAGGTTCAATCTCCTGCCAAAGAAGAAACA
NAT2-590 TP_R1 (SEQ ID NO: 326) CGAACAATTGTGGAGACGTCTGCAGGTATGTA
NAT2-590 TP_R2 (SEQ ID NO: 327) CGAACAATTGTTGGAGACGTCTGCAGGTATGT
NAT2-590 TP_R7 (SEQ ID NO: 332) CGAACAATTGAGATGTTGGAGACGTCTGCAGG
NAT2-590 TP_R8 (SEQ ID NO: 333) CGAACAATTGAAGATGTTGGAGACGTCTGCAG
NAT2-590 (SEQ ID NO: 317)
ATCAGGAGAGAGCAGTATATTACAAACAAAGAATTTCTTAATTCTCATCTCCTGCCAAAGAAGAAACACCAAAAAATATACTTATTTACGCTTGAACCTCGAACAATTGAAGATTTTGAGTCTATGAATACATACCTGCAGACGTCTCCAACATCTTCATTTATAACCACATCATTTTGTTCCTTGCAGACCCCAGAAGGG

G2 fTP4-7 (SEQ ID NO: 107) CCGCCACAGGTGTATGGCTCTGGAGCCCGT
G2 rTP4-4 (SEQ ID NO: 112) ACAAAACGTAATCAGGGGCTTGTACAAAAT
G2 rTP4-6 (SEQ ID NO: 114) AAAACGTAATTGACCAGGGGCTTGTACAAA
G2_template (SEQ ID NO: 52)
GGCATGGATTTTTACGTGCCCAGACAAGAGCCAATGTTCAGATGGATGAGATTCTCAGATCTGAGCACGTGGGAGGGCGATCGCAATCTGGCTCCCAGCTTTGTGAATGAAGATGGCGTCGAATGACGCCAACCCATCTGATGGGTCCGCAGCCAACCTCGTCCCAGAGGTCAATAATGAGGTTATGGCTCTGGAGCCCGTTGTTGGTGCCGCTATTGCGGCACCTGTGGCGGGCCAACAAAACGTAATTGACCCCTGGATTAGAAACAATTTTGTACAAGCCCCTGGTGGAGAGTTCACAGTGTCCCCTAGAAACGCTCCAGGTGAGATACTGTGGAGCGCGCCCTTGGGCCCTGATCTGAACCCCTATCTTTCTCATTTGTCCAGAATGTACAATGGTTATGCAGGTGG

KCNQ1_rs2237897 TP-F1 (SEQ ID NO: 402) CGTCCCCAGCTCCCTAGGGACGGAGGTGGCCAGAG
KCNQ1_rs2237897 TP-F2 (SEQ ID NO: 403) CGTCCCCAGCTCCCTGGGACGGAGGTGGCCAGAGG
KCNQ1_rs2237897 TP-F4 (SEQ ID NO: 405) CGTCCCCAGCTCCCTGACGGAGGTGGCCAGAGGCC
KCNQ1_rs2237897 TP-F8 (SEQ ID NO: 409) CGTCCCCAGCTCCCTGAGGTGGCCAGAGGCCTCTC
KCNQ1_rs2237897 TP-R7 (SEQ ID NO: 416) ACGAGGGGCCTCAGCCACTCCCTGGGCCTGAAG
KCNQ1_rs2237897_template (SEQ ID NO: 436)
catggtgtgcgtgtttgggagcaccctgggcagcctctcgagtgtccagagagaggacaccgtcttgagccagcagggacagccaagagcgcccggggagAGGCCTGGGTGGAGACAGAACACAGCAGGCTGTGGGCGTGGCCAATGTTGAGGGACGGAGGTGGCCAGAGGCCTCTCAGTGGTGCCCAGGGAGCTGGGGACGAGGGGCCTCATCCTTCCCCTGAGCCCCTCCCTTCAGGCCCAGGGAGTGGCCTGGGGCCCAGGAGAGCCCGCTCCGCGCTCACAGCCTCCGTTCCCAGACacgcccgggcctgagcccccaggctgcactgtgccacagaacagactccccggcccctccctgctcgagaggtgaccaaggcccagggtctcccagcgag

ABCG2 TP_F3 (SEQ ID NO: 169) TGTAAGTTTTCTTAAGGATGATGTTGTGA
ABCG2 TP_F4 (SEQ ID NO: 170) TGTAAGTTTTCCTTAAGGATGATGTTGTG
ABCG2 TP_F5 (SEQ ID NO: 171) TGTAAGTTTTGCCTTAAGGATGATGTTGT
ABCG2 TP_F7 (SEQ ID NO: 173) TGTAAGTTTTTTGCCTTAAGGATGATGTT
ABCG2 TP_F8 (SEQ ID NO: 174) TGTAAGTTTTTTTGCCTTAAGGATGATGT
ABCG2 TP_R1 (SEQ ID NO: 175) ACAGTTCTCAATGATTCGTCATAGTTGTT
ABCG2 TP_R2 (SEQ ID NO: 176) ACAGTTCTCACATGATTCGTCATAGTTGT
ABCG2 TP_R3 (SEQ ID NO: 177) ACAGTTCTCATCATGATTCGTCATAGTTG
ABCG2 TP_R5 (SEQ ID NO: 179) ACAGTTCTCATTTCATGATTCGTCATAGT
ABCG2 TP_R8 (SEQ ID NO: 182) ACAGTTCTCATTTTTTCATGATTCGTCAT
ABCG2_template (SEQ ID NO: 437)
ggagttaactgtcatttgcagaactgcaggttcatcattagctagaactttaccttagttatgttatctttgtggattatgttatgtatactaaacagtcATGGTCTTAGAAAAGACTCATTATCATTATGTCTCATTAAAATGCTATTTGCCTTAAGGATGATGTTGTGATGGGCACTCTGACGGTGAGAGAAAACTTACAGTTCTCAGCAGCTCTTCGGCTTGCAACAACTATGACGAATCATGAAAAAAACGAACGGATTAACAGGGTCATTCAAGAGTTAGGTCTGGATAAAGTGGCagactccaaggtaatgtggaaaaactgaaagcatcatgatcagcataagtaggactttccctgtgtggataaaatgactctgattcagaagtttcctgta

FTO_2 TP_F1 (SEQ ID NO: 234) CTAAATTCACAGCAGTTATGCATTTAGAATGTCTGA
FTO_2 TP_F3 (SEQ ID NO: 236) CTAAATTCACAGATCAGTTATGCATTTAGAATGTCT
FTO_2 TP_F4 (SEQ ID NO: 237) CTAAATTCACAGCATCAGTTATGCATTTAGAATGTC
FTO_2 TP_R1 (SEQ ID NO: 242) AGTGATGCACTTCCATTTCTGACTGTTACCTATTAA
FTO_2 TP_R6 (SEQ ID NO: 247) AGTGATGCACTTCCACTCCATTTCTGACTGTTACCT
FTO_2_template (SEQ ID NO: 435)
tatcttttggcagatcagaacataatgaaaataaaataaaaaaattcaaaactggctcttgaatgaaataggattcagaagagatgatctcaaatctactTTATGAGATAATGTCCTTTTTAAAAATAAACACTAACATCAGTTATGCATTTAGAATGTCTGAATTATTATTCTAGGTTCCTTGCGACTGCTGTGAATTTAGTGATGCACTTGGATAGTCTCTGTTACTCTAAAGTTTTAATAGGTAACAGTCAGAAATGGAGTGGGAGAGCATAAAAGCAAACTGAAATGCAAATAGCTGgtaccctgaagccattaactttaagctggttattcctgacctactgtttggacataagatggtagagaggctgagtgtgacttgaacatttgttccttag

  The present invention relates to a primer for nucleic acid amplification, a method for designing a primer for nucleic acid amplification, a method for producing a primer for nucleic acid amplification, and a method for amplifying nucleic acid. According to the primer obtained by the method for designing a primer for nucleic acid amplification of the present invention, the amplification rate of nucleic acid and the specificity of nucleic acid amplification are improved in an isothermal nucleic acid amplification method using a primer set containing TP. For this reason, the method for designing a primer for nucleic acid amplification, the method for producing a primer for nucleic acid amplification, the primer set, and the method for amplifying a nucleic acid of the present invention can be said to be extremely useful tools in the field of genetic engineering, for example.

Claims (11)

A method for designing a primer used for amplification of a target nucleic acid sequence,
A sequence (Ac ′) complementary to the sequence (A) on the 3 ′ end side of the target nucleic acid sequence on the 3 ′ end side of the primer;
The 5 ′ terminal side of the primer contains the same sequence (B ′) as the sequence (B) present 5 ′ to the sequence (A) of the target nucleic acid sequence,
Primer design method condition [1] characterized by satisfying the following condition [1] and / or condition [2]: a sequence of 3 bases from the 5 ′ end side of the sequence (B ′) of the primer (B The sequence complementary to '5'-3) should be 2 or less in the primer sequence.
Condition [2]: A sequence complementary to the sequence (Ac′3′-3) of 3 bases from the 3 ′ end side of the sequence (Ac ′) of the primer does not exist in the primer (0) Like that. A method for producing a primer used for amplification of a target nucleic acid sequence,
Including a primer design process and a primer synthesis process,
The primer production method, wherein the primer set design step is performed by the primer design method according to claim 1. A method for designing a primer set used for amplification of a target nucleic acid sequence in a double-stranded template nucleic acid comprising a first template nucleic acid and a second template nucleic acid,
The primer set includes a first primer and a second primer,
The first primer is designed by the design method according to claim 1,
A sequence (Ec ′) complementary to the sequence (E) on the 3 ′ end side of the target nucleic acid sequence in the second template nucleic acid on the 3 ′ end side of the second primer;
On the 5 ′ end side of the second primer, the same sequence (F ′) as the sequence (F) present on the 5 ′ side of the sequence (E) of the target nucleic acid sequence in the second template nucleic acid Including
A sequence complementary to the 3 base sequence (Ac′3′-3) from the 3 ′ end of the sequence (Ac ′) of the first primer does not exist in the second primer (0 Pieces),
A sequence complementary to the sequence of 3 bases (Ec′3′-3) from the 3 ′ end side of the sequence (Ec ′) of the second primer does not exist in the first primer (0 pieces) ) A primer set design method characterized by: The first primer satisfies the condition [2],
A sequence complementary to the 3 base sequence (Ec′3′-3) from the 3 ′ end of the sequence (Ec ′) of the second primer does not exist in the second primer (0 The method for designing a primer set according to claim 3, which is designed as follows. A method for producing a primer set used for amplification of a target nucleic acid sequence in a double-stranded template nucleic acid comprising a first template nucleic acid and a second template nucleic acid,
Including primer set design process and primer set synthesis process,
The method for producing a primer set, wherein the primer set design step is performed by the primer set design method according to claim 3 or 4. A method for amplifying a complementary nucleic acid comprising synthesizing a nucleic acid complementary to a target nucleic acid sequence in a template nucleic acid, comprising the following steps (S1) to (S5).
(S1) A step of producing a primer by the production method according to claim 2.
(S2) A step of providing a template nucleic acid.
(S3) A step of annealing the primer to the template nucleic acid and performing a primer extension reaction to synthesize a complementary nucleic acid containing a complementary sequence of the target nucleic acid sequence.
(S4) The sequence (B ′) present on the 5 ′ side of the complementary nucleic acid synthesized in the step (S3) is hybridized to the sequence (Bc) present on the complementary nucleic acid, thereby A step of making the portion of the sequence (A) a single strand.
(S5) Annealing another primer having the same sequence as the primer to the portion of the sequence (A) on the template nucleic acid made into a single strand by the step (S4), thereby carrying out a strand displacement reaction, A step of replacing the complementary nucleic acid synthesized in the step (S3) with a complementary nucleic acid newly synthesized by the other primer.   The nucleic acid amplification method according to claim 6, wherein the double-stranded nucleic acid obtained in step (S5) is repeatedly used in step (S4).   The method for amplifying a nucleic acid according to claim 6 or 7, wherein the step (S3), the step (S4) and the step (S5) are performed isothermally. A method for amplifying a target nucleic acid sequence in a double-stranded template nucleic acid, comprising the following steps (S′1) to (S′5):
(S'1) The process of manufacturing a primer set by the manufacturing method of Claim 5.
(S′2) A step of providing a double-stranded template nucleic acid comprising a first template nucleic acid and a second template nucleic acid.
(S′3) The first and second primers containing the complementary sequences of the target nucleic acid sequences by annealing the first and second primers to the first and second template nucleic acids, respectively, and performing primer extension reactions, respectively. Synthesizing complementary nucleic acids;
(S′4) The sequences (B ′) and (F ′) present on the 5 ′ side of the first and second complementary nucleic acids synthesized by the step (S′3) are present on the complementary nucleic acids. A step of hybridizing to the sequence (Bc) and the sequence (Fc), respectively, so that the portions of the sequence (A) and the sequence (E) on the first and second template nucleic acids become single-stranded.
(S′5) The sequence (A) and the sequence (E) on the first and second template nucleic acids made into single strands by the step (S′4) have the same sequence as the primer. By performing the strand displacement reaction by annealing the other primer, the first and second complementary nucleic acids synthesized in the step (S′3) are replaced with the complementary nucleic acid newly synthesized by the other primer. Process.   The nucleic acid amplification method according to claim 9, wherein the double-stranded nucleic acid obtained in step (S'5) is repeatedly used in step (S'4).   The method for amplifying a nucleic acid according to claim 9 or 10, wherein the step (S'3), the step (S'4) and the step (S'5) are performed isothermally.

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