JP2008161111A - Method for discriminating repeated motif in protein - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method capable of discriminating repeated motives in a protein clearly and in a good reproducibility, and a reagent therefor. <P>SOLUTION: This method for discriminating the repeated motives in the protein is provided by performing a nucleic acid amplification reaction by using a primer (D1) having 3'-terminal in a specific sequence (D), a primer (P1) of which mutual elongated products have a property of becoming mutual templates with the primer (D1), having 3'-terminal within any of two regions at the outside of the specific sequence and being common in nucleic acid sequences encoding the family of the proteins, and a primer (C1) of which mutual elongated products have a property of becoming the mutual templates with the primer (P1) and having a 3'-terminal within a specific sequence (C) which is a variant of the specific sequence (D). <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a method for identifying a protein having a repetitive motif. The present invention is particularly useful for classification of toxin proteins produced by bacteria, classification of the bacteria based on the classification, or estimation of pathogenicity.

  Among proteins, there is a region called a motif containing a characteristic amino acid sequence expected to have a certain function, and the function of the protein can be estimated by the presence or absence of the region. As motifs, DNA binding (leucine zipper) motif consisting of repeating leucine-unspecified 6 amino acids, glycosaminoglycan binding motif consisting of serine-glycine-unspecified 1 amino acid-glycine, cells consisting of arginine-glycine-aspartic acid It consists of dozens of amino acids such as homeodomain, zinc finger, helix-loop-helix, and hairpin β sheet, which are often found in gene regulatory proteins, from small ones composed of several amino acids such as adhesion (RGD) motifs. Many are known to big ones.

An example in which the function is enhanced by repeating the motif has been reported. For example, in the carboxyl terminal (C-terminal) region of cytoplasmic toxin related protein (CagA), which is one of the pathogenic factors of Helicobacter pylori, there is a motif that undergoes phosphorylation named C sequence and D sequence. . The C sequence is mainly present in Helicobacter pylori that infects many Westerners, and the D sequence is a similar sequence (variant) present in Helicobacter pylori that mainly infects East Asians. CagA protein is phosphorylated by Src when sent into cells by Helicobacter pylori infected with stomach epithelial cells, and binds to Src homology phosphatase-2 (SHP-2), thereby mediated by Ras and ERK. Dephosphorylation of focal adhesion kinase (FAK) associated with abnormal cell growth and cell adhesion causes improvement in cell motility. SHP-2 is known to bind to the C or D sequence of CagA protein, and when CagA protein has multiple C or D sequences, the binding power to SHP-2 is strong and the toxicity is strong. It is known that it becomes easy to cause cancer (see, for example, Non-Patent Documents 1 to 4).

Thus, the function of a protein can be estimated by analyzing a specific motif. In the above example, by analyzing the type and / or number of repeats of the C sequence or D sequence of the CagA protein produced by Helicobacter pylori, it is possible to classify the bacterium or to infer pathogenicity.

As a conventional technique for analyzing a protein motif, for example, there is a sequencing method for determining the sequence of a nucleic acid encoding the protein. In the sequencing method, information necessary for the analysis of the amino acid sequence can be obtained by analyzing the nucleic acid sequence. However, since the length of the determinable nucleic acid sequence is substantially about 500 to 700 bases, it is approximately 200. It is difficult to analyze motifs longer than amino acids, especially repetitive motifs. In addition, it takes a lot of labor and time to prepare template nucleic acid, DNA polymerase reaction, polyacrylamide gel electrophoresis, analysis of nucleic acid sequence, etc., and labor can be saved by using a recent automatic sequencer. However, there is a problem that an expensive device is required. As another conventional technique, it is possible to directly analyze the sequence of amino acids constituting the protein. However, since the determinable length is as short as about 10 amino acids, there is a defect that only a very small motif can be analyzed.
Asahi et al., J. Exp. Med. 191, 593-602, (2000) Higashi et al., Proc. Natl. Acad. Sci. USA 99, 14428-14433, (2002) Azuma et al., J. Infect. Dis. 189, 820-827, (2004). Azuma et al., J. Clin. Microbiol. 42, 2508-2517, (2004).

  An object of the present invention is to solve the above problems and provide a method capable of clearly and reproducibly identifying a protein motif.

  In view of the above circumstances, the present inventors have completed the present invention as a result of intensive studies.

That is, the present invention has the following configuration.
1. A method for identifying a repetitive motif in a protein, comprising the steps of (a) to (b) below, wherein the number of repetitions of a specific sequence in a nucleic acid sequence encoding the protein is determined.
(A) A step of performing nucleic acid amplification using the following primers (i), (ii) and (iii):
(i) Primer (D1) having a 3 ′ end in the specific sequence (D)
(ii) The primer (D1) and the extension products of each other serve as templates for each other, and have a 3 ′ end in any one of the two regions outside the specific sequence, and encode the family of the protein Primer common to nucleic acid sequences to be performed (P1)
(iii) Primer (C1) having the property that the extension product of the primer (P1) and each other serves as a template, and having a 3 ′ end in the specific sequence (C) which is a variant of the specific sequence (D)
(B) Analyzing the type of the amplified nucleic acid fragment obtained from the one or more primers (D1) obtained in step (a) and the type of the amplified nucleic acid fragment obtained from the primer (C1) A method for identifying a repetitive motif in a protein, comprising the steps of (a) to (b) below, wherein the number of repetitions of a specific sequence in a nucleic acid sequence encoding the protein is determined.
(A) A step of performing nucleic acid amplification using the following primers (i), (ii) and (iii):
(i) Primer (D1) having a 3 ′ end in the specific sequence (D)
(ii) The primer (D1) and the extension products of each other serve as templates for each other, and have a 3 ′ end in any one of the two regions outside the specific sequence, and encode the family of the protein Primer common to nucleic acid sequences to be performed (P1)
(iii) The primer (P1) and the extension products of each other serve as templates for each other. Of the two regions outside the specific sequence, 3 ′ is present in the other region different from the region where the primer (P1) is set. Primer with ends and common to the family of proteins (P2)
(iv) Primer (C2) having the property that primer (P2) and the extension products of each other serve as templates for each other, and having 3 ′ end in specific sequence (C) which is a variant of specific sequence (D)
(B) the type of amplified nucleic acid fragment obtained from one or more primers (D1) obtained in step (a), the type of amplified nucleic acid fragment obtained from primer (C2), the primer (P1) and 2. Analyzing presence or absence of amplified nucleic acid fragment obtained from (P2) The identification method according to 1 or 2, wherein at least one nucleotide after the 3 'end of the primer (D1) is labeled with a ligand.
4). 4. The identification method according to any one of 1 to 3, wherein at least one nucleotide after the 3 ′ end of the primer (D1) and the primer (C1 or C2) is labeled with a different ligand.
5. 3. The identification method according to 3 or 4, wherein the ligand is at least one selected from the group consisting of an antigen, an antibody, a fluorescent substance, a luminophore, and an enzyme.
6). 3-5, wherein the number of repeats of the specific sequence is determined by detecting a ligand bound to at least one nucleotide after the 3 ′ end of the primer having a 3 ′ end in the specific sequence. Either identification method.
7). 7. The identification method according to any one of 1 to 6, wherein the repetitive motif is a C sequence or a D sequence present in the C-terminal region of the CagA protein of Helicobacter pylori.

  The present invention provides a method that can clearly and easily identify a motif possessed by a protein. Further, according to the method of the present invention, when a protein has a repeated motif, the number of repetitions can be determined, and the motif and protein can be identified in more detail.

Hereinafter, the present invention will be described in detail.
In the present invention, a primer is an oligonucleotide having a sequence complementary to a nucleic acid encoding a protein to be identified, a chromosome containing the nucleic acid, or a fragment thereof, and is generally used for nucleic acid amplification. As long as it has the characteristics to be achieved, it may be modified as necessary. The primer chain length may be 9 to 35 bases, and preferably 11 to 30 bases. The amplification method using these primers is not particularly limited. A known amplification method may be used. Known amplification methods include, for example, PCR, NASBA, LCR, SDA, RCA, TMA, LAMP, ICAN, and UCAN methods.

In the present invention, nucleic acid amplification refers to amplification of a nucleic acid to be amplified from a nucleic acid in a sample (target nucleic acid) using complementarity of its inherent sequence. As a nucleic acid amplification method applicable to the method for identifying a repetitive motif of a protein in the present invention, it can be basically performed using a conventional method, and is usually an object of identification that has been denatured into a single strand. A target nucleic acid by allowing 4 types of deoxynucleoside triphosphates (dNTP) and DNA polymerase and 2 or more types of oligonucleotides or primers to act on a nucleic acid encoding the protein to be obtained or a chromosome containing the nucleic acid, or a fragment thereof The sequence between the primers using as a template is amplified. In addition, when the target nucleic acid does not contain an amount sufficient for detection, the nucleic acid encoding the protein to be identified or the chromosome containing the nucleic acid or a fragment thereof is amplified by the amplification reaction shown below. It is also possible to keep it.

Examples of nucleic acid amplification methods include PCR (Japanese Patent Publication No. 4-67960, Japanese Patent Publication No. 4-67957), NASBA (Nucleic acid sequence-based amplification method; Nature 350, 91 (1991)), LCR ( International Publication No. 89/12696, JP-A-2-2934), SDA (Strand Displacement Amplification: Nucleic Acids Res. 20, 1691 (1992)), RCA (International Publication No. 90/1069) TMA (Transcription mediated amplification method; J. Clin. Microbiol. Vol. 31, p. 3270 (1993)), LAMP (loop-mediated isothermal amplification method: J. Clin Microbiol. Vol. 42: p. 1, 956) (2004)), ICAN (isothermal and chimeric primer-initiated amplification of nucleic acids: Kekkaku. 78, 533 (2003)), UCA N (see the Japanese Cancer Association (H13.9.26 to H13.9.28, held in Yokohama) or the Japan Society for Expertise on Science and Technology (H13.11.8 to H13.11.9, held in Tokyo)).

In particular, the PCR method repeats a cycle consisting of three steps of denaturation, annealing, and extension in the presence of a sample nucleic acid, four types of deoxynucleoside triphosphates, a pair of primers, and a heat-resistant DNA polymerase. In this method, the region of the sample nucleic acid sandwiched between the primers is exponentially amplified. That is, the sample nucleic acid is denatured in the denaturation step, each primer is hybridized with a region on the single-stranded sample nucleic acid complementary to each in the subsequent annealing step, and DNA is started from each primer in the subsequent extension step. A DNA strand complementary to each single-stranded sample nucleic acid serving as a template is extended by the action of the polymerase to form double-stranded DNA. By this one cycle, one double-stranded DNA is amplified into two double-stranded DNAs. Therefore, if this cycle is repeated n times, the region of the sample DNA sandwiched between the pair of primers is theoretically amplified to 2 n times. Since the amplified DNA region exists in a large amount, it can be easily detected by a method such as electrophoresis. Therefore, if a gene amplification method is used, it is possible to detect even a very small amount of sample nucleic acid that could not be detected in the past (one molecule is acceptable), which is a very widely used technique recently. .

The sample containing the nucleic acid used for amplification can be prepared from any material such as bacteria, animal or plant tissue, lysates from individual cells. The method for preparing the sample is not particularly limited. For example, the sample may be prepared from a patient's blood or tissue by a known method. Typical examples include phenol / chloroform extraction method (Biochimica et Biophysica acta 72, 619-629, 1963), alkaline SDS method (Nucleic Acids Res. 7, 1513-1523, 1979). ) Etc. in the liquid phase. As a system using a nucleic acid binding carrier for nucleic acid isolation, a method using glass particles and a sodium iodide solution (Proc. Natl. Acad. Sci. USA, Vol. 76-2, pages 615 to 619, 1979) and a method using hydroxyapatite (Japanese Patent Laid-Open No. 63-263093). Other methods include a method using silica particles and chaotropic ions (J. Clin. Microbiol. Vol. 28-3, pages 495-503, 1990, JP-A-2-289596). Moreover, the nucleic acid may be dissolved in the sample, or may be fixed to a solid phase.

In the present invention, the specific sequence (D) is a nucleic acid sequence encoding a motif carried by a protein or a repeating unit contained in the motif, or a nucleic acid sequence complementary to the sequence. The specific sequence (C) is a nucleic acid sequence (variant) similar to the specific sequence (D) and is complementary to the nucleic acid sequence encoding a motif possessed by a protein or a repeating unit contained in the motif, or the sequence. Nucleic acid sequence.

As a method for analyzing an amplified nucleic acid fragment applicable to the method for identifying a repetitive motif in the protein of the present invention, a conventionally known nucleic acid analysis method can be used, and is not particularly limited. Gel electrophoresis is preferred. When using agarose gel electrophoresis, as an example, it contains a primer (D1) having a 3 ′ end in a specific sequence (D) and a nucleic acid sequence that can be a pair of primers in a nucleic acid amplification reaction together with the primer (D1). Contains an amplified nucleic acid fragment generated by a primer, a primer (C1 or C2) having a 3 ′ end in a specific sequence (C), and a nucleic acid sequence that can be a pair of primers in a nucleic acid amplification reaction together with the primer (C1 or C2) Each primer is designed such that the length of the amplified nucleic acid fragment generated by the primer to be different is different. By mixing these primers and amplifying the nucleic acid, the presence or absence of the motif corresponding to the specific sequence (D) and / or (C) and the number of repetitions can be quickly determined from the pattern of the amplified nucleic acid fragment. Further, if the primer (D1) and the primer (C1 or C2) are labeled with different ligands, the amplified nucleic acid fragment generated by the primer (D1) and the primer (C1 or C2) are generated. Whether the amplified nucleic acid fragment is derived from the specific sequence (D) or (C) by detecting each ligand after agarose gel electrophoresis even if the chain length of the amplified nucleic acid fragment is the same Can do.

One of the important disclosures of the present invention is a primer (D1) having a 3 ′ end in a specific sequence (D) and a 3 ′ end in any one of two regions outside the specific sequence, A primer (P1) common to the nucleic acid sequence encoding the protein family, a primer (C2) having a 3 ′ end in a specific sequence (C) which is a variant of the specific sequence (D), and two outside the specific sequence Among the two regions, a primer (P2) having a 3 ′ end in the other region different from the region where the primer (P1) is set and common to the protein family is set as shown in FIG. 1 as an example. Then, a nucleic acid amplification reaction is performed, and a remarkable effect is found such that the presence or absence of the motif corresponding to the specific sequence (D) and / or (C) and the number of repetitions can be clearly determined in the target protein. It lies in the fact.

For example, when one specific sequence (D) is present in a nucleic acid encoding a protein, as shown in FIG. 1 (A), a primer (D1) having a 3 ′ end in the specific sequence (D) and the specific sequence One amplified nucleic acid fragment is generated by a primer (P1) having a 3 ′ end in one of the two outer regions and common to the nucleic acid sequence encoding the protein family. When two specific sequences (D) are present in the nucleic acid encoding the protein, as shown in FIG. 1 (B), the primer (D1) having a 3 ′ end in the specific sequence (D) and the specific sequence Two kinds of amplified nucleic acid fragments having different chain lengths are generated by a primer (P1) having a 3 ′ end in any one of the two outer regions and a common nucleic acid sequence encoding the protein family. Furthermore, when one specific sequence (C), which is a variant of the specific sequence (D), is present in the nucleic acid encoding the protein, as shown in FIG. 1 (C), the 3 ′ end in the specific sequence (C) Primer (C2) having a 3′-end in the other region different from the region where primer (P1) is set out of two regions outside the specific sequence and primer (P2) common to the protein family Produces one type of amplified nucleic acid fragment. In addition, when two specific sequences (C) are present in the nucleic acid encoding the protein, as shown in FIG. 1 (D), the primer (C2) having a 3 ′ end in the specific sequence (C) and the specific sequence Two types of amplifications that have 3 ′ ends in the other two regions different from the region where the primer (P1) is set, and have different chain lengths by using the primer (P2) common to the protein family Nucleic acid fragments are generated.

The primers (D1, C1, C2, P1, P2) used in the present invention need only have properties generally used for nucleic acid amplification, and may be modified as necessary. The ligand is preferably bound to a position that does not inhibit the nucleic acid amplification reaction. As a position for binding the ligand, it is preferable to bind the ligand to at least one nucleotide after the 3 ′ end of the primer (D1, C1, C2, P1, P2). More preferably, a ligand is bound to the 5 ′ end of C1, C2, P1, P2).

Another important disclosure of the present invention is that the first ligand is bound and the primer (D1) having a 3 ′ end in the specific sequence (D) is bound to the second ligand. A primer (C1) having a 3 ′ end in a specific sequence (C) which is a variant of the sequence (D), and a 3 ′ end in any one of two regions outside the specific sequence, The primer (P1) common to the nucleic acid sequence encoding the family is set as shown in FIG. 2 as an example, a nucleic acid amplification reaction is performed, and the first ligand and the second ligand are detected, respectively. And the presence of the motif corresponding to the specific sequence (D) and / or (C) and the number of repetitions have been found to be remarkable.

For example, when a specific sequence (D) and a specific sequence (C) that is a variant of the specific sequence (D) are arranged in a nucleic acid encoding a protein as shown in FIG. 2, as shown in FIG. A primer (D1) having a first ligand bound thereto and having a 3 ′ end in the specific sequence (D) and a 3 ′ end in any one of the two regions outside the specific sequence; A primer (C1) having a 3 ′ end in a specific sequence (C), wherein a single amplified nucleic acid fragment is generated by a primer (P1) common to a nucleic acid sequence encoding a family, Two kinds of amplified nucleic acid fragments are generated by a primer (P1) having a 3 ′ end in any one of two regions outside the specific sequence and common to the nucleic acid sequence encoding the protein family. By performing agarose gel electrophoresis and detecting each ligand, it can be determined whether the amplified nucleic acid fragment is derived from the specific sequence (D) or (C).

  The ligand is not particularly limited as long as it does not interfere with nucleic acid amplification, but is preferably an antibody, oligonucleotide, receptor, nucleic acid-specific binding substance, avidin, biotin, digokesigenin, fluorescent chemical, luminophore , An enzyme, a fluorescent protein, a photoprotein, a magnetic substance, and a conductive substance. Avidin, biotin, digokesigenin, fluorescent chemicals, fluorescent proteins, luminophores, enzymes, and conductive substances are preferred, avidin, biotin, digokesigenin, and fluorescent chemicals are more preferred, and fluorescent chemicals are particularly preferred.

  The detection of the ligand is not particularly limited as long as it is a known method. However, when the ligand is a fluorescent chemical substance or fluorescent protein, light of a specific wavelength is irradiated to excite the fluorescent chemical substance or fluorescent protein to bring it to the ground state. It is possible to measure the amount of fluorescence of a specific wavelength generated when converted. Examples of the fluorescent chemical substance used in these include FITC, FAM, TAMRA, Texas Red, VIC, Cy3, Cy5, HEX, and the like, and examples of the fluorescent protein include GFP, YFP, and RFP. When the label is an enzyme, it can be measured by detecting the reaction product produced by adding the enzyme substrate. Examples of combinations of enzymes and substrates used in these include alkaline phosphatase and paranitrophenyl phosphate, CDP-star, AMPPD, DDAOphosphate, BCIP-NBT, etc., peroxidase and TMB, Lumi-Light (Roche Diagnostics) ), Combinations of SAT-1 (Dojindo), combinations of diaphorase and NTB, various oxidases and substrates, combinations of various dehydrogenases and substrates, etc. It can be used without any problems. When the label is a magnetic substance, measurement can be performed by detecting magnetism. The label can be measured by detecting a conductive substance and a current value.

The method for identifying repetitive motifs in the protein of the present invention can be used, for example, to predict the risk of carcinogenesis caused by Helicobacter pylori. Examples of the cancer include stomach cancer. As described above, if the identification method of the present invention is used, the presence or absence of a specific sequence and the number of repeats can be clearly determined in the repeat motif of the target protein, thereby detecting the polymorphism of the C-terminal region of the CagA protein. can do. More specifically, by using the identification method of the present invention, it is possible to detect the presence or absence of C sequences and / or D sequences that are closely related to carcinogenesis, and also detect the number of C sequences and / or D sequences. be able to. The presence or absence of carcinogenesis can be predicted by the presence or absence of the C sequence and / or the D sequence, and the possibility of carcinogenesis (the C sequence is determined by the number of the C and / or D sequences). And / or the greater the D sequence, the greater the risk of carcinogenesis). As an example, for Helicobacter pylori that frequently infects Westerners, the possibility of carcinogenesis can be predicted by the presence or absence of the C sequence, and the possibility of carcinogenesis (the larger the C sequence, the more The risk of cancer is high) can be predicted by the number of C sequences. In addition, for Helicobacter pylori that infects many East Asians, the possibility of carcinogenesis can be predicted by the presence or absence of the D sequence, and the possibility of carcinogenesis (the more the D sequence, the more The risk of cancer is high) can be predicted by the number of D sequences.

  Hereinafter, based on an Example, this invention is demonstrated more concretely. However, the present invention is not limited to the following examples.

Example 1
Identification of repetitive motifs in the CagA protein of Helicobacter pylori (1) Synthesis of oligonucleotides Oligos having the nucleotide sequences shown in SEQ ID NOs: 1 to 3 by the phosphoamidite method using DNA synthesizer type 392 manufactured by PerkinElmer Nucleotides (hereinafter referred to as oligos 1 to 3) were synthesized. The synthesis was performed according to the manual, and the deprotection of each oligonucleotide was carried out overnight at 55 ° C. with ammonia water. Oligonucleotide purification was carried out on a Perkin Elmer OPC column. Alternatively, DNA commissioned companies (Nippon Bioservice, Sigma-Aldrich Japan, Operon Biotechnology, etc.) were requested.
Oligo 1 is a sense strand, a nucleic acid sequence encoding the D sequence of the CagA protein, that is, a primer (D1) having a 3 ′ end in the specific sequence (D), and combined with oligo 3 of the antisense strand for amplification reaction Used as an oligonucleotide. Oligo 2 is a nucleic acid sequence encoding C sequence of CagA protein, that is, a primer (C1) having a 3 ′ end in a specific sequence (C), and is used as an oligonucleotide for amplification reaction in combination with oligo 3 of the antisense strand. The Oligo 3 is a primer (P1) containing a nucleic acid sequence that can be a pair of primers in a nucleic acid amplification reaction together with a primer common to a nucleic acid sequence encoding a family of CagA proteins, ie, primer (D1) or (C1). Oligo 1 is labeled with FITC at the 5 ′ end, and oligo 2 is labeled with TAMRA at the 5 ′ end.

(2) Analysis of Helicobacter pylori cagA gene by PCR method Using DNA solutions extracted from Helicobacter pylori bacteria of sample numbers 1 to 6 in Table 1 by the phenol / chloroform method as samples, the following reagents were added. Then, the cagA gene of Helicobacter pylori by PCR was analyzed under the following conditions.

Reagents A 25 μl solution containing the following reagents was prepared.
KOD DNA polymerase reaction solution Oligo 1 (5 ′ end labeled with FITC) 10 pmol,
Oligo 2 (5 ′ end labeled with TAMRA) 10 pmol,
Oligo 3 10 pmol,
X10 buffer 2.5 μl,
2.5 μl of 2 mM dNTP,
1 μl of 25 mM MgCl ₂ ,
KOD DNA polymerase 0.5U,
Extracted DNA solution 100ng

Amplification conditions 94 ° C, 5 minutes 94 ° C, 15 seconds,
60 ° C for 30 seconds,
68 ℃, 30 seconds (35 cycles)
25 ° C for 15 minutes.

(3) Detection Using Agarose Gel Electrophoresis 5 μl of the amplification reaction solution was electrophoresed on a 3% agarose gel and detected with a fluorescent light-emitting LED light source device VISEIRAISE AE-6935 (manufactured by ATTO). FITC, which is a label for oligo 1, was observed under a blue light source and under a TAMRA green (long) light source, which was a label for oligo 2, and the fluorescence of the amplified band was visually confirmed. In Table 2, Sample No. The number of amplification bands clearly confirmed for the samples indicated by 1 to 6 is shown.

(4) Results As is clear from the sample numbers 1 to 6 in Table 2 (and Table 1), according to the method for identifying a repeated motif in the protein of the present invention, the types of repeated motifs in the CagA protein of Helicobacter pylori It is possible to determine the number of repetitions. These results indicate that the six strains were identified using the sequencing method, which can obtain information necessary for discrimination by directly analyzing the nucleic acid sequence, but has the disadvantages that a great deal of labor and time are required. Consistent with (Table 1).

That is, the identification method of the repeated motif in the protein of the present invention achieves the same identification ability as the conventional method, while greatly reducing the number of work steps required for the analysis, and easily and quickly the presence or absence of the motif in the protein. It is possible to clearly determine the number of repetitions of the motif.

According to the present invention, it is possible to clearly determine the presence or absence of a motif in a protein and the number of repetitions of the motif, and it is possible to quickly and easily obtain a reproducible result without requiring a complicated operation as in the conventional methods. It is expected that this will contribute greatly to the industry.

An example of how to set each primer An example showing how to set each primer, and a detection example using the first and second ligands

Claims (7)

A method for identifying a repetitive motif in a protein, comprising the steps of (a) to (b) below, wherein the number of repetitions of a specific sequence in a nucleic acid sequence encoding the protein is determined.
(A) A step of performing nucleic acid amplification using the following primers (i), (ii) and (iii):
(i) Primer (D1) having a 3 ′ end in the specific sequence (D)
(ii) The primer (D1) and the extension products of each other serve as templates for each other, and have a 3 ′ end in any one of the two regions outside the specific sequence, and encode the family of the protein Primer common to nucleic acid sequences to be performed (P1)
(iii) Primer (C1) having the property that the extension product of the primer (P1) and each other serves as a template, and having a 3 ′ end in the specific sequence (C) which is a variant of the specific sequence (D)
(B) analyzing the type of the amplified nucleic acid fragment obtained from the one or more primers (D1) obtained in step (a) and the type of the amplified nucleic acid fragment obtained from the primer (C1) A method for identifying a repetitive motif in a protein, comprising the steps of (a) to (b) below, wherein the number of repetitions of a specific sequence in a nucleic acid sequence encoding the protein is determined.
(A) A step of performing nucleic acid amplification using the following primers (i), (ii) and (iii):
(i) Primer (D1) having a 3 ′ end in the specific sequence (D)
(ii) The primer (D1) and the extension products of each other serve as templates for each other, and have a 3 ′ end in any one of the two regions outside the specific sequence, and encode the family of the protein Primer common to nucleic acid sequences to be performed (P1)
(iii) The primer (P1) and the extension products of each other serve as templates for each other. Of the two regions outside the specific sequence, 3 ′ is present in the other region different from the region where the primer (P1) is set. Primer with ends and common to the family of proteins (P2)
(iv) Primer (C2) having the property that primer (P2) and the extension product of each other serve as a template for each other, and having 3 ′ end in specific sequence (C) which is a variant of specific sequence (D)
(B) the type of amplified nucleic acid fragment obtained from one or more primers (D1) obtained in step (a), the type of amplified nucleic acid fragment obtained from primer (C2), the primer (P1) and Step of analyzing presence or absence of amplified nucleic acid fragment obtained from (P2) The identification method according to claim 1 or 2, wherein at least one nucleotide after the 3 'end of the primer (D1) is labeled with a ligand. The primer (D1) and at least one nucleotide from the 3 'end of the primer (C1 or C2) after the 3' end are labeled with different ligands, respectively. Identification method. The identification method according to claim 3 or 4, wherein the ligand is at least one selected from the group consisting of an antigen, an antibody, a fluorescent substance, a luminophore, and an enzyme. The number of repetitions of the specific sequence is determined by detecting a ligand bound to at least one nucleotide after the 3 'end of the primer having a 3' end in the specific sequence. The identification method in any one of -5. The identification method according to any one of claims 1 to 6, wherein the repetitive motif is a C sequence or a D sequence present in the C-terminal region of the CagA protein of Helicobacter pylori.