Genotyping method

Abstract

The present invention relates to a genotyping method, and more particularly to an ID sequence, which is assigned to each genotype, and a multiplex genotyping method which uses the ID sequence. When pyrosequencing is performed using the ID sequence, a unique and simple pyrogram can be obtained for each genotype. Thus, the use of the ID sequence makes it possible to genotype viral genes, disease genes, bacterial genes and identification genes in a simple and efficient manner. In addition, a genotyping primer of the invention can be used in various genotyping methods which are performed using dispensation orders and sequencing methods.

1 . An ID sequence for genotyping which consists of (ID−S)n−E, wherein ID is an ID mark which is a single nucleotide selected from among A, T, C and G; S is a signpost which is a nucleotide linked with the adjacent ID mark and different from that of the adjacent ID mark;
 E is an endmark which is a nucleotide different from that of the signpost; and n is a natural number ranging from 1 to 32. 
 
     
     
         2 . An ID sequence for genotyping which consists of ID−S, wherein ID is an ID mark which is a single nucleotide selected from among A, T, C and G; and S is a signpost which is a nucleotide linked with the adjacent ID mark and different from that of the adjacent ID mark. 
     
     
         3 . A genotyping primer comprising a gene-specific sequence for genotyping linked to the ID sequence of  claim 1 . 
     
     
         4 . The genotyping primer of  claim 3 , wherein the gene-specific sequence for genotyping is a sequence specific for a gene selected from the group consisting of viral genes, disease genes, bacterial genes, and identification genes. 
     
     
         5 . A genotyping method which comprises using the genotyping primer of  claim 3 . 
     
     
         6 . The genotyping method of  claim 5 , wherein the genotyping uses pyrosequencing methods or semiconductor sequencing methods. 
     
     
         7 . The genotyping method of  claim 5 , the method comprising the steps of:
 (a) designing an ID sequence for genotyping according to genotyping target gene, the ID sequence consisting of (ID−S)n−E, wherein ID is an ID mark which is a nucleotide selected from among A, T, C and G, S is a signpost which is a nucleotide linked with the adjacent ID mark and different from that of the adjacent ID mark, E is an endmark which is a nucleotide different from that of the signpost, and n is a natural number ranging from 1 to 32;   (b) amplifying the template of the genotyping target gene by PCR using a genotyping primer comprising a gene-specific sequence for genotyping linked to the designed ID sequence, thereby obtaining a PCR product; and   (c) pyrosequencing the PCR product to obtain a pyrogram for the ID sequence.   
     
     
         8 . A method for genotyping HPV, the method comprising the steps of:
 (a) designing an ID sequence for genotyping according to the genotype of each HPV virus, the ID sequence consisting of (ID−S)n−E, wherein ID is an ID mark which is a nucleotide selected from among A, T, C and G; S is a signpost which is a nucleotide linked with the adjacent ID mark and different from that of the adjacent ID mark, E is an endmark which is a nucleotide different from that of the signpost, and n is a natural number ranging from 1 to 32;   (b) constructing a genotyping primer composed of a pyrosequencing primer sequence, the ID sequence, and a sequence specific to a virus genotype corresponding to the ID sequence;   (c) amplifying an HPV virus-containing sample by PCR using the genotyping primer; and   (d) subjecting the amplified PCR product to pyrosequencing to obtain a pyrogram for the ID sequence, and distinguishing the genotype of HPV according to the ID sequence.   
     
     
         9 . The method of  claim 8 , wherein the sequence specific to a virus genotype is selected among nucleotide sequences shown by SEQ ID NOS: 1 to 15. 
     
     
         10 . A method for detecting KRAS gene mutation, the method comprising the steps of:
 (a) designing an ID sequence for genotyping according to each gene mutation of KRAS, the ID sequence consisting of (ID−S)n−E wherein ID is an ID mark which is a nucleotide selected from among A, T, C and G; S is a signpost which is a nucleotide linked with the adjacent ID mark and different from that of the adjacent ID mark, E is an endmark which is a nucleotide different from that of the signpost, and n is a natural number ranging from 1 to 32;   (b) constructing a detection primer composed of a pyrosequencing primer sequence, the ID sequence, and a sequence specific for a KRAS gene mutation corresponding to the ID sequence;   (c) amplifying a KRAS gene-containing sample by PCR using the detection primer; and   (d) subjecting the amplified PCR product to pyrosequencing to obtain a sequence for the ID sequence, and detecting the KRAS gene mutation according to the ID sequence.   
     
     
         11 . The method of  claim 10 , wherein the sequence specific for the KRAS gene mutation is selected among nucleotide sequences shown by SEQ ID NOS: 34 to 35. 
     
     
         12 . A method for detecting respiratory virus, the method comprising the steps of:
 (a) designing an ID sequence for genotyping according to the genotype of each of influenza A virus, influenza B virus, RSV B, rhinovirus, and coronavirus OC43, the ID sequence consisting of (ID−S)n−E wherein ID is an ID mark which is a nucleotide selected from among A, T, C and G; S is a signpost which is a nucleotide linked with the adjacent ID mark and different from that of the adjacent ID mark, E is an endmark which is a nucleotide different from that of the signpost, and n is a natural number ranging from 1 to 32;   (b) constructing a detection primer composed of a pyrosequencing primer sequence, the ID sequence, and a sequence specific to each respiratory virus gene corresponding to the ID sequence;   (c) amplifying a sample, which contains a respiratory virus selected from the group consisting of influenza A virus, influenza B virus, RSV B, rhinovirus, and coronavirus OC43, by PCR using the detection primer; and   (d) subjecting the amplified PCR product to pyrosequencing to obtain a sequence for the ID sequence, and detecting the respiratory virus according to the ID sequence.   
     
     
         13 . The method of  claim 12 , wherein the sequences specific to the respiratory virus genotypes are nucleotide sequences shown by SEQ ID NO: 41 for influenza A virus, SEQ ID NO: 42 for influenza B virus, SEQ ID NO: 43 for RSV B, SEQ ID NO: 44 for rhinovirus, and SEQ ID NO: 45 for coronavirus OC43.