Method of detecting genetic polymorphism

#Incorporated by reference in its entirety herein is a computer-readable nucleotide/amino acid sequence listing submitted concurrently herewith and identified as follows: 54,734 bytes ASCII (Text) file named “702743SequenceListing.txt,” created Mar. 27, 2008.

#The present invention relates to a method of detecting genetic polymorphism based on the invader assay, particularly to a method suitable for the detection of copy number variation (CNV).

#A genomic region where the copy number of a gene per cell differs among individuals in a certain population is referred to as copy number variation (CNV). Other known forms of polymorphism in genomic DNA number include, for example, single nucleotide polymorphism (SNP), variable number of tandem repeat (VNTR) and microsatellite polymorphism. These forms of polymorphism all involve regions of 1 kbp or shorter (each repeat unit is usually about several to several tens of bases for VNTR, and about two to four bases for microsatellite polymorphism), whereas copy number variation involves longer regions not less than 1 kb and is known to lead to a change in the copy number of the whole gene. As with SNP and the like, a type of copy number variation that occurs at frequencies of 1% or more in a population is also especially referred to as copy number polymorphism (CNP) (hereinafter, the abbreviation “CNV” is used to refer to copy number variation of any frequency).

#FIG. 1 shows the detection of allele asymmetry by PCR-RETINA using an artificial template. All experiments were performed in quadruplicate. (a) Real-time detection of fluorescence signals during invader assay. Plotted are reporter fluorescence signal values (FAM and Yakima Yellow) after normalization with Rox. The ordinate indicates normalized reporter signals (FAM or Yakima Yellow); the abscissa indicates reaction time at 30-second intervals. (b) Two-dimensional AD plot of PCR-RETINA after 3 minutes of the invader reaction. The ordinate indicates normalized FAM allele signals; the abscissa indicates normalized Yakima Yellow allele signals. (c) Two-dimensional AD plots at 0.5, 3, 10 and 30 minutes of the invader reaction. (d) Changes over time in cluster pattern showing allele asymmetry during 30 minutes of the invader reaction.

#FIG. 2 shows the detection of multiplication of the CYP2D6 gene by PCR-RETINA. Two-dimensional AD plots of PCR-RETINA using CEU individuals (a) and YRI individuals (b). The alphanumeric characters in boxes indicate rs numbers and RETINA time points. Shown are two-dimensional AD plots at the point of inflection just before fluorescence intensity reaches a plateau. The ordinate of the AD plots indicates normalized FAM allele signals; the abscissa indicates normalized Yakima Yellow allele signals. ● represents an individual with allele asymmetry. X represents an individual plotted in a cluster of normal homozygotes or heterozygotes, or several individuals with undetermined type. ▪ represents no template control (NTC). Copy number analysis of CYP2D6 by Taqman assay in CEU (c) and YRI (d). All reactions were performed in duplicate; mean copy number values are shown. Each open diamond (⋄) represents an individual with allele asymmetry in PCR-RETINA.

#FIG. 3 shows the detection of multiplication of the MRGPRX1 gene by PCR-RETINA. Shown are 2-dimensional AD plots of PCR-RETINA using CEU individuals (a) and YRI individuals (b). ● represents an individual with allele asymmetry. X represents an individual plotted in a cluster of normal homozygotes or heterozygotes, or several individuals with undetermined type. ▪ represents no template control (NTC). Results of copy number analysis by Taqman assay in CEU (c) and YRI (d).

#The present invention provides a method of determining the genotype of a subject in a genomic region comprising an SNP site by real-time invader assay (RETINA) (hereinafter, also referred to as “the genotyping method of the present invention”). The subject in the genotyping method of the present invention is not particularly limited, as long as it has a genomic DNA comprising an SNP site, and the subject is preferably a mammal, particularly a human, a mouse or the like, for which mammals data on SNP have been accumulated. The genomic region comprising an SNP site to be subjected to typing, is also not particularly limited. In the case of humans, for example, such genomic regions include genomic regions comprising any SNP site registered with the NCBI SNP database [www.ncbi.nlm.nih.gov/SNP/] and JSNP database [snp.ims.u-tokyo.acjp/index.html].

#Preferably, the genomic region comprising an SNP site in the present invention exists within a copy number variation (CNV) region. To date, CNV regions in the human genome have been identified by a variety of methods, and are summarized in the Database of Genomic Variants [projects.tcag.ca/variation/]. For example, the CNV region comprising the MRGPRX1 gene, used in Example 1 below, is registered as Locus 2220 with the Database of Genomic Variants, which region spans an about 290 kbp of the base number 18,762,138-19,051,811 of chromosome 11, in which the 26 known SNP sites specified by RefSNP ID in FIG. 6 are contained.

#Examples of polymorphism include SNP as well as Indel and gene conversion polymorphism. Indel is an abbreviation of Insertion/Deletion. The gene conversion polymorphism is a polymorphism in which a DNA sequence has been converted into another DNA sequence. Herein, the descriptions concerning SNPs may be interpreted as ones concerning Indel or gene conversion polymorphism, as necessary.