title: Allele-specific long-range PCR/sequencing method for allelic assignment of multiple single nucleotide polymorphisms.
abstract: We report an allele-specific sequencing method using allele-specific long-range polymerase chain reaction (PCR) to determine if multiple (specifically, more than three) single nucleotide polymorphisms (SNPs) are located on the same allele. We sequenced the glucocorticoid receptor (GR) gene as a model and detected four nucleotide changes, including two novel variations, in intron 4 and exons 6, 8, and 9 alpha in four of the investigated cell lines. The terminal SNPs (intron 4 and exon 9 alpha) were separated by 19 kb. Following SNP identification, the first round PCR allele-specific primers are designed at the both distal SNP sites (intron 4 and exon 9 alpha), placing the SNP positions at the primer 3'-end. Using these first round PCR products as template, the second round PCR was performed to separately amplify exons 6 and 8. These second round PCR products were subsequently sequenced. The sequencing results showed that the four SNPs were located on the same allele, i.e., forming a haplotype. This allele-specific long-range PCR/sequencing (ALP/S) method is rapid and applicable to the allelic assignment for more than three SNPs.
authors.affiliation: Project Team for Pharmacogenetics, National Institute of Health Sciences, 1-18-1, Kamiyoga, Setagaya, Tokyo 158-8501, Japan.
authors.forename: Michiyo
authors.initials: M
authors.lastname: Nagano
authors.forename: Takahiro
authors.initials: T
authors.lastname: Nakamura
authors.forename: Shogo
authors.initials: S
authors.lastname: Ozawa
authors.forename: Keiko
authors.initials: K
authors.lastname: Maekawa
authors.forename: Yoshiro
authors.initials: Y
authors.lastname: Saito
authors.forename: Jun-ichi
authors.initials: J
authors.lastname: Sawada
mesh_terms: D000483:Alleles; D002460:Cell Line; D004247:DNA / Q000378:metabolism; D005091:Exons; D006239:Haplotypes; D006801:Humans; D007438:Introns; D008957:Models, Genetic; D016133:Polymerase Chain Reaction / Q000379:methods*; D020641:Polymorphism, Single Nucleotide; D011965:Receptors, Glucocorticoid / Q000235:genetics
chemical_list: D011965:Receptors, Glucocorticoid; D004247:DNA