title: Estimation of single-nucleotide polymorphism allele frequency by alternately binding probe competitive polymerase chain reaction.
abstract: Estimation of single-nucleotide polymorphism (SNP) allele frequency in pooled DNA samples is a promising approach to clarify the relationships between SNPs and diseases. Here, we present a simple, accurate, and cost-effective method for estimating SNP allele frequency, called alternately binding probe (ABProbe) competitive polymerase chain reaction (ABC-PCR) that entails no expensive devices for real-time fluorescence measurement and complex post-PCR steps. We prepared DNA pools of PCR products derived from homozygous samples of three different SNPs (ALDH2, GNB3, and HTR2A) in different portions, and the allele frequencies of these samples were estimated by ABC-PCR. Two alleles were coamplified by PCR with a fluorescent probe that binds to either alleles, and then fluorescence intensity was measured using a simple fluorometer. The ratio of the two alleles was calculated from the fluorescence intensity of the probe at the end-point. The estimated allele frequencies strongly correlated to the expected ratios for all three SNPs with high accuracy. When the allele frequencies were more than 5%, the relative standard deviations (R.S.D.s) of ABC-PCR were less than 20%. Moreover, we also confirmed that this method was applicable to SNP genotyping.
authors.affiliation: Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan.
authors.forename: Naohiro
authors.initials: N
authors.lastname: Noda
authors.forename: Hidenori
authors.initials: H
authors.lastname: Tani
authors.forename: Nao
authors.initials: N
authors.lastname: Morita
authors.forename: Shinya
authors.initials: S
authors.lastname: Kurata
authors.forename: Kazunori
authors.initials: K
authors.lastname: Nakamura
authors.forename: Takahiro
authors.initials: T
authors.lastname: Kanagawa
authors.forename: Satoshi
authors.initials: S
authors.lastname: Tsuneda
authors.forename: Yuji
authors.initials: Y
authors.lastname: Sekiguchi
mesh_terms: D001667:Binding, Competitive; D015342:DNA Probes / Q000032:analysis / Q000235:genetics* / Q000378:metabolism*; D005787:Gene Frequency / Q000235:genetics*; D005838:Genotype; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*
chemical_list: D015342:DNA Probes