Estimation of single-nucleotide polymorphism allele frequency by alternately binding probe competitive polymerase chain reaction

Naohiro Noda, Hidenori Tani, Nao Morita, Shinya Kurata, Kazunori Nakamura, Takahiro Kanagawa, Satoshi Tsuneda, Yuji Sekiguchi

Research output: Contribution to journalArticle

7 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)211-216
Number of pages6
JournalAnalytica Chimica Acta
Volume608
Issue number2
DOIs
Publication statusPublished - 2008 Feb 11

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Keywords

  • Allele frequency
  • Alternately binding probe competitive polymerase chain reaction
  • Competitive polymerase chain reaction
  • Fluorescence quenching
  • Real-time polymerase chain reaction
  • Single-nucleotide polymorphism

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Environmental Chemistry
  • Spectroscopy

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