Mutation detection by TaqMan-allele specific amplification: Application to molecular diagnosis of glycogen storage disease type Ia and medium-chain Acyl-CoA dehydrogenase deficiency

Kunihiro Fujii, Yoichi Matsubara, Jun Akanuma, Kazutoshi Takahashi, Shigeo Kure, Yoichi Suzuki, Masue Imaizumi, Kazuie Iinuma, Osamu Sakatsume, Piero Rinaldo, Kuniaki Narisawa

Research output: Contribution to journalArticlepeer-review

54 Citations (Scopus)

Abstract

We have devised an allele-specific amplification method with a TaqMan fluorogenic probe (TaqMan-ASA) for the detection of point mutations. Pairwise PCR amplification using two sets of allele-specific primers in the presence of a TaqMan probe was monitored in real time with a fluorescence detector. Difference in amplification efficiency between the two PCR reactions was determined by 'threshold' cycles to differentiate mutant and normal alleles without post-PCR processing. The method measured the efficiency of amplification rather than the presence or absence of end-point PCR products, therefore allowing greater flexibility in designing allele-specific primers and an ample technical margin for allelic discrimination. We applied the TaqMan-ASA method to detect a prevalent 727G>T mutation in Japanese patients with glycogen storage disease type Ia and a common 985A>G mutation in Caucasian patients with medium-chain acyl-CoA dehydrogenase deficiency. The method can be automated and may be applicable to the DNA diagnosis of various genetic diseases. (C) 2000 Wiley-Liss, Inc.

Original languageEnglish
Pages (from-to)189-196
Number of pages8
JournalHuman mutation
Volume15
Issue number2
DOIs
Publication statusPublished - 2000 Feb 14

Keywords

  • ACADM
  • Acyl-CoA dehydrogenase
  • Allele-specific amplification
  • G6PC
  • Glucose-6-phosphatase
  • Glycogen storage disease type Ia
  • Medium- chain
  • Mutation detection
  • TaqMan

ASJC Scopus subject areas

  • Genetics
  • Genetics(clinical)

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