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

研究成果: Article査読

54 被引用数 (Scopus)

抄録

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.

本文言語English
ページ(範囲)189-196
ページ数8
ジャーナルHuman mutation
15
2
DOI
出版ステータスPublished - 2000 2 14

ASJC Scopus subject areas

  • Genetics
  • Genetics(clinical)

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