title: Mutation detection by TaqMan-allele specific amplification: application to molecular diagnosis of glycogen storage disease type Ia and medium-chain acyl-CoA dehydrogenase deficiency.
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.
authors.affiliation: Department of Medical Genetics, Tohoku University School of Medicine, Sendai, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Fujii
authors.forename: Y
authors.initials: Y
authors.lastname: Matsubara
authors.forename: J
authors.initials: J
authors.lastname: Akanuma
authors.forename: K
authors.initials: K
authors.lastname: Takahashi
authors.forename: S
authors.initials: S
authors.lastname: Kure
authors.forename: Y
authors.initials: Y
authors.lastname: Suzuki
authors.forename: M
authors.initials: M
authors.lastname: Imaizumi
authors.forename: K
authors.initials: K
authors.lastname: Iinuma
authors.forename: O
authors.initials: O
authors.lastname: Sakatsume
authors.forename: P
authors.initials: P
authors.lastname: Rinaldo
authors.forename: K
authors.initials: K
authors.lastname: Narisawa
mesh_terms: D042964:Acyl-CoA Dehydrogenase; D044944:Acyl-CoA Dehydrogenases / Q000172:deficiency / Q000235:genetics*; D000483:Alleles; D020137:Base Pair Mismatch / Q000235:genetics; D004252:DNA Mutational Analysis / Q000379:methods*; D017931:DNA Primers; D015342:DNA Probes; D004305:Dose-Response Relationship, Drug; D005456:Fluorescent Dyes; D005820:Genetic Testing / Q000379:methods; D005838:Genotype; D005953:Glycogen Storage Disease Type I / Q000175:diagnosis / Q000235:genetics*; D006801:Humans; D007564:Japan; D017354:Point Mutation / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity; D019914:Taq Polymerase / Q000378:metabolism*; D013698:Templates, Genetic; D013997:Time Factors; D044465:White People / Q000235:genetics
chemical_list: D017931:DNA Primers; D015342:DNA Probes; D005456:Fluorescent Dyes; D044944:Acyl-CoA Dehydrogenases; D042964:Acyl-CoA Dehydrogenase; D019914:Taq Polymerase