Mutation Detection by TaqMan-allele specific amplification: Application tomolecular diagnosis of glycogen storage disease type Ia and medium-chain acyl-CoA dehydrogenase deficiency
K. Fujii et al., Mutation Detection by TaqMan-allele specific amplification: Application tomolecular diagnosis of glycogen storage disease type Ia and medium-chain acyl-CoA dehydrogenase deficiency, HUM MUTAT, 15(2), 2000, pp. 189-196
We have devised an allele specific amplification method with a TaqMan fluor
ogenic probe (TaqMan-ASA) for the detection of point mutations. Pairwise PC
R amplification using two sets of allele specific primers in the presence o
f a TaqMan probe was monitored in real time with a fluorescence detector. D
ifference in amplification efficiency between the two PCR reactions was det
ermined by "threshold" cycles to differentiate mutant and normal alleles wi
thout post-PCR processing. The method measured the efficiency of amplificat
ion rather than the presence or absence of end-point PCR products, therefor
e allowing greater flexibility in designing allele specific primers and an
ample technical margin for allelic discrimination. We applied the TaqMan-AS
A method to detect a prevalent 727G>T mutation in Japanese patients with gl
ycogen storage disease type Ia and a common 985A>G mutation in Caucasian pa
tients with medium-chain acyl-CoA dehydrogenase deficiency. The method can
be automated and may be applicable to the DNA diagnosis of various genetic
diseases. Hum Mutat 15:189-196, 2000. (C) 2000 Wiley-Liss, Inc.