Mutation Detection by TaqMan-allele specific amplification: Application tomolecular diagnosis of glycogen storage disease type Ia and medium-chain acyl-CoA dehydrogenase deficiency

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.