Mutation detection by TaqMan‐allele specific amplification: Application to molecular 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 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. Hum Mutat 15:189–196, 2000. © 2000 Wiley‐Liss, Inc.