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A fluorogenic allele‐specific amplification method for DNA‐based screening for inherited metabolic disorders
Author(s) -
Matsubara Y,
Fujii K,
Rinaldo P,
Narisawa K
Publication year - 1999
Publication title -
acta pædiatrica
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.772
H-Index - 115
eISSN - 1651-2227
pISSN - 0803-5253
DOI - 10.1111/j.1651-2227.1999.tb01162.x
Subject(s) - taqman , point mutation , polymerase chain reaction , microbiology and biotechnology , allele , mutation , medicine , genomic dna , cold pcr , genetics , multiplex , dna , biology , gene
Rapidly growing mutation databases for various inherited metabolic diseases raise the possibility of neonatal screening by DNA‐based diagnosis. It is therefore important to develop a simple DNA diagnostic method which is suitable for processing a large number of samples. We have devised an allele‐specific amplification (ASA) method with a fluorogenic probe (TaqMan probe) to detect point mutations. A pairwise PCR amplification with two sets of allele‐specific primers was performed in the presence of the TaqMan probe with real‐time fluorescence monitoring on an ABI PRISM 7700 sequence detector. The difference in the amplification efficiency between two PCR reactions was determined by “threshold” cycles to differentiate mutant and normal alleles. The method, TaqMan‐ASA, does not require post‐PCR processing, thus obviating potential PCR contamination. Since the entire procedure can be carried out in a 96‐well microtiter plate format, it is easy to automate. We successfully applied TaqMan‐ASA to detect a common g727t mutation in Japanese patients with glycogen storage disease type Ia and a prevalent a985g mutation in Caucasian patients with medium‐chain acyl‐CoA dehydrogenase deficiency.