A fluorogenic allele-specific amplification method for DNA-based screening for inherited metabolic disorders.
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.
Matsubara
Y
Y
Department of Medical Genetics, Tohoku University School of Medicine, Aobaku, Sendai, Japan. ymats@mail.cc.tohoku.ac.jp
Fujii
K
K
Rinaldo
P
P
Narisawa
K
K
D000483:Alleles; D001483:Base Sequence; D004252:DNA Mutational Analysis / Q000379:methods*; D005260:Female; D030342:Genetic Diseases, Inborn / Q000175:diagnosis; D006801:Humans; D007231:Infant, Newborn; D008297:Male; D008661:Metabolism, Inborn Errors / Q000175:diagnosis* / Q000235:genetics; D008969:Molecular Sequence Data; D015997:Neonatal Screening / Q000379:methods; D021141:Nucleic Acid Amplification Techniques; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity