Detection of point mutations in human tyrosinase gene by improved allele-specific amplification.

Allele-specific amplification (ASA) is a simple and non-radioactive technique for detecting known point mutations that produce genetic diseases. Although this technique is based on the specific amplification of the target allele by a polymerase chain reaction (PCR) with allele-specific primers, the specificity of the amplification may depend on various PCR conditions. To avoid non-specific amplification which leads to false-positive results in ASA, we modified both the normal and mutant allele-specific primers so that they would have one constant base mismatch, located at the penultimate 3' position. We confirmed that our modification could inhibit such unfavorable amplification by using as templates genomic DNAs of patients affected with tyrosinase-negative oculocutaneous albinism (OCA). We then analyzed new patients affected with tyrosinase-negative OCA, and based the diagnosis on both the results of a clinical examination and those of a hair bulb test using ASA with the modified allele-specific primers. The results indicated that more than 3 alleles of the tyrosinase gene with a pathological mutation existed in Japanese patients.