Polymerase Chain Reaction-Single Strand Conformation Polymorphism (PCR-SSCP)解析法を用いたAdenine Phosphoribosyltransferase遺伝子異常の検出

Abstract

PCR-SSCP法はAPRT遺伝子の変異の検出に有用な検査法であり保因者の頻度を推定するためのスクリーニングにも応用できる.さらにこの方法を赤血球の酵素活性測定法と併用すれば, APRT欠損症を引き起こす新しい変異遺伝子を検出することも可能であろう

Polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis is a rapid and sensitive method to identify point mutations in a given sequence of genomic DNA. We tried to apply the PCR-SSCP to the diagnosis of adenine phosphoribosyltransferase (APRT) deficiency, which is an autosomal recessive hereditary disease leading to 2, 8-dihydroxyadenine urolithiasis. Genomic APRT genes, with or without mutations, were amplified and labeled simultaneously with 32P-dCTP by PCR. When run in a 6% polyacrylamide gel containing 10% glycerol, two types of mutant genes, APRT*Q0 and APRT*J, gave bands clearly distinct from those of the respective normal APRT genes. Since heterozygotes as well as homozygotes for these mutant APRT genes can be detected in 2 days, PCR-SSCP should be a valuable method in the diagnosis of APRT deficiency and in screening a large population for APRT mutant genes.