The procedure of polymerase chain reaction — Restriction fragment — Single strand conformation polymorphism analysis by Hha I/Hinc II to detect mitochondrial DNA mutations

Abstract

We reported 56 mitochodrial DNA (mtDNA) mutations in Japanese compared with the Cambridge Sequence by polymerase chain reaction — restriction fragment — single strand conformation polymorphism (PCR-RF-SSCP) analysis. Here we report the principle and the detailed procedures of PCR-RF-SSCP analysis. Restriction map of the 15673 bp PCR product of mtDNA was designed by MacMolly Tetra ™, and Hha I and Hinc II were selected. Restriction fragment length polymorphism (RFLP) analysis of 15673 bp PCR products by Hha I/Hinc II revealed one common pattern and 10 polymorphic patterns compared with the prediction based on the Cambridge Sequence. When the samples were electrophoresed by SSCP analysis, DNA fragments of 912 bp, 772 bp, 761 bp, 634 bp, 431bp, 327 bp, 226 bp and 171 bp showed 6, 4, 5, 4, 5, 7, 2 and 3 polymorphic patterns, respectively. The determinations of nucleotide sequence of these 46 polymorphic patterns revealed 56 mutations compared with the Cambridge Sequence and 7 of these mutations were common in Japanese. Among these, 20 mutations have not been reported before.