Development of a universal primer for PCR in mtDNA D-loop region of salamanders from Japan

Abstract

This study developed a set of universal primer of the PCR in mitochondrial DNA D-loop region that is applicable to almost all species of Japanese salamanders. As the first step, the sequence of 1316 base pairs (including a part of unknown sequences of about 340bp) in the region of tRNA^pro-D-loop-tRNA^phe-12SrRNA was determined for the mitochondrial DNA extracted from muscle tissue of the Oita salamander (Hynobius dunni). Using the sequence, we developed a set of PCR primer of HDL-F1 (5'-GGC ACC CAA AGC CAA AAT TC-3') and HDL-R 4 (5' TAG ATC CAG ACC AGA TGC CGG-3') to target 222 bp in the former part of the D-loop region. This primer set should be useful for studies to analyze the intra-specific genetic variations because this part of the D-loop region shows a high rate of base displacement. In this study, we also show sequences of the former part of the D-loop region (222 bp) for 5 Japanese salamander species cut by the present primer set.