Note PCR Error and Molecular Population Genetics Norio Kobayashi, 1±3 Koichiro Tamura, 2 and Tadashi Aotsuka 2 Received 2 Apr. 1999ÐFinal 15 June 1999 INTRODUCTION The polymerase chain reaction (PCR) technique (Saiki et al., 1988) is a very useful tool for population genetic studies at the DNA level. However, there is an inherent problem in the PCR technique because the DNA fragments ampli® ed are subject to nucleotide substitution during the reaction processes. The rate and pattern of arti® cial substitution have already been estimated by several authors (e.g., Lundberg et al., 1991), and its in¯ uence on phylogenetic studies has been also discussed (Kwiatowski et al., 1991). However, the in¯ uence of PCR error on population genetic studies has not been well documented. Determinations of the sequences from PCR products were performed by two methods, cloning and direct sequencing. Assuming that the sequences obtained by the direct sequencing method are correct, we discuss the in¯ uence of the error on the estimates of various parameters commonly used in molecular population genetics. In addition, we evaluate the efficiency of Pfu polymerase to improve the ® delity of PCR. MATERIALS AND METHODS Four species of Epilachna ladybird beetles, E. vigintioctomaculata, E. niponica, E. pustulosa, and E. yasutomii, were used in this study. To estimate the rate and pattern of PCR error using Taq polymerase and Taq 1 Pfu polymerase, 33 and 22 beetles, respectively, were used. According to Steller (1990), total DNA for PCR template was extracted individually from fresh or alcohol-preserved specimens. For PCR ampli® cation, primer designs were as described by Kobayashi et al. 1 Division of Biological Science, Graduate School of Science, Hokkaido University, Sapporo, Hokkaido 060-0810, Japan. 2 Department of Biology, Graduate School of Science, Tokyo Metropolitan University, 1-1 Minami- ohsawa, Hachioji-shi, Tokyo 192-0397, Japan. 3 To whom correspondence should be addressed at Division of Biological Science, Graduate School of Science, Hokkaido University, Sapporo, Hokkaido 060-0810, Japan. Fax: 1 81-11-746-0862. Biochemical Genetics, Vol. 37, Nos. 9/10, 1999 317 0006-2928/99/1000-03 17$16.00/0 r 1999 Plenum Publishing Corporation