efficiency of peptide nucleic acid-directed pcr clamping and its application in the investigation of natural diets of the japanese eel leptocephali肽核酸的效率acid-directed pcr夹及其应用在日本鳗鱼leptocephali自然饮食的调查.pdfVIP

Efficiency of Peptide Nucleic Acid-Directed PCR Clamping and Its Application in the Investigation of Natural Diets of the Japanese Eel Leptocephali 1¤ 2 2 3 4 Takeshi Terahara , Seinen Chow , Hiroaki Kurogi , Sun-Hee Lee , Katsumi Tsukamoto , Noritaka 5 6 1 Mochioka , Hideki Tanaka , Haruko Takeyama * 1 Department of Life Science and Medical Bioscience, Waseda University, Shinjuku-ku, Tokyo, Japan, 2 National Research Institute of Aquaculture, Yokosuka, Japan, 3 Dong-A University, Saha-gu, Busan, Korea, 4 Atmosphere and Ocean Research Institute, The University of Tokyo, Chiba, Japan, 5 Kyushu University, Fukuoka, Japan, 6 National Research Institute of Aquaculture, Mie, Japan Abstract Polymerase chain reaction (PCR)-clamping using blocking primer and DNA-analogs, such as peptide nucleotide acid (PNA), may be used to selectively amplify target DNA for molecular diet analysis. We investigated PCR-clamping efficiency by studying PNA position and mismatch with complementary DNA by designing PNAs at five different positions on the nuclear rDNA internal transcribed spacer 1 of the Japanese eel Anguilla japonica in association with intra-specific nucleotide substitutions. All five PNAs were observed to efficiently inhibit amplification of a fully complementary DNA template. One mismatch between PNA and template DNA inhibited amplification of the template DNA, while two or more mismatches did not. DNA samples extracted from dorsal muscle and intestine of eight wild-caught leptochephalus larvae were subjected to this analysis, followed by cloning, nucleotide sequence analysis, and database h