イ．遺伝子解析による同定方法 - maff.go.jp

1 17 PCR-RFLP PCR-RFLP DNA PCR-RFLP PCR-RFLP PCR-RFLP DNA PCR PCR-RFLP A B 18 A LepF1 (short) 3 5 -ATTCAACCAATCATAAAGATAT-3 Xsp I Ase I Sac I LepR1 (short) 3 5 -TAAACTTCTGGATGTCCAAAAA-3 B C1-J-2797 4 5 -CCTCGACGTTATTCAGATTACC-3 Alu I Dra I Nla III C2-N-3400 5 5 -TCAATATCATTGATGACCAAT-3 COI 710 COI-COII 610 Hebert et al.

2 (2004) Simon et al. (1994) Taylor et al. (1997) PCR-RFLP a DNA 1 1 SNET 100 l K 5mg/ml l SNET 50 l 55 C60 95 C 5 4 C 10 l ( ) ml PCR 90 l TE 19 Lysis buffer for PCR URL 60 C 98 C DNA < > C SNET 1M Tris-HCl (pH ) 2ml (20mM) 5M NaCl 8ml (400mM) EDTA 1ml (5mM) 10% SDS 3ml ( ) 86ml (100ml ) 100ml b PCR ( )ml PCR 19 l DNA l PCR A B Ampdirect@Plus l EX Taq HS l l F (10 M) l R (10 M) l DNA l l Ampdirect@Plus (Shimazu) SNET Ampdirect@Plus < > EX Taq HS < > 20 PCR A B 94 C 94 C 94 C 94 C 45 45 C 30 46 C 45 72 C 15 72 C 45 94 C 72 C 51 C 30 72 C 1 15 72 C c PCR PCR l 1 100V 40 PCR 100bp TA E TBE PCR Gel Red 2 Gel Red 1 S < > 2 Gel Red < > d ( )

3 Ml PCR l PCR l 37 C 35 36 21 l l l PCR l l A Xsp I Ase I Sac I B Alu I Dra I Nla III e 10 l DNA 50bp c PCR f A PCR Xsp I Ase I Sca I A F Sac I A, B 10 XspI AseI SacI 11 , Xsp I (kbp) A B C D E F 22 Ase I (kbp) A B C D E F Sac I (kbp) A ( ) B 23 10 , , M 50bp (Xsp I , Ase I), (Sac I).

4 11 M (kbp) M Xsp I Ase I Sac I A B A B A A A A B A A A B C D D E F F F A A B C D C C C E F 24 11 A F Ase I C D Sac I B PCR Alu I Dra I Nla III 10 11 Alu I Dra I Nla III 10 10 12 10 Lewter and Szalanski, 2007 (kbp) Alu I Dra I Nla III Lewter and Szalanski (2007) Nla III 25 12 14 Alu I Dra I Nla III M 100bp M