An insect cell line discrimination method by RAPD-PCR.
RAPD-PCR with a tenmar single primer for discrimination of insect cell lines was devised. The base sequence of the primers used were TTCGAGCCAG, CGGCATCTAC, GAACGGACTC, and TGAGTGGGTG (GC contents were 60%). Genome DNA was extracted by modified Landry et al. (1993) method. The reaction mixture consisted of 10 microliters buffer, 8 microliters dNTP mixture (2.5 mM each), 4 microliters primer (50 microM), Taq DNA polymerase (2.5 units), 1 microliter template DNA; and the reaction was run at 94 degrees C for 2 min (denaturation), followed by 31 cycles of 94 degrees C for 1 min, 42 degrees C for 1 min (annealing), and 72 degrees C for 2 min (extension) and terminated with 72 degrees C for 7 min. By developing the reaction products with agarose gel electrophoresis, it became evident that DNA fragments were amplified with all the primers used. Among four primers, the second primer was selected as a suitable primer for distinguishing cell lines. With this method, cell lines derived from different species were clearly distinguished.
Kawai
Y
Y
Faculty of Agriculture, Tokyo University of Agriculture and Technology, Japan.
Mitsuhashi
J
J
D000818:Animals; D002460:Cell Line; D004247:DNA / Q000032:analysis*; D017931:DNA Primers; D004175:Diptera / Q000166:cytology; D004587:Electrophoresis, Agar Gel; D007313:Insecta / Q000166:cytology* / Q000235:genetics*; D007915:Lepidoptera / Q000166:cytology; D016133:Polymerase Chain Reaction / Q000379:methods*; D011110:Polymorphism, Genetic
D017931:DNA Primers; D004247:DNA