中国生物工程杂志

To amplify microsatellite loci, one fluorescently labeled multiplex-PCR was constructed and optimized using capillary electrophoresis. First, according to the expected length, 9 microsatellite loci were divided into 2 groups , 5 labeled with FAM (Blue) and 4 labeled with HEX (Green). Different fluorescent groups were optimized separately using agar gel electrophoresis. Second, 8 Chinese mitten crabs were was amplified by one set of fluorescently labeled primers（9 loci multiplex-PCR. The Multiplex-PCR products were detected through capillary electrophoresis with ROX500 as size standard and analysised using Genemapper3.5. The results show that capillary electrophoresis has 1 bp precision and can differentiate the main peak and the stutter bands caused by slippage. Uniform PCR products were obtained by adjusting the ratio of primer concentration. Finally, the fundamentals parameters （dNTP concentration、 PCR program and template DNA concentration） were tested one by one. This study showed it was precise, efficient and stable to genotype microsatellite by the detecting the fluorescent multiplex-PCR with capillary electrophoresis.