Abstract:

Genomic DNA was isolated from the musculature of Macrobrachium nipponensis to establish a reproducible and stable PCR technical system. The ingredients and programs of amplified reactions were optimized respectively. The results showed that the profiles of PCR products could be improved obviously by using the 25 μL solution of 10×Buffer 2.5 μL, Mg⁺² 1.5 mmol/L, 4×dNTP 0.2 mmol/L, 1 U Taq DNA polymerase, 0.4 μmol/L of each primer and 200~300 ng of DNA template as well as the programs of 5 min at 95 ℃, 35 cycles of 1 min at 95 ℃, 50 s at 56 ℃, 1 min at 72 ℃ and finally, 10 min at 72 ℃. The optimized conditions could result in satisfactory specificity and reproducibility in amplifying 18S rRNA,COI,ND5 gene fragments, and consequently provide a useful means for the research of genetic diversity, molecular marker and germplasm identification in Macrobrachium nipponensis.

Key words: polymerase chain reaction, optimization, Macrobrachium nipponensis, polymerase chain reaction, optimization