Optimization of PCR conditions to amplify COI gene fragment of Anostraca (Branchinecta orientalis) mt DNA

Optimization of PCR conditions to amplify COI gene fragment of Anostraca (Branchinecta orientalis) mt DNA

PCR is a technique used in molecular biology to amplify a single copy or a few copies of a segment of DNA. A number of parameters such as annealing temperature, concentration of primer pairs and number of cycles of denaturation and annealing temperature are very important influencing the quality of PCR product. Therefore, the DNA extracted from Branchinecta orientalis, mt DNA (COI), and 6 different annealing temperatures 47.1, 49.7, 51.9, 53.1, 54.1 and 54.7°C in 80 secs were considered to achieve the purpose by gradient PCR. Concentration of primers, 5 µM and 10 µM and the number of cycles was considered 35 and 37 cycles. The gene was successfully amplified, giving the correct fragment lengths as assigned for both forward and reverse primers. The results show that the optimal conditions were determined to be 5 µM primer, 37c of both denaturation and annealing cycles, and annealing temperature of 49.7 (Fig.1). In conclusion, this study provides updated data to improve amplification quality of gene fragment of Branchinecta orientalis mt DNA (COI=710bp). Our findings can be also used as useful modified method to study of the other anostracan species.

PCR is a technique used in molecular biology to amplify a single copy or a few copies of a segment of DNA. A number of parameters such as annealing temperature, concentration of primer pairs and number of cycles of denaturation and annealing temperature are very important influencing the quality of PCR product. Therefore, the DNA extracted from Branchinecta orientalis, mt DNA (COI), and 6 different annealing temperatures 47.1, 49.7, 51.9, 53.1, 54.1 and 54.7°C in 80 secs were considered to achieve the purpose by gradient PCR. Concentration of primers, 5 µM and 10 µM and the number of cycles was considered 35 and 37 cycles. The gene was successfully amplified, giving the correct fragment lengths as assigned for both forward and reverse primers. The results show that the optimal conditions were determined to be 5 µM primer, 37c of both denaturation and annealing cycles, and annealing temperature of 49.7 (Fig.1). In conclusion, this study provides updated data to improve amplification quality of gene fragment of Branchinecta orientalis mt DNA (COI=710bp). Our findings can be also used as useful modified method to study of the other anostracan species.