DNA Extraction and Optimization of ISSR-PCR Reaction System for Pyracantha

4th International Conference on Advances in Energy Resources and Environment Engineering

能源学;生态环境科学

Deng, Qian^1 ; Deng, Qunxian^1 ; Liu, Lu^1 ; Tao, Lian^1

Pomology, College of Horticulture, Sichuan Agricultural University, Chengdu, Sichuan

611130, China^1

关键词: Annealing temperatures;    DNA extraction;    DNA polymerase;    Fresh leaves;    High quality;    Orthogonal design;    PCR reactions;    Taq polymerase;   

Others  :  https://iopscience.iop.org/article/10.1088/1755-1315/237/5/052025/pdf
DOI  :  10.1088/1755-1315/237/5/052025

学科分类：环境科学（综合）

来源: IOP

【 摘 要 】

Three different DNA extraction methods (CTAB, improved CTAB, and nuclear DNA method) were compared in order to isolate high-quality genomic DNA from fresh leaves of Pryacantha. The concentration of Mg2+, Taq polymerase, dNTPs, primer, and template DNA, greatly influencing ISSR-PCR of Pyracantha, were optimized by orthogonal design in this study, and the annealing temperature was also improved. The results showed that the high-quality genomic DNA was obtained by the improved CTAB method and was suitable for ISSR study. The optimal PCR system for ISSR analysis was as follows: total volume 25 μL, 2.5 μL 10 buffer, 1.0 mmol•L-1 Mg2+, 0.15 mmol•L-1 dNTPs, 0.1 umol•L-1 primer, 1.2 U Taq DNA polymerase, and 80 ng template DNA. The reaction procedure was as follows: pre-degeneration at 94 °C for 5 min, degeneration at 94 °C for 1 min, annealing at 51.0 °C∼59.2 °C for 1 min (annealing temperature depend on different primer), extension at 72 °C for 1 min, 40 cycles, final extension at 72 °C for 5 min and preservation at 4 °C.