DNA Extraction and Optimization of ISSR-PCR Reaction System for Pyracantha. Issue 5 (February 2019)

• Record Type: Journal Article
• Authors: Deng, Qian; Deng, Qunxian; Liu, Lu; Tao, Lian
• Is Part Of: IOP conference series. Volume 237:Issue 5(2019)
• Publication Date: 2019-02
• Subjects: Earth sciences -- Periodicals; Environmental sciences -- Congresses; Environmental sciences -- Periodicals; 550.5
• DOI: 10.1088/1755-1315/237/5/052025
• Languages: English
• ISSNs: 1755-1307

Abstract

Abstract: 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 Mg 2+, 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 Mg 2+, 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.