巴东木莲DNA的提取及SRAP-PCR反应体系的正交优化

Isolation of Genomic DNA and Optimization of the SRAP-PCR Reaction System by Orthogonal Design in Manglietia patungensis

研究确定了提取巴东木莲基因组DNA的方法,采用正交试验设计法对影响SRAP-PCR反应的引物浓度、Taq DNA聚合酶的用量、Mg2+和dNTPs浓度及PCR扩增程序中的退火温度及循环次数进行了比较、优化,同时对DNA模板浓度进行了筛选。结果表明,Mg2+、dNTPs、Taq酶及引物的不同水平均对PCR反应结果有显著的影响。建立了巴东木莲20μL SRAP-PCR的反应体系为1×buffer、Mg2+浓度为2.0 mmol·L-1、dNTPs浓度为0.25 mmol·L-1、引物浓度为0.45μmol·L-1、Taq酶为0.5 U和模板DNA 40ng。适宜的扩增程序为94℃预变性1 min,94℃变性1 min,33℃复性1 min,72℃延伸1 min,10个循环;94℃变性1 min,55℃复性1 min,72℃延伸1 min,30个循环;最后72℃延伸5min。试验表明,该体系重复性好、稳定性强。

An efficient method for isolation of high quality genomic DNA from Manglietia patungensis was determined. Some factors involving primer, Taq DNA polymerase, Mg^2＋, dNTPs, annealing temperature and cycle times affecting the SRAP-PCR researching system were optimized. And the concentration of genomic DNA templates were filtrated at the same time. The results showed that Mg^2＋, dNTPs, Taq polymerase and primer in different levels had the marked influence on the result of PCR. The optimal PCR reaction system for M. patungnesis was 2μL 10xbuffer, 2.0mmol·L^-1 Mg^2＋, 0.25mol·L^-1 dNTPs, 0.45 μmol·L^-1 primers, 0.5U Taq polymerase and 40ng template DNA in a total volume of 20μL SRAP-PCR reaction system. The most suitable protocol was initially denaturing at 94℃ for 1 min, then the first ten cycles were run at 94℃, for 1 rain, 33℃ for l min, and 72℃ for 1 rain, for denaturing, annealing and extension, respectively. In the following 30 cycles the annealing temperature was raised to 55℃, with a final elongation step of 5 min at 72℃. The new established SRAP-PCR system of M. patungensis was with good repetition and stablility.