三叶木通RAPD最佳反应体系的研究

Optimization of Akebia.trifoliata(Thunb) Koidz.Genomic DNA Isolation and RAPD Condition

RAPD技术是一种新的分子标记技术,DNA质量是保证RAPD分析成功的关键。为获得高质量的木通属植物基因组DNA,本文采用SDS法、改良CTAB法、高盐低pH值法提取三叶木通总DNA。结果表明,高盐低pH值法不适用于木通属植物DNA的提取;改良CTAB法是合适的DNA提取方法,它们获得DNA模板纯度高、质量好,可直接用于RAPD分析。同时,对RAPD反应条件中的一些重要参数进行摸索和优化试验,建立适合木通属植物RAPD分析应用的体系,其组成为:反应液总体积为25μL,2.5μL 10×PCR缓冲液、0.16 mmol/L dNTPs2、.0 mmol/L Mg2+、0.64μmol/L引物、1 U/μL Taq酶4、0 ng/25μLDNA模板。PCR循环体系为:94℃,4 min-[-94℃,30 s-37℃,90 s-72℃,60 s-]-72℃,5 min,40个循环。

RAPD technique is a new method for detecting genetic variation. The quality of DNA sample is important in RAPD analysis. To explore method for high-quality DNA extract from Akebia. trifoliate, seven different methods were tested in isolation of Akebia. trifoliata （ Thunb ） Koidz. Genomie DNAs. The results show that the high salt-low pH method is not applicable to Akebia. trifoliata while the reformed CTAB is more effective. The isolated DNA is proved suitable for RAPD- PCR amplification. Moreover, an optimal reaction system suitable for the assay and usage of RAPD in Akebia. trifoliate was established. This optimal of 25 μL reaction solution contains 2.5 μL 10 × PCR buffer,0.16 mmol/L each dNTP,2.0 mmol/L Mg^2＋,0.4μmol/L S1 random primer, 1 U Taq polymerase and 40 ng/25μL template DNA. After pre-denaturing at 94 ℃ for 4 min, under the condition of denaturing at 94μ for 30 s, annealing at 37 μ for 90 s, extension at 72 μ for 1 min, amplifing for 40 cycles, and extension at 72μ for 5 min at last.