兜兰ITS-PCR反应体系的建立及优化

Construction and Optimization of ITS-PCR Reaction System for Paphiopedilum

为优化兜兰ITS-PCR反应体系,以兜兰属植物为试材,用改进的CTAB法提取总DNA,并采用单因子试验设计,对影响兜兰DNA ITS-PCR扩增反应的主要因素即Taq DNA聚合酶的用量、Mg2+浓度、dNTP浓度、引物退火温度、模板DNA用量和引物浓度等进行优化研究,建立兜兰最佳ITS扩增反应体系。结果表明:最佳反应体系为25μL体系中,添加10×PCR buffer 2.5μL、Taq DNA酶1.25U、Mg2+1.5mmol/L、dNTP 0.15mmol/L、引物0.6μmol/L和模板DNA 40ng,反应程序为94℃预变性4min,1个循环;94℃变性30s,54℃退火45s,72℃延伸1min,35个循环;72℃延伸7min后终止反应,4℃保存。利用此反应体系可得到预期大小的ITS基因片段。

To optimize the ITS-PCR reaction system for Paphiopedigurn, taking Paphiopedilum as the material, the total DNA was extracted by improved CTAB, and the effects of factors, including TaqDNA polymerase dosage, Mg^2＋ concentration, dNTP concentration, annealing temperature, DNA template dosage and primer concentration, on the ITS-PCR reaction system. The results showed that the optimum conditions were as follows： reaction system 25 μL, 10 ×PCR buffer 2. 5 μL, TaqDNA polymerase 1.25 U, Mg^2＋ 1.5 mmol/L, dNTP 0.15 mmol/L, primer 0.6 μmol/L and DNA template 40 ng. The PCR procedures were described as follows： 94℃ for 4 min, followed by 35 cycles of denaturing at 94℃ for 30 s, annealing at 54℃ for 45 s, and extending at 72℃ for one minute,35 circulation in total; finally extended at 72℃ for 7 min. The expectant ITS gene section could be obtained in this reaction system.