兜兰ITS-PCR反应体系的建立及优化

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25 μL体系中,添加10×PCR buffer 2.5 μL、Taq DNA酶1.25U、Mg2+ 1.5mmol/L、dNTP 0.15 mmol/L、引物0.6μmol/L和模板DNA 40 ng,反应程序为94℃预变性4 min,1个循环；94℃变性30 s,54℃退火45 s,72℃延伸1 min,35个循环；72℃延伸7 min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.

Construction and Optimization of ITS-PCR Reaction System for Paphiopedilum

To optimize the ITS-PCR reaction system for Paphiopedilum,taking Paphiopedilum as the material,the total DNA was extracted by improved CTAB,and the effects of factors,including TaqDNA polymerase dosage,Mg2+ 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,Mg2+ 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.