正交设计优化莲藕ISSR-PCR反应体系研究

Optimization for ISSR-PCR Reaction System in Lotus Root Using Orthogonal Design

利用正交实验设计的方法,对莲藕ISSR-PCR反应的四因素(TaqDNA聚合酶浓度、dNTP浓度、引物浓度、Mg2+浓度)三水平进行优化分析,并在此基础上对模板DNA浓度、PCR反应过程中的退火温度进行梯度检测。结果表明:20μL的ISSR-PCR反应体系中各因素的最佳浓度为:10×PCRbuffer 2.0μL、dNTP150μmol/L、引物1.2μmol/L、Mg2+2.0 mmol/L和TaqDNA聚合酶4 U,最佳模板DNA浓度为40～60 ng,引物U826的最佳退火温度为48.7℃。

The orthogonal design was used to optimize the ISSR-PCR amplification system of Lotus root in four factors（the concentration of）TaqDNA polymerase,dNTP,Primers and Mg2＋） at three levels.Then,based on the optimal ISSR-PCR amplification system,the concentration of template DNA and annealing temperature were proposed by gradient PCR.The results showed that 10×PCR buffer 2.0 μL,dNTP 150 μmol/L,primers1.2 μmol/L,Mg2＋ 2.0 mmol/L,Taq DNA polymerase 4 U and 40～60 ng template DNA in a total volume of 20 μL were suitable for ISSR-PCR amplification system of lotus root.The optimal annealing temperature for primers U826 was 48.7℃.