连翘ISSR-PCR反应体系的建立及优化

Establishment and optimization of Forsythia suspensa ISSR—PCR reaction system

通过正交设计的方法优化连翘ISSR—PCR反应体系,PCR结果用MINITAB 16数据处理软件分析,建立了适用于连翘ISSR反应的最适反应体系(25μL):Taq DNA聚合酶1.2U、Mg2+浓度1.5 mmol·L-1、模板DNA 20～80 ng、dNTPs 0.3 mmol·L-1、引物0.3μmol·L-1。进行了梯度退火试验,得到连翘ISSR—PCR反应体系最佳退火温度为55℃。

The ISSR-PCR amplification system of Forsythia suspensa was optimized by using orthogonal design method with five factors （Taq DNA polymerase, Mg^2＋, DNA template, dNTPs and primer） and four levels respectively. The results of PCR was analyzed by software MINITAB. A suitable ISSR reaction system was established. It is 25 μL reaction system, containing 1.2 U Taq DNA polymerase, 1.5 m mol·L^-1 Mg^2＋, 20-80 ng DNA template, 0.3 m mol·L^-1 dNTPs and 0.3 μmol·L^-1 primer. Through the gradient PCR test, the optimal annealing temperature for ISSR-PCR reaction turned out to be 55 ℃.