丹参ISSR-PCR反应体系的建立与正交优化

Establishment and orthogonal optimization of ISSR-PCR amplification system in Salvia miltiorrhiza

关键词:  丹参  ISSR-PCR  反应体系  正交优化

Key words:  Salvia miltiorrhiza  ISSR-PCR  amplification system  orthogonal optimization

摘要:

利用正交试验设计的方法,从引物浓度、Taq DNA聚合酶浓度、Mg²⁺浓度、dNTP浓度4种因素3个水平,对丹参ISSR-PCR反应体系进行优化分析,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:20 μL ISSR-PCR反应体系中各因素的最佳浓度为1×PCR buffer、200 μmol/L dNTP、1.0 μmol/L引物、1.5 mmol/L Mg²⁺和1 U Taq DNA聚合酶,最佳模板DNA浓度为20～60 ng,引物UBC 835的最佳退火温度为51.7 ℃。

Abstract:

In this paper,the orthogonal design was used to optimize the ISSR-PCR amplification system of Salvia miltiorrhiza in four factors(the concentration of dNTP,Primers,Mg²⁺ and Taq DNA polymerase)at three levels respectively. 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: a suitable ISSR-PCR amplification system of S.miltiorrhiza was established. In a total volume of 20 μL ISSR-PCR amplification system,it contains 1×PCR buffer,200 μmol/L dNTP,1.0 μmol/L primer,1.5 mmol/L Mg²⁺,1 U Taq DNA polymerase and 20～60 ng template DNA. The optimal annealing temperature for primer UBC 835 is 51.7 ℃.