甘草RAPD-PCR反应体系正交优化研究

Optimization of RAPD-PCR Reaction System for Glycyrrhiza uralensis Based on Orthogonal Design

[目的]建立一套适合甘草分子学研究的RAPD-PCR反应体系。[方法]以甘草种质为试材,采用正交试验法设计,对影响RAPD-PCR扩增的主要因素dNTPs、引物、Taq酶和DNA模板进行优化筛选。[结果]总体积25μl的甘草RAPD-PCR最佳反应体系为:10×PCR缓冲液(含MgCl2)2.5μl,10 mmol/L dNTPs 2.5μl,50 ng DNA 2.0μl,10μmol/L引物2.0μl,5 U/μl Taq酶0.4μl。对引物的退火温度进行了梯度筛选,34℃时扩增效果较好。[结论]进行甘草RAPD-PCR反应体系的正交优化非常有效。

[Objective] The aim was to obtain the optimum RAPD-PCR reaction system for Glycyrrhiza uralensis.[Method] Orthogonal design was adopted to screen the suitable concentration of major factors（dNTPs,primer,Taq polymerase,DNA template） in PCR reaction system.[Result] The optimal protocol was accomplished by orthogonal design in 25 μl reaction volume containing 10×PCR buffer solution（include MgCl2） 2.5 μl,10 mmol/L dNTPs 2.5 μl,50 ng DNA template 2.0 μl,10 μmol/L primer 2.0 μl,5 U/μl Taq polymerase 0.4 μl;the optimum annealing temperature was 34 ℃.[Conclusion] Orthogonal design was an effective method for the optimization of RAPD-PCR reaction system for G.uralensis.