甘草RAPD-PCR反应体系正交优化研究

摘要

[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,10mmol/LdNTPs2.5μl,50ng DNA2μl,10μmol/L引物2μl,5UTaq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 four major factors（dNTPs,primers,Taq polymerase and DNA template） in PCR reaction system.[Result] The optimal reaction system obtained by orthogonal design was 25 μl in total volume,containing 2.5 μl of 10×PCR buffer solution（include MgCl2）,2.5 μl of 10 mmol/L dNTPs,2 μl（100 ng） of DNA template,2 μl of 10 μmol/L primers,0.4 μl（5 U） of Taq polymerase;the optimum annealing temperature was 34 ℃.[Conclusion] Orthogonal design was an effective method for the optimization of RAPD-PCR reaction system for G.uralensis.