中药艾总DNA的提取及ISSR-PCR反应体系的优化

Total DNA Extraction from Artemisia argyi and Optimization on ISSR-PCR Reaction System

为探索艾的遗传多样性,采用正交试验方法 L16(45)与单因素分析法相结合对其PCR反应的5个主要因素(Mg2+、引物、模板DNA、dNTPs以及Taq酶)在4个水平上分别进行优化。结果表明:得到最佳的反应体系(20μL)为模板DNA 60ng,Mg2+2.5mmol/L,引物0.8μmol/L,dNTPs 0.15mmol/L,Taq酶1.0U。通过统计数据分析,对艾ISSR-PCR反应的影响程度依次为Mg2+浓度>引物>模板DNA>dNTPs浓度>Taq酶。

In order to explore the genetic diversity of A.argyi,an orthogonal experimental design was employed combined with single factor analysis to optimize the five main factors（Mg^2＋,primers,template DNA,dNTPs and Taq DNA enzyme）of PCR reaction in four levels.Results：The optimal ISSR-PCR reaction system：20μL system,template DNA 60 ng,Mg^2＋2.5 mmol/L,primer 0.8μmol/L,dNTPs0.15mmol/L,Taq DNA enzyme 1.0 U.The results of range analysis showed that the order of the influence degree to ISSR was Mg^2＋ primer〉template DNA 〉dNTPs〉 Taq DNA enzyme.