马蹄金ISSR反应体系的正交优化

Optimization of the ISSR Reaction System of Dichondra repens by Orthogonal Design

以CTAB法提取的马蹄金叶片DNA为模板,应用L16(45)正交表系统分析了DNA、Mg2+、dNTP、Taq酶、引物5种ISSR反应成分浓度变化对扩增结果的影响。量化分析结果表明:Taq酶、dNTP不同水平对PCR反应结果有显著影响,正交设计可以应用于ISSR-PCR反应体系的建立,用这种方法建立的马蹄金IS-SR优化反应体系为:1×buffer,25ng模板DNA,1.75mmol/LMg2+,225μmol/LdNTP,0.9μmol/L引物,1.25UTaqDNA聚合酶,总体积20μl。这一优化系统的建立为今后利用ISSR标记技术,研究马蹄金的地理变异提供一个标准化程序。

An orthogonal diagram as experimental design was applied to study the effects of different levels of DNA, Mg^2＋, dNTP, Taq polymerase and primer on ISSR results for D. repens in order to optimize the ISSR-PCR system of the plant species. The results indicated that Taq DNA polymerase and dNTP in different levels were the effective factors to the result of PCR, and the orthogonal design could be used to optimize the ISSR-PCR system. The suitable PCR reaction system for D. repens was included by 1 xbuffer, 1.75mmol/L Mg^2＋, 225μmol/L dNTP, 0.9μmol/L primer, 1.25U Taq DNA polymerase and 25ng genomic DNA templates with total 20μl reaction solution. The result provided a standardizing ISSR-PCR program for the analysis of geographic variation of D.repens.