正交优化枣树ISSR-PCR反应体系的研究

Research on Orthogonal Optimization of ISSR-PCR System in Chinese Jujube

为建立适用于枣树ISSR-PCR的最佳反应体系,本研究针对Mg2+、dNTPs、TaqDNA聚合酶和引物共4个因素分别设置4个浓度水平进行正交优化设计。采用极差、方差和多重比较的方法进行分析,以期获得最佳优化体系。研究结果表明:20μLISSR-PCR反应体系中4个因素的最佳浓度分别为2.0mmol/LMg2+、0.2mmol/LdNTPs、0.05U/μLTaqDNA聚合酶和0.6μmol/L引物。采用该优化体系对枣树不同品种进行扩增,扩增条带清晰、锐利,进一步证实了该体系的稳定性。

The four factors such as Mg2 ＋ ,dNTPs, Taq DNA polymerase and primers at four different concentration levels were designed by orthogonal optimization set for establishing ISSR-PCR reaction system in Chinese Jujube. The methods such as range, variance and multiple comparison were used to obtain the best optimized system. The results showed that the best concentrations of these factors in the 20 μL reaction system were 2. 0 mmol/L Mg2 ＋ ,0. 2 mmol/L dNTPs,0. 05 U/μL Taq DNA polymerase and 0.6 μmol/L primers respectively. The optimized system was applied to amplify different varieties of Chinese Jujube the obtained clear sharp lanes proved the stability of them in advance.