Optimization of ISSR-PCR reaction system of Tussilago farfara based on orthogonal design / 中国药学杂志

ABSTRACT

OBJECTIVE: To analyze the influence of main factors and second order interaction between main factors in Tussilago farfara ISSR-PCR systems and build ISSR reaction system with high stability and repeatability. METHODS: Based on the analysis of variance, an orthogonal table L27(313) was used to optimize the ISSR-PCR amplification system of T. farfara by four factors at three concentration levels, respectively. In addition, analysis of variance and range analysis were used to analyze the result of the experiment. RESULTS: A highly stable and repeatable ISSR-PCR reaction system was constructed. The factor Mg2+ had the most significant effect on T. farfara ISSR-PCR system (P<0.01), followed by Taq polymerase (P<0.05) and dNTPs (P<0.05). Among the second order interactions, the interaction of Mg2+ and dNTPs had the largest influence on the system (P<0.1). CONCLUSION: When building ISSR-PCR reaction system, special attention should be paid to the influence of the main factors and interaction between main factors on the whole ISSR-PCR system.