正交设计优化半夏相关序列扩增多态性-聚合酶链式反应体系

摘要

目的 采用正交设计优化半夏SRAP - PCR的反应体系.方法 以半夏基因组DNA为模板,应用L16 (45)正交表,研究了Taq酶、Mg2+、随机引物、dNTPs和模板DNA 5种SRAP反应组分浓度变化对扩增结果的影响.结果 优化反应体系为:25μL反应体系中含有buffer2.5μL、Taq酶1.0U、Mg2+2.5mmol/L、引物0.8μmol/L、dNTPs 0.1 mmol/L、模板DNA 50 ng.结论 正交设计能客观高效的优化半夏SRAP反应体系.%Objective To optimize the SRAP - PCR reaction system of Pinellia ternata with orthogonal design method. Methods The L16(45) orthogonal diagram was applied to evaluate the effects of different concentrations of Taq polymerase, Mg2+ ,dNTPs,primer and DNA on Pinellia ternata SRAP - PCR results. Results A suitable SRAP reaction system was developed, I. E. 2. 5 μL Taq polymerase reaction buffer ,2.5 mmol/L Mg2 + , 1.0 U Taq DNA polymerase ,0.1 mmol/L dNTPs ,0.8 μmol/L primer and 50ng genomic DNA templates with total 25 μL reaction solution. Conclusion The results showed that the orthogonal design could be used successfully to optimize the SRAP - PCR system.