高丹草SRAP-PCR反应体系优化及验证

Optimization and verification of a simple sequence-related amplified polymorphism(SRAP)-PCR system in Sorghum-Sudan hybrid grass

【目的】筛选高丹草SRAP-PCR反应的最佳体系。【方法】采用单因素和正交试验设计相结合的方法,对影响高丹草SRAP-PCR反应体系的5个因素(dNTP、DNA、引物、Taq DNA聚合酶、Mg^(2+))进行优化;并对高丹草SRAP-PCR优化体系进行验证。【结果】高丹草SRAP-PCR的最佳反应体系为:dNTP浓度275μmol/L、模板DNA浓度3.0 ng/μL、引物浓度1.1μmol/L、Taq DNA聚合酶用量1.00 U、Mg^(2+)浓度2.25 mmol/L,用ddH_(2)O补足至20μL。【结论】筛选出的SRAP-PCR最佳体系扩增条带数目多、多态性丰富、重复性好。

【Objective】To find the optimal Sorghum-Sudan hybrid grass SRAP-PCR reaction system.【Methods】The five components(dNTP,template DNA concentration,primers,Taq DNA polymerase,and Mg^(2+))were optimized using single-factor and orthogonal-design approaches.The optimized SRAP-PCR reaction system was verified.【Results】The best SRAP-PCR system for Sorghum-Sudan hybrid grass contained 275μmol/L dNTP,3.0 ng/μL template DNA,1.1μmol/L for each primer,1.00 U Taq DNA polymerase,and 2.25 mmol/L Mg^(2+),making up to 20μL with ddH_(2)O.【Conclusion】The optimized SRAP-PCR reaction system amplified many bands,with abundant polymorphism and good repeatability.