人参SRAP-PCR体系优化条件的建立

Establishment of the Optimization Conditions of SRAP-PCR System in Ginseng

[目的]研究人参基因组SRAP-PCR的扩增条件,建立其优化扩增体系。[方法]采用单因子实验方法探讨模板DNA、引物浓度、dNTPs浓度、Mg2+浓度等因素对PCR结果的影响。[结果]优化后的扩增程序为:94℃预变性2 min;94℃变性30 s,48℃复性30 s,72℃延伸1 min,共40次循环;72℃延伸7 min。最佳反应体系为:DNA模板30 ng,上下游引物浓度2.0μmol/L,dNTPs浓度0.3 mmol/L,Mg2+2.5 mmol/L,总体积25μl。[结论]建立了满足人参SRAP-PCR的优化扩增体系,为人参亲缘关系和遗传多样性SRAP分析提供快速、简便、重复性好的实验方法。

[ Objective ] The research aimed to study the amplification conditions of SRAP- PCR of ginseng genome and set up the optimized amplification system. [ Method ] The effects of template DNA, primer, dNTPs and Mg^2＋ on PCR results were disussed by single factor experiments. [ Result ] The optimized amplification procedure was as follows： pre-denaturing at 94℃ for 2 min; denaturing at 94 ℃ for 30 s, annealing at 48℃ for 30 s, extending at 72℃ for 1 min, 40 cycles ; extending at 72 ℃ for 7 min. The optimum reaction system was as follows： 30 ng DNA template, 2.0μmol/L upstream primer and downstream primer, 0. 3 mmol/L dNTPs, 2.5 mmol/L Mg^2 ＋ , the total volume was 25 μl. [ Conclusion] The optimization amplification system for SRAP-PCR of ginseng was set up, which provided rapid and simplified test methods with good repeatability for SRAP analysis of the genetic relationship and genetic diversity of ginseng.