人参SRAP-PCR体系优化条件的建立

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、Mg^2＋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,Mg^2＋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。

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

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 Mg2+ 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,2.0 μmol/L upstream primer and downstream primer,0.3 mmol/L dNTPs,2.5 mmol/L Mg2+,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.