华山松SRAP-PCR反应体系的优化

Optimization of SRAP-PCR System for Pinus armandii

为建立华山松SRAP-PCR反应体系,研究先采用L16(45)正交设计对影响华山松SRAP反应的5个因子(DNA模板浓度、Mg2+浓度、dNTPs浓度、引物浓度、Taq酶)在4个水平上进行优化试验。结果表明:各因素对SRAP反应的影响依次为:Mg2+>dNTPs>Taq酶>DNA模板>引物;对SRAP反应结果影响较大的3个因子(Mg2+浓度、dNTPs浓度、Taq酶浓度)进行单因素试验;确立华山松SRAP反应最佳体系为:20μL的PCR体系中含有Mg2+2.2mmol/L、dNTPs0.25mmol/L、DNA模板60ng、Taq酶0.8mol/s、引物0.8μmol/L。将该体系用于华山松的SRAP扩增能获得稳定、清晰的多态性条带。

To establish appropriate SRAP reaction system for Pinus armandii, the orthogonal design was used to optimize the concentration of Mg2＋ , DNA template, dNTPs, primer and Taq DNA polymerase concentration at four levels. The results showed that the effect degrees of the factors for the SRAP reac- tion were in the order of Mg2＋〉dNTPs〉Taq DNA polymerase〉DNA template〉Primer. Then the sin- gle-factor test was used to choose an appropriate concentration of Mg2＋ , dNTPs and Taq DNA polymerase which had a greater impact on the results of SRAP reaction. The optimum concentrations of components in 20 μL reaction mixture were as follows： Mg2＋ 2.2 mmol/L,dNTPs 0.25 retool/L, Taq DNA polymerase 0.8 mol/s,primer 0.8 μmol/L and DNA template 60 ng, respectively. The stable and legible polymorphic bands were recorded when amplified with this system.