利用正交设计优化桃SRAP-PCR反应体系

Optimization of SRAP-PCR Reaction System for Prunus persica by Using Orthogonal Design

以桃基因组DNA为模板,通过正交试验设计,从Mg2+、Taq酶、dNTP、引物、模板5种因素4个水平对桃SRAP反应体系进行优化,建立了适合于桃的SRAP-PCR优化反应体系,该25μL反应体系:模板DNA50 ng,MgCl22.5 mmol/L,dNTP200μmol/L,上下引物各0.4μmol/L,Taq DNA聚合酶1.5 U,以灭菌双蒸水补齐至25μL。PCR反应程序为:94℃预变性5min;94℃变性1 min,35℃复性1 min,72℃延伸1 min,5个循环;94℃变性1 min,50℃复性1 min,72℃延伸1 min,35个循环,72℃延伸10 min。

By using the genomic DNA of peach(Prunus persica) as template,the major components of SRAP,such as the concentration of Mg2+,dNTPs,Taq DNA polymerase,primers and template,were optimized by orthogonal design(five factors × four levels) in this study.The results showed that the optimum SRAP reaction system included DNA template 50 ng,MgCl2 2.5 mmol/L,dNTPs 200 μmol/L,primer 0.4 μmol/L and Taq DNA polymerase 1.5 U in the 25 μL reaction system.The most suitable protocol of PCR was initial denaturing at 94 ℃ for 5 min;preamplifying at 94 ℃ for 1 min,35 ℃ 1 min and 72 ℃ 1 min for five cycles;then amplifying at 94 ℃ for 1 min,50 ℃ 1 min,72 ℃ 1 min for 35 cycles;finally extending at 72 ℃ for 10 min.