正交设计优化狭叶坡垒ISSR-PCR反应体系

Optimization for ISSR Reaction System of Hopea chinensis by Orthogonal Design

以狭叶坡垒DNA为模板,利用正交试验分别对ISSR-PCR反应的MgCl2浓度、dNTPs浓度、Taq聚合酶浓度、引物浓度、模板DNA浓度进行了优化,并通过梯度PCR确定最佳退火温度和循环次数,最终确定狭叶坡垒最佳反应体系及扩增条件为:25μL体系中1×PCRbuffer,2mmol/LMgCl2,0.25mmol/LdNTPs,0.04U/μLTaq聚合酶,0.2μmol/L引物,4ng/μLDNA模板;最佳扩增程序为:94℃预变性5min;94℃变性45s,53℃退火45s,72℃延伸1.5min,共35个循环;72℃最后延伸7min。

To optimize the inter simple sequence repeat（ISSR） reaction condition for Hopea chinensis genomic DNA,the concentrations of MgCl 2,dNTPs,Taq polymerase,primers and template DNA were studied with an orthogonal experimental design,and the optimal anneal temperature of primer and cycles were determined through gradient PCR.The optimal PCR system for ISSR analysis was 1 × PCR buffer,2 mmol/L MgCl 2,0.25 mmol/L dNTPs,0.04 U/μL Taq polymerase,0.2 μmol/L primer,4 ng/μL template DNA in 25 μL reaction solution.And the augmentation procedure was pre-denaturation at 94℃ for 5 min,denaturation at 94℃ for 45 s,annealing at 53℃ for 45 s,extension at 72℃ for 1.5 min,reaction with 35 cycles,and extension at 72℃ for 7 min.