正交设计优化广西火桐ISSR-PCR反应体系

Optimization for ISSR Reaction System of Erythropsis kwangsiensis by Orthogonal Design

以广西火桐DNA为模板,利用正交试验分别对ISSR-PCR反应的MgCl2浓度、dNTPs浓度、TaqDNA聚合酶浓度、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,最终确定广西火桐最佳反应体系及扩增条件。25μL体系中1×PCR buffer,2.5 mmol/L MgCl2,0.15 mmol/L dNTPs,0.06 U/μL TaqDNA聚合酶,3 ng/μL DNA模板,0.2μmol/L引物;最佳扩增程序为:94℃预变性5 min;94℃变性45 s,52℃退火45 s,72℃延伸1.5 min,共30个循环;72℃最后延伸7 min。扩增后的PCR产物在4℃保存。应用该ISSR体系对10个广西火桐样品进行了扩增,证实了该体系的适用性和稳定性。

To optimize the inter simple sequence repeat（ISSR）reaction condition for Erythropsis kwangsiensis genomic DNA,the concentrations of MgCl2,dNTPs,Taq polymerase 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,2.5 mmol/L MgCl2,0.15 mmol/L dNTPs,0.06 U/μL Taq polymerase,3 ng/μL template DNA,0.2 μmol/L primer in 25 μL reaction solution.The augmentation procedure was pre-denaturation at 94℃ for 5 min,denaturation at 94℃ for 45 s,annealing at 52℃ for 45 s,extension at 72℃ for 1.5 min,reaction with 30 cycles,and extension at 72℃ for 7 min and holding the samples at 4℃.The system was applied in the amplification of 10 varieties of Erythropsis kwangsiensis,and indicated the suitability and stability of the system.