正交设计优化苜蓿ISSR-PCR反应体系的研究

Optimization of ISSR-PCR amplification system in Afalfa

以苜蓿基因组DNA为模板,利用正交设计,对影响ISSR反应体系的主要因素（Taq酶、Mg2＋、dNTP、引物和模板）从4个水平上进行筛选和优化,建立了适合苜蓿的最佳ISSR反应体系：20μL反应体系中加83.4 nkat Taq酶,2.0 mmol/L Mg2＋,10×PCR Buffer 2.0μL,0.4 mmol/L dNTP,0.2 mmol/L引物,2.5 ng DNA模板.PCR扩增反应程序为：94℃预变性4 min（94℃变性1 min,45~53℃退火1 min,72℃延伸1 min）40个循环,于72℃延伸7 min,4℃保存.在此反应体系下,可以得到重复性好、稳定且多态性高的条带.

Template DNA used for ISSR was extracted from Alfalfa leaf tissue.The orthogonal design was applied to optimize ISSR amplification system at four levels of five factors（Taq DNA polymerase,Mg2＋,dNTP,primer and template）respectively.Anoptimal reaction system was established,20 μL reaction system contained 83.4 nkat Taq DNA,2.0 mmol/L Mg2＋,10×PCRBuffer 2.0 μL,0.4 mmol/L dNTP,0.2 mmol/L primer,2.5 ng DNA template.Amplification program included pre-denaturation at 94℃for 4 min（denaturation at 94℃for 1 min,annealing at 45~53℃ for 1 min,extending at 72℃for 1 min）after 40 cycles,the product was extended at 72℃for 7 min and kept at 4℃ clear,steady and higher polymorphic bands were obtained through this optimized system.