高原鼠兔ISSR引物反应体系的优化与筛选

目的]筛选和优化高原鼠兔(Ochotona curzoniae)适宜的ISSR反应体系,以在对高原鼠兔进行ISSR分析时获得清晰和多态性好的扩增结果。方法]以高原鼠兔基因组DNA为模板,通过单因素试验,对体系中的模板浓度、Mg2+浓度、dNTPs浓度、Taq酶用量、引物用量、退火温度进行探讨。结果]结果表明,高原鼠兔ISSR-PCR扩增的最佳条件为:25μl PCR反应体系,其中4lμDNA模板,1.5 mmol/LMgCl2,0.2 mmol/L dNTPs,1.25 UTaq聚合酶,1.5μmol/L引物,复性温度4560℃(退火温度随引物不同而确定)。用11条引物进行了PCR扩增,筛选出效果较好的6条引物。结论]该反应体系的建立为鼠兔遗传多样性和分子系统学研究提供技术支持。

Optimization and Screening of ISSR Primer Reaction System for Ochotona curzoniae

Objective] The research aimed to study the suitable ISSR reaction system for Ochotona curzoniae,to obtain high resolution and multiple polymorphic bands.Method] Based on the genomic DNA extracted from Ochotona curzoniae,the effects of the main reaction system elements such as Mg2+,dNTPs,primer,template DNA,Taq DNA polymerase and annealing temperature were presented by single factor experiment.Result] The results showed that the optimum conditions of ISSRPCR amplification were as follow:The total volume of the reaction system was 25 μl,consisted of 4 μl DNA template,1.5 mmol/L MgCl2,0.2 mm/L dNTPs,1.25 U Taq DNA polymerase,1.5 μmol/L primer and annealing temperature of 45-60 ℃.The 11 ISSR primers were used to screen the suitable primers,of which 6 ISSR primers were screened.Conclusion] This optimal system offered a technical system of ISSR analysis suitable to Ochotona curzoniae.