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目的建立穿龙薯蓣ISSR-PCR扩增的稳定体系。方法利用单因素及正交试验设计的方法对影响穿龙薯蓣ISSR-PCR扩增效果的d NTPs浓度、引物浓度、模板DNA用量Taq DNA聚合酶用量、Mg~(2+)浓度进行优化。结果穿龙薯蓣的ISSR-PCR的20μl最佳反应体系为:2.0μl 10×Taq Buffer,2.0 mmol/L Mg~(2+),0.225mmol/L d NTPs,1.5 UTaq DNA聚合酶,1.0μmol/L引物,2.0 ng/μl DNA模板,dd H2O补齐。应用该反应体系筛选出来适用于穿龙薯蓣ISSR扩增的12条引物,并确定了各引物的最佳退火温度。结论确立了一个适用于穿龙薯蓣ISSR-PCR扩增的体系。
Objective To establish a stable ISSR-PCR amplification system for Dioscorea nipponica. Methods The concentration of dNTPs, primer concentration, dosage of Taq DNA polymerase and concentration of Mg 2+ were optimized by single factor and orthogonal design. Results The optimal reaction system of ISSR-PCR for Dioscorea nipponica: 2.0μl 10 × Taq Buffer, 2.0 mmol / L Mg 2+, 0.225mmol / L dNTPs, 1.5 UTaq DNA polymerase, 1.0μmol / L primer, 2.0 ng / μl DNA template, dd H2O. Using this reaction system, 12 primers suitable for ISSR amplification of Dioscorea mandshurica were screened and the optimal annealing temperature of each primer was determined. Conclusion A system suitable for amplification of ISSR-PCR amplification of Dioscorea nipponica was established.