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通过单因子试验和正交设计,对影响太子参ISSR-PCR扩增效果的因素,如模板DNA浓度、Taq酶浓度、dNTPs浓度、引物浓度、退火温度、延伸时间和循环数进行优化。确立了可用于太子参ISSR-PCR分析最适宜的反应体系:20μL反应体系中含80 ng模板DNA,0.5U Taq酶,0.4μmol·L~(-1)引物,0.18 mmol·L~(-1)dNTPs;PCR扩增延伸时间为70 s,循环数为35。稳定性试验表明,该体系能在不同太子参品种中扩增出清晰明亮、稳定性、多态性好的条带。
Factors affecting DNA amplification efficiency, such as template DNA concentration, Taq enzyme concentration, dNTPs concentration, primer concentration, annealing temperature, extension time and number of cycles, were optimized by single factor experiments and orthogonal design. The most suitable reaction system for ISSR-PCR analysis was established: 20μL reaction system contained 80 ng template DNA, 0.5 U Taq enzyme, 0.4 μmol·L -1 primer, 0.18 mmol·L -1 ) dNTPs. The extension of PCR amplification was 70 s and the number of cycles was 35. The stability test showed that the system can amplify the bands with clear, bright, stable and polymorphic in different heterophylla species.