【摘要】 以兴安落叶松针叶DNA为模板,对影响落叶松RAPD-PCR扩增的重要参数进行了优化试验,以期建立兴安落叶松RAPD-PCR反应的最佳体系。通过采用正交设计L16(45)对兴安落叶松RAPD-PCR反应的5因素(Taq酶、Mg2+、dNTP、模板DNA、引物)在4个水平上进行优化试验,结果表明兴安落叶松最佳的RAPD-PCR的反应体系(20μL)中含有模板90ng,0.5μmol·L-1的引物,1×反应缓冲液,DNTP各为0.25mmol·L-1,1U的TaqDNA聚合酶,Mg2+2.5mmol·L-1。在此基础上筛选出20个扩增稳定、多态性丰富的RAPD引物,并通过梯度PCR试验,确定了引物最佳退火温度。更多还原

【Abstract】 The orthogonal design was used to optimize RAPD amplification system of Larix gmelnii(Rupr.)Rupr with five factors(Taq polymerase,Mg2+,dNTP,primer,DNA templet)at four levels,respectively.Through the deep analysis,a suitable RAPD-PCR reaction system was established,namely 20 μL reaction system containing 1.0 U Taq DNA polymerase,2.5 mmol·L-1 Mg2+,0.25 mmol·L-1 dNTP,0.5 μmol·L-1 primer,1×PCR buffer,90 ng DNA template.20 primers with stable amplification and rich polymorphism for RAPD were screened.The optimal annealing temperature for RAPD-PCR reaction was proposed by gradient PCR.The result provided a standardizing RAPD-PCR program for the analysis of genetic diversity of L.gmelnii(Rupr.)Rupr.更多还原