利用正交设计优化兴安落叶松RAPD-PCR反应体系

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(20 μL)中含有模板90 ng，0.5 μmol·L^-1的引物，1×反应缓冲液，DNTP各为0.25 mmol·L^-1，1 U的Taq DNA聚合酶，Mg²⁺ 2.5 mmol·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。

Establishment and Optimization of RAPD-PCR Reaction System for Larix gmelnii(Rupr.) Rupr Using Orthogonal Design

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.