利用正交设计优化异色瓢虫SRAP-PCR反应体系

利用正交设计L16(45)对异色瓢虫Harmonia axyridis(Pallas)SRAP-PCR反应体系的5个因素(Taq酶、Mg2+、模板DNA、dNTPs、引物)在4个水平上进行优化试验,试验结果用DPS和MINITAB软件进行分析,建立了异色瓢虫SRAP-PCR反应的最佳体系,即在20μL体系中模板50~100ng、引物0.25μmol/L、dNTPs0.1mmol/L、Taq DNA聚合酶1.5U、Mg2+0.375~0.625mmol/L。并对反应体系进行梯度退火试验,得到最佳退火温度为50.3℃。这一优化系统的建立,为今后利用SRAP技术进行瓢虫遗传图谱的构建、多态性分析和基因定位奠定了技术基础。

Optimization of SRAP-PCR reaction system for Harmonia axyridis using orthogonal design

The orthogonal design was used to optimize SRAP-PCR reaction system for Harmonia axyridis (Pallas) using five factors (Taq DNA polymerase, Mg~ 2+ , DNA template, dNTPs and primer) at four levels, respectively. The results of PCR were analyzed by DPS and MINITAB and a most suitable SRAP-PCR system for H. axyridis was established. The 20 μL volume system contained 50～100 ng DNA template, 0.25 μmol/L primers, 0.1 mmol/L dNTPs, 1.5 U Taq DNA polymerase and 0.375～0.625 mmol/L Mg~ 2+ . The optimal annealing temperature for SRAP-PCR was 50.3℃ based on gradient PCR. This system provided the opportunity for analysis of diversity, construction of genetic map and gene location in H. axyridis.