海南龙血树RAPD-PCR反应体系的优化

Optimization of RAPD-PCR reaction systemfor Dracaena cambodiana

关键词:  海南龙血树  RAPD  反应条件优化  有效引物筛选

Key words:  Dracaena cambodiana  RAPD  optimization of reaction condition  screening of effective primers

摘要:

采用单因子试验和正交试验设计,对影响海南龙血树RAPD-PCR反应的模板DNA量、Mg²⁺、dNTP和引物浓度,Taq聚合酶用量等因子进行研究,分析各因子对RAPD-PCR扩增结果的影响,确立了海南龙血树RAPD-PCR反应的最佳条件,即在25 μL反应体系中,包含10×buffer 2.5 μL,2.0 mmol/L MgCl₂,300 μmol/L dNTPs,Taq酶1.5U,引物0.8 μmol/L和DNA模板20 ng。以此条件为基础,筛选出适用于海南龙血树RAPD-PCR分析的18条有效引物,为采用RAPD技术分析海南龙血树种群遗传变异水平和遗传结构打下了基础。

Abstract:

Both single factor test and orthogonal design were applied to study the effects of main factors on the RAPD-PCR system for Dracaena cambodiana,in which the main factors included the content of template DNA,the concentration of Mg²⁺、dNTPs and primers and the content of Taq DNA polymerase,and an optimal 25μL RAPD-PCR reaction system for D.cambodiana was established,including 2.5 μL 10×PCR buffer,2.0 mmol/L MgCl₂,300 μmol/L dNTPs,1.5 U Taq polymerase,0.8 μmol/L primers,and 20 ng DNA template. 18 primers were selected for RAPD-PCR analysis for D.cambodiana using the optimized amplification system above,which can be employed for the analysis of genetic variation and structure of D.cambodiana population.