非洲菊基因组DNA提取及ISSR-PCR扩增模板浓度优化

Optimization of the Genomic DNA Extraction and Concentration of DNA Template for ISSR-PCR Amplification of Gerbera jamesonii Cultivars

以改良的CTAB法为基础 ,对非洲菊基因组DNA提取进行了下述优化 :在第二次用氯仿 异戊醇抽提时 ,加入“PCN”溶液 (0 0 6 75gPVP ,4 5 μl 10 %CTAB +4%NaCl) ,可明显去除多糖 ;在材料研磨时加入化学物质M (0 10 0 0gNa2 SO3 ,0 0 5 0 0gVC)及N (0 0 2 0 0gVE ,0 10 0 0gNa2 B4O7,0 0 2 0 0gPVP ,2 0 0 μlβ -巯基乙醇 ) ,都可去除酚类物质 ,明显提高DNA及ISSR PCR扩增的质量 ,但N的效果稍优于M ;同时加入M、N及“PCN” ,具有明显的优化效果 ,所提 5个样品DNA ,平均A2 6 0 A2 80、A2 6 0 A2 30分别为 1 81、 2 0 2 ,浓度为 0 6 49μg μl,片段大小约为 4 9kb ,ISSR扩增带多且清晰、明亮、稳定性及多态性高 ,表明DNA纯度很高。对扩增反应的最佳模板浓度进行研究表明 ,所提DNA模板必须稀释 30倍 (浓度为 15～30ng μl时 ) ,才能扩增出清晰的谱带。

It was found that CTAB method was more suitable for DNA extraction of Gerbera jamesonii than SDS method.Thus an optimized protocol for Gerbera genomic DNA extraction based on the improved CTAB method was proposed as following below:Polysaccharides could be better removed,while the“PCN”solution (0.0675?g PVP,45?μl 10% CTAB+4% NaCl) was added during the second extraction with chloroform-isopentanol.While both chemicals M(0.1000?g Na 2SO 3,0.0500?g V C) and N(0.0200?g V E,0.1000?g Na 2B 4O 7,0.0200?g PVP,200?μl β-mercaptoethanol)were respectively recommended for removing phenolic terpenoids during sample grinding,good quality of DNA template and ISSR amplification bands were received,but it was better adding N than adding M.The DNA extracted from 5 Gerbera cultivars by adding M,N and “PCN” altogether,on an average,had 1.81 of A260/A280 ratio,2.02 of A260/A230 ratio,0.649?ng μl^(-1) of concentration,49?kb of fragment length,and had more amplification bands that were clear,bright,stable and polymorphic,and it showed high purity of DNA and distinct effect of improved method.The studies of optimum concentration of templates used in ISSR-PCR amplification showed these DNA templates gave better band patterns when they were diluted to 15-30?ng μl^(-1).