Contamination problems on DNA isolation from “recalcitrant plant taxa" which is rich in polysaccharides have been commonly encountered in a wide range of research fields such as plant population biology, biodiversity, and molecular marker assisted breeding. Here we present an improved protocol to extract DNA efficiently from dry or fresh leaves of a “recalcitrant plant taxa", Betula alnoides Buch. Ham. ex D. Don in which three key steps are involved: 1) washing out most of polysaccharides and other secondary compounds with CTAB free buffer from homogenate; 2) adoption of 3% CTAB rather than 2% CTAB in the exaction medium; and 3) using of high concentration of salt prior to DNA precipitation with isopropanol to remove residual polysaccharides. The isolated DNA has been proved suitable for RAPD PCR amplification and restriction digestion. This modified procedure is simple, inexpensive and reliable, and is also applicable to many other plant taxa with high polysaccharides.
顽拗植物类群的总DNA 制备
曾杰1 邹喻苹2*! 白嘉雨1 郑海水1
（1. 中国林业科学院热带林业研究所，广州F510520；2. 中国科学院植物研究所系统与进化植物学开放研究实验室，北京100093）
摘要： 从富含多糖的顽拗植物类群提取与纯化DNA是许多研究领域例如居群生物学、生物多样性、分子标记辅助育种研究普遍遇到的难题.以西南桦( Betula alnoides )为例发展了一套改进的方案,有效地从这种顽拗植物的干叶和鲜叶中制备了DNA.此方案包括3个关键步骤:首先从植物细胞匀浆中用不含CTAB的缓冲液洗去大部分多糖和其他次生物质;在提取介质中采用3% CTAB而不是通常用的2% CTAB;将常用的高盐去糖的纯化操作提前到用异丙醇沉淀DNA之前进行.从西南桦提取的DNA已成功地用于RAPD-PCR扩增和限制性酶切.这个简单、经济和可靠的改进方案也适用于许多其他的顽拗植物类群.
关键词： 西南桦；多糖；DNA 制备；RAPD；限制性酶切
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