PLANT DNA ISOLATION - A METHOD TO EFFICIENTLY REMOVE POLYPHENOLICS, POLYSACCHARIDES, AND RNA
PLANT DNA ISOLATION - A METHOD TO EFFICIENTLY REMOVE POLYPHENOLICS, POLYSACCHARIDES, AND RNA
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作者：
JOBES, DV;HURLEY, DL;THIEN, LB
从植物中分离DNA已经被许多作者描述过，每个作者都贡献了不同的方法来克服提取DNA时出现的问题。至少有三种与植物DNA相关的主要污染物，在进行限制性内切酶分析和PCR（聚合酶链式反应）实验时会造成相当大的困难：多酚化合物，多糖和RNA。下面描述的程序通过利用PVP（聚乙烯吡咯烷酮）结合多酚化合物、高摩尔浓度的氯化钠抑制多糖和DNA的共沉淀以及通过用氯化锂选择性沉淀除去RNA的改进方法，来减少CTAB（十六烷基三甲基溴化铵）和氯化铯方法的时间和费用。分离的DNA很容易用限制性内切酶消化，并通过PCR从广泛不同的植物物种中扩增。
The isolation of DNA from plants has been described by numerous authors, each contributing a different method to overcome the problems that arise when extracting DNA. There are at least three main contaminants associated with plant DNA that can cause considerable difficulties when conducting restriction enzyme analyses and PCR (polymerase chain reaction) experiments: polyphenolic compounds, polysaccharides, and RNA. The procedure described below alleviates the time and expense of CTAB (cetyltrimethylammonium bromide) and caesium chloride methods by utilizing PVP (polyvinylpyrrolidone) to bind the polyphenolic compounds, a high molar concentration of sodium chloride to inhibit co-precipitation of the polysaccharides and DNA, and an improved method for removing RNA by selective precipitation with lithium chloride. Isolated DNA was easily digested with restriction enzymes and amplified by the PCR from widely different plant species.