DNA Extraction Protocol for Plants with High Levels of Secondary Metabolites and Polysaccharides without Using Liquid Nitrogen and Phenol.
DNA Extraction Protocol for Plants with High Levels of Secondary Metabolites and Polysaccharides without Using Liquid Nitrogen and Phenol.
复制标题
DOI：
10.5402/2012/205049
复制
发表时间：
2012
期刊：
影响因子：
--
通讯作者：
Kathiresan K
中科院分区：
文献类型：
--
作者：
Sahu SK;Thangaraj M;Kathiresan K
已知红树林和盐沼物种合成广谱的多糖和多酚，包括类黄酮和其他干扰纯基因组DNA提取的次级代谢物。虽然存在过多的植物DNA分离方案，但从红树林和盐沼物种中提取DNA是一项具有挑战性的任务。本研究描述了一种快速可靠的十六烷基三甲基溴化铵（CTAB）方案，特别适合于从富含多糖和次生代谢产物的植物中提取DNA，并且该方案也不使用昂贵的液氮和有毒酚。提取的DNA的纯度是优异的，如范围为1.78至1.84的A260/A280比率和A260/A230比率>2所证明的，这也表明制剂充分不含蛋白质和多酚/多糖化合物。DNA浓度范围为8.8至9.9 μg μL−1。提取的DNA适合于RAPD、限制性酶切和植物条形码基因（matK和rbcl）的PCR扩增。优化后的方法对干叶和鲜叶均适用。该方法在获得高质量基因组DNA方面的成功证明了该方法的广泛适用性。
Mangroves and salt marsh species are known to synthesize a wide spectrum of polysaccharides and polyphenols including flavonoids and other secondary metabolites which interfere with the extraction of pure genomic DNA. Although a plethora of plant DNA isolation protocols exist, extracting DNA from mangroves and salt marsh species is a challenging task. This study describes a rapid and reliable cetyl trimethylammonium bromide (CTAB) protocol suited specifically for extracting DNA from plants which are rich in polysaccharides and secondary metabolites, and the protocol also excludes the use of expensive liquid nitrogen and toxic phenols. Purity of extracted DNA was excellent as evident by A260/A280 ratio ranging from 1.78 to 1.84 and A260/A230 ratio was >2, which also suggested that the preparations were sufficiently free of proteins and polyphenolics/polysaccharide compounds. DNA concentration ranged from 8.8 to 9.9 μg μL−1. The extracted DNA was amenable to RAPD, restriction digestion, and PCR amplification of plant barcode genes (matK and rbcl). The optimized method is suitable for both dry and fresh leaves. The success of this method in obtaining high-quality genomic DNA demonstrated the broad applicability of this method.