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首页> 外文期刊>African Journal of Biotechnology >A simple and rapid DNA extraction method from leaves of grapevine suitable for polymerase chain reaction analysis
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A simple and rapid DNA extraction method from leaves of grapevine suitable for polymerase chain reaction analysis

机译：一种简单快速的从葡萄叶中提取DNA的方法，适用于聚合酶链反应分析

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摘要

The genomic grapevine (Vitis vinifera?L.) DNA extraction is difficult because of secondary metabolites that interfere with DNA isolation procedures and subsequent applications. We developed a simple, rapid and efficient method for the extraction of genomic DNA from asymptomatic and pathogen-infected grape leaves. The protocol reported, based on a modified cetyl trimethylammonium bromide (CTAB) extraction procedure, allowed the rapid DNA extraction from little amounts of leaf material without employment of liquid nitrogen for initial tissue grinding. The protocol included polyvinylpyrrolidone (PVP) to bind phenolic compounds,?β-mercaptoethanol to inhibit the oxidation of polyphenols, and a high concentration of NaCl (2.5 M) to increase the solubility of polysaccharides, thus reducing their co-precipitation with DNA. Final DNA solution did not contain polysaccharides, polyphenols and other major contaminants. The purity of genomic DNA was confirmed by A260/280?and A260/230?ratios calculated from the spectrophotometric readings. In addition, the quality of the DNA extracted from asymptomatic,?Oidium tuckeri- and?Plasmopara viticola-infected leaves of?V. vinifera?L. was evaluated in polymerase chain reaction (PCR) analyses by using different set of primers to be able to amplify vegetal, fungal and bacterial DNA.
机译：由于次级代谢产物会干扰DNA分离程序和后续应用，因此难以提取基因组葡萄（Vitis vinifera？L。）DNA。我们开发了一种简单，快速且有效的方法，用于从无症状和病原体感染的葡萄叶片中提取基因组DNA。该协议报告基于改良的十六烷基三甲基溴化十六烷基溴化铵（CTAB）提取程序，可从少量叶片中快速提取DNA，而无需使用液氮进行初始组织研磨。该方案包括聚乙烯吡咯烷酮（PVP）结合酚类化合物，β-巯基乙醇抑制多酚氧化，高浓度NaCl（2.5 M）增加多糖的溶解度，从而减少了它们与DNA的共沉淀。最终的DNA溶液不含多糖，多酚和其他主要污染物。由分光光度法读数计算出的A260 / 280和A260 / 230比值证实了基因组DNA的纯度。另外，从无症状的，ΔOidiumtuckeri-和ΔPlasmoparaviticola感染的ΔV叶片中提取的DNA的质量。 vinifera？L。通过使用不同的引物组在聚合酶链反应（PCR）分析中评估DNA能够扩增植物，真菌和细菌DNA。

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