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首页> 外文期刊>American Journal of Plant Sciences >Standardization of DNA Extraction Method from Mature Dried Leaves and ISSR-PCR Conditions for Melia dubia Cav. —A Fast Growing Multipurpose Tree Species
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Standardization of DNA Extraction Method from Mature Dried Leaves and ISSR-PCR Conditions for Melia dubia Cav. —A Fast Growing Multipurpose Tree Species

机译：苦瓜成熟叶片DNA提取方法的标准化和ISSR-PCR条件。 —快速生长的多用途树种

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摘要

Melia dubia Cav. of family Meliaceae is a fast growing, high value tree species native to India. Isolating DNA from matured dried leaves of M. dubia was difficult due to accumulation of secondary metabolites, majorly polyphenolics, which resulted in dark brown to black colour of the pellet. In this study, a modified STE-(Sucrose, Tris-HCl and Ethylene Diamine Tetra Acetic Acid) CTAB (hexadecyltrimethylammonium bromide) method was standardized for removal of polyphenolics. The protocol developed yielded 200 - 1000 ng/μl of quality DNA without any impurities as evident by A260/280 ratio ranging from 1.75 - 2.0. It was also suitable for extracting quality DNA from other members of Meliaceae like Azadirachta indica and Melia azedarach. In downstream applications, the extracted DNA was used for PCR amplification by using ISSR and SSR markers. ISSR PCR conditions were optimized in a reaction volume of 25 μl, consisting of 30 ng of template DNA, 1.5 mM MgCl2, 200 μM of each of dNTPs and 2 U of Taq polymerase. The best amplification was observed and the same was applicable for SSR markers.
机译：Melia dubia骑士Meliaceae科的一员是印度原产的一种快速生长的高价值树种。由于次生代谢产物（主要是多酚类）的积累，很难从杜鹃花的成熟干叶中分离DNA，从而导致沉淀物呈深棕色至黑色。在这项研究中，改良的STE-（蔗糖，Tris-HCl和乙二胺四乙酸）CTAB（十六烷基三甲基溴化铵）方法被标准化用于去除多酚。开发的方案可产生200-1000 ng /μl的优质DNA，不含任何杂质，A260 / 280比在1.75-2.0范围内可明显看出。它也适用于从Mel科的其他成员中提取高质量的DNA，例如印度的Azadirachta和Melia azedarach。在下游应用中，使用ISSR和SSR标记将提取的DNA用于PCR扩增。在25μl的反应体积中优化了ISSR PCR条件，该反应体积由30 ng模板DNA，1.5 mM MgCl2，每种dNTP 200μM和2 U Taq聚合酶组成。观察到最好的扩增，同样适用于SSR标记。

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