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一种从枣椰树中高效回收高质量DNA的改进方法。

An improved method for efficient recovery of high quality DNA from date palm ( L; ).

作者信息

Safeena M I S, Dissanayake Y, Zakeel M C M, Warnakula L, Cooray R, Dayarathna D A R K

机构信息

Department of Biological Science, Faculty of Applied Sciences, South Eastern University of Sri Lanka, Sammanthurai, Sri Lanka.

Section of Genetics, Institute for Research and Development, Colombo, Sri Lanka.

出版信息

MethodsX. 2021 May 21;8:101384. doi: 10.1016/j.mex.2021.101384. eCollection 2021.

Abstract

Date palm ( L; ) is one of a few fruit trees that can remarkably grow in dessert agroecosystems that are characterized by extreme temperature fluctuations. Due to increasing demands for dates in the global market and commercial cultivation in many countries, the tree is currently under extensive research in many countries, particularly to improve the germplasm using different molecular tools. Most molecular techniques largely depend on good quality DNA in significant quantities, which are highly compromised by the presence of various contaminants in DNA. The traditional cetyltrimethylammoniumbromide (CTAB) based method has failed to produce good quality DNA from date palm due to hard fibrous nature of tissue. On the basis of previous studies, commercial DNA extraction kits are not economical although they are very effective. Therefore, we have developed an improved DNA extraction protocol by modifying the original CTAB method to produce extra pure DNA in large quantities. The novel method has been validated using different quality testing approaches. This cost-effective method can be used successfully for DNA extraction from date palm. Moreover, this improved method may have potential for DNA extraction from other palms that have similar leaf texture to date palm leaves, but this method needs to be tested for other palms before being used. The improved method has following key modifications:•Grinding of plant tissue in liquid nitrogen and subsequent lysis of cells in CTAB buffer that has increased concentration of ß-mercaptoethanol•Repeated steps of chloroform: IAA extraction and ethanol washing•Addition of RNase A before the DNA precipitation step.

摘要

海枣(椰枣)是少数能够在以极端温度波动为特征的沙漠农业生态系统中显著生长的果树之一。由于全球市场对椰枣的需求不断增加以及许多国家的商业化种植,目前许多国家都在对这种树进行广泛研究,特别是利用不同的分子工具来改良种质。大多数分子技术在很大程度上依赖于大量高质量的DNA,而DNA中各种污染物的存在严重影响了DNA的质量。基于传统十六烷基三甲基溴化铵(CTAB)的方法由于组织的坚硬纤维性质,未能从椰枣中提取出高质量的DNA。根据以往的研究,商业DNA提取试剂盒虽然非常有效,但并不经济。因此,我们通过修改原始的CTAB方法,开发了一种改进的DNA提取方案,以大量生产超纯DNA。该新方法已通过不同的质量检测方法得到验证。这种经济高效的方法可成功用于从椰枣中提取DNA。此外,这种改进方法可能对从与椰枣叶质地相似的其他棕榈树中提取DNA具有潜力,但在用于其他棕榈树之前需要进行测试。改进后的方法有以下关键改进:

  • 在液氮中研磨植物组织,随后在含有增加浓度的β-巯基乙醇的CTAB缓冲液中裂解细胞

  • 重复氯仿:异戊醇提取和乙醇洗涤步骤

  • 在DNA沉淀步骤之前添加核糖核酸酶A。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/cbf9/8374510/720c74d7fb8c/fx1.jpg

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