A simple and rapid DNA microextraction method for plant, animal, and insect suitable for RAPD and other PCR analyses.
A simple and rapid DNA microextraction method for plant, animal, and insect suitable for RAPD and other PCR analyses.
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一种简单快速的植物、动物和昆虫 DNA 微提取方法，适用于 RAPD 和其他 PCR 分析。
DOI：
10.1101/gr.3.1.69
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发表时间：
1993
期刊：
影响因子：
--
通讯作者：
B. Landry
中科院分区：
文献类型：
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作者：
W. Cheung;N. Hubert;B. Landry
PCR 和特定的随机扩增多态性 DNA (RAPD) 分析允许在获得纯化的 DNA 后每天分析数百个 DNA 样本。从选定的组织中制备 DNA，无论是动物、植物还是昆虫来源的组织，都是充分利用该技术潜力的时间限制因素。已经发表了许多解决这个问题的 DNA 分离方法，但其中大多数虽然简单，但都是为动物或植物设计的，或者更常见的是为特定物种或组织设计的，并且没有报道可以普遍应用，包括微小昆虫 (< 500~ m)。在这里，我们报告了一种简单的总 DNA 微提取方法，适用于所有测试物种的基于 PCR 的 DNA 分析。两项关键改进是在提取缓冲液中使用非常高的盐浓度，可能会“盐析出”几种 PCR 抑制剂，以及使 DNA 提取过程简单、快速且廉价的组织破坏技术。它提供高度可重复的结果和均匀的 DNA 产量。我们通常使用它从芸苔属植物的叶组织和微小昆虫（小于 500~ m）中提取 DNA，进行位点特异性 PCR 扩增和 RAPD 分析。操作步骤如下：将用于植物组织的 5 毫米直径叶盘、10-20 mg 动物组织或单个完整昆虫（小膜翅目；赤眼蜂属物种）浸入 160 tzl 提取缓冲液 [200 mM Tris-HC1 (pH 8.0)、70 mM EDTA、2 M NaC1、20 mM 焦亚硫酸钠]在微量离心管 (Kontes) 中。使用电动马达驱动的 Kontes 杵通过物理研磨来实现组织破损，直到
PCR and specifically random amplified polymorphic DNA (RAPD) analyses permit the analysis of hundreds of DNA samples per day after purified DNA is available. DNA preparation from the selected tissues, whether they are of animal, plant, or insect origins, is the timelimiting factor to fully exploit the potential of the technology. Numerous DNA isolation methods addressing this problem have been published,(ls) but most of them, although simple, are designed either for animal or plant, or more often for specific species or tissues, and none has been reported for universal applications, including minute insects (< 500~ m). Here, we report a simple microextraction method for total DNA suitable for PCR-based DNA analyses in all species tested. The two key improvements are the use of a very high salt concentration in the extraction buffer that presumably" salts out" several PCR inhibitors, and the tissue disruption technique that renders the DNA extraction process simple, fast, and inexpensive. It provides highly repeatable results and uniform DNA yield. We use it routinely for extracting DNA from leaf tissues of Brassica plant species and from minute insects (smaller than 500~ m) for both site-specific PCR amplification and RAPD analyses.The procedure is as follows: A 5-mmdiameter leaf disc for plant tissues, 10-20 mg of animal tissues, or a single intact insect (microhymenoptera; Trichogramma species) was immersed in 160 tzl of extraction buffer [200 mM Tris-HC1 (pH 8.0), 70 mM EDTA, 2 M NaC1, 20 mM sodium metabisulfite] in a microcentrifuge tube (Kontes). Tissue breakage was achieved by physical grinding using an electric motor-driven Kontes pestle until