Preparation of megabase-sized DNA from a variety of organisms using the nuclei method for advanced genomics research
Preparation of megabase-sized DNA from a variety of organisms using the nuclei method for advanced genomics research
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DOI：
10.1038/nprot.2011.455
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发表时间：
2012-03-01
期刊：
影响因子：
14.8
通讯作者：
Zhang, Hong-Bin
中科院分区：
文献类型：
--
作者：
Zhang, Meiping;Zhang, Yang;Zhang, Hong-Bin
兆级大小的DNA对所有生物体的现代基因组学研究至关重要。在已开发的制备方法中，细胞核法是制备高质量的巨型DNA的最简单和最广泛使用的方法。在这种方法中，首先通过物理研磨源组织来分离细胞核。通过离心和洗涤去除非靶细胞质细胞器基因组和代谢物，从而最大限度地提高了方法的实用性，并大大提高了所得DNA的消化率和可克隆性。然后将细胞核嵌入含有许多孔的琼脂糖基质中，允许限制性内切酶进入，同时防止DNA被物理剪切。从细胞核中提取DNA，纯化并随后在琼脂糖基质中进行处理。在这里，我们描述了细胞核方法，我们已经成功地从数百种植物，动物，鱼类，昆虫，藻类和微生物中制备高质量的兆级DNA。整个过程类似于3d。
Megabase-sized DNA is crucial to modern genomics research of all organisms. Among the preparation methods developed, the nuclei method is the simplest and most widely used for preparing high-quality megabase-sized DNA from divergent organisms. In this method, nuclei are first isolated by physically grinding the source tissues. The nontarget cytoplast organellar genomes and metabolites are removed by centrifugation and washing, thus maximizing the utility of the method and substantially improving the digestibility and clonability of the resultant DNA. The nuclei are then embedded in an agarose matrix containing numerous pores, allowing the access of restriction enzymes while preventing the DNA from physical shearing. DNA is extracted from the nuclei, purified and subsequently manipulated in the agarose matrix. Here we describe the nuclei method that we have successfully used to prepare high-quality megabase-sized DNA from hundreds of plant, animal, fish, insect, algal and microbial species. The entire protocol takes similar to 3 d.