Lee Patricia N, McFall-Ngai Margaret J, Callaerts Patrick, de Couet H Gert
Department of Zoology, University of Hawaii at Manoa, Honolulu, HI 96822, USA.
Cold Spring Harb Protoc. 2009 Nov;2009(11):pdb.prot5319. doi: 10.1101/pdb.prot5319.
This procedure describes the extraction of genomic DNA from adult bobtail squid (Euprymna scolopes) tissues by cesium chloride (CsCl) gradient centrifugation. There are numerous generic methods and commercial kits for the preparation of genomic DNA based on proteolytic digestion of chromatin components, followed by selective binding of nucleic acids to ion-exchange affinity media, but many of these do not yield DNA that can be readily restricted. Also, molluscan tissues contain mucopolysaccharides, which tend to copurify with DNA under certain conditions. Although nucleic acids prepared this way can serve as a template for polymerase chain reaction (PCR), other enzymatic modifications of nucleic acids are inhibited by these contaminants. The method described here yields high-molecular-weight DNA that can be readily restricted for Southern hybridization. The procedure uses brain tissue under the assumption that its genome is unlikely to be rearranged in any way, has a high nucleic acid:protein ratio, and avoids potential sources of enzymatic contaminants and parasites from the intestinal sac. However, the method can be applied to other tissue sources and works well with other species. The purification of DNA by gradient centrifugation is an established method based on the specific buoyant density of double-stranded nucleic acids and the ability of CsCl solutions to form a salt gradient in a centrifugal field. It can also be adapted to the purification of RNA, which has a higher buoyant density than DNA. Unfortunately, this method is somewhat involved and expensive and produces large amounts of ethidium bromide waste.
本实验方法描述了通过氯化铯（CsCl）梯度离心从成年短尾鱿鱼（Euprymna scolopes）组织中提取基因组DNA的过程。基于对染色质成分进行蛋白水解消化，随后使核酸选择性结合到离子交换亲和介质上，有许多通用方法和商业试剂盒可用于制备基因组DNA，但其中许多方法无法得到易于酶切的DNA。此外，软体动物组织含有粘多糖，在某些条件下往往会与DNA共纯化。虽然以这种方式制备的核酸可以作为聚合酶链反应（PCR）的模板，但这些污染物会抑制核酸的其他酶促修饰。这里描述的方法可产生高分子量DNA，可易于酶切用于Southern杂交。该实验方法使用脑组织，是基于这样的假设：其基因组不太可能以任何方式重排，核酸与蛋白质的比例高，并且避免了来自肠囊的酶污染物和寄生虫的潜在来源。然而，该方法也可应用于其他组织来源，并且对其他物种也适用。通过梯度离心纯化DNA是一种基于双链核酸的特定浮力密度以及CsCl溶液在离心场中形成盐梯度能力的成熟方法。它也可用于纯化RNA，RNA的浮力密度比DNA高。不幸的是，这种方法有些复杂且昂贵，并且会产生大量溴化乙锭废物。
