ADAPTER-BASED URACIL DNA GLYCOSYLASE CLONING SIMPLIFIES SHOTGUN LIBRARY CONSTRUCTION FOR LARGE-SCALE SEQUENCING
ADAPTER-BASED URACIL DNA GLYCOSYLASE CLONING SIMPLIFIES SHOTGUN LIBRARY CONSTRUCTION FOR LARGE-SCALE SEQUENCING
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DOI：
10.1006/abio.1994.1182
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发表时间：
1994-05-01
影响因子：
2.9
通讯作者：
GIBBS, RA
中科院分区：
文献类型：
--
作者：
ANDERSSON, B;POVINELLI, CM;GIBBS, RA
报道了一种制备DNA随机测序文库的改进策略。该方案是对先前基于衔接子的策略的修改，并且利用长（11个碱基）突出端，这消除了载体插入连接的不可靠步骤。通过接头连接制备随机插入物，而M13载体如聚合酶链式反应（PCR）产物的尿嘧啶DNA糖基化酶（UDG）克隆所述制备，使用含尿嘧啶的引物进行PCR，然后进行UDG处理以产生突出端。已发现该方法可靠地产生大量克隆。由于载体的重新连接，没有背景，并且所有克隆都含有插入片段。此外，该方法简单，适合出口到其他调查人员。从含有人DNA和人cDNA的cDNA构建文库，以表征用于多个较短片段的鸟枪测序的策略。(C)1994年出版社出版。
An improved strategy for the preparation of libraries for the random sequencing of DNA is reported. The protocol is a modification of a previous adaptor-based strategy, and utilizes long (11 base) overhangs, which eliminates the unreliable step of vector-insert ligation. The random inserts are prepared by adaptor ligation, while the M13 vector is prepared as described for uracil DNA glycosylase (UDG) cloning of polymerase chain reaction (PCR) products, using PCR with uracil-containing primers, followed by UDG treatment to produce overhangs. This method has been found to reliably yield large numbers of clones. There is no background due to religation of the vector, and all clones contain inserts. In addition, the method is simple and suitable for export to other investigators. Libraries were constructed from cosmids containing human DNA and from human cDNAs in order to characterize a strategy for shotgun sequencing of multiple shorter fragments. (C) 1994 Academic Press, Inc.