Andersson B, Povinelli C M, Wentland M A, Shen Y, Muzny D M, Gibbs R A
Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas 77030.
Anal Biochem. 1994 May 1;218(2):300-8. doi: 10.1006/abio.1994.1182.
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.
报道了一种改进的用于DNA随机测序文库制备的策略。该方案是对先前基于衔接子的策略的改进，利用长（11个碱基）突出端，消除了载体-插入片段连接这一不可靠步骤。随机插入片段通过衔接子连接制备，而M13载体按照用于聚合酶链反应（PCR）产物的尿嘧啶DNA糖基化酶（UDG）克隆的方法制备，使用含尿嘧啶的引物进行PCR，随后进行UDG处理以产生突出端。已发现该方法能可靠地产生大量克隆。不存在由于载体重新连接导致的背景，并且所有克隆都含有插入片段。此外，该方法简单且适合传授给其他研究人员。为了表征对多个较短片段进行鸟枪法测序的策略，从含有人DNA的黏粒和人cDNA构建了文库。
