Webb R, Reddy K J, Sherman L A
Division of Biological Sciences, University of Missouri, Columbia 65211.
DNA. 1989 Jan-Feb;8(1):69-73. doi: 10.1089/dna.1.1989.8.69.
A strategy is presented for the efficient construction of lambda ZAP genomic expression libraries. Procedures are described for the evaluation of the status of vector DNA at each stage of library construction to facilitate troubleshooting. Ligation of lambda ZAP cohesive ends and preparation of the multiple cloning site were verified by restriction enzyme digestion of vector DNA. Sonication was a rapid way of producing random chromosomal fragments of a size range ideal for expression library construction. The advantages of cloning into the Not I site of the lambda ZAP polylinker are discussed. The choice of this site eliminated the need to perform the methylation of chromosomal DNA, which is required when the conventional Eco RI site is used. This method also facilitates restriction mapping of cloned inserts. Genomic expression libraries were constructed using this approach for Synechococcus sp. PCC7942, Synechocystis sp. PCC6803, and Prochlorothrix hollandica. The utility of expression libraries and in vivo excision was demonstrated by verifying the identity of clones coding for Synechococcus sp. PCC7942 cytochrome f, since the correct reading frames of these cloned inserts were determined unambiguously.
本文介绍了一种高效构建λZAP基因组表达文库的策略。文中描述了在文库构建的每个阶段评估载体DNA状态的程序，以利于故障排除。通过对载体DNA进行限制性酶切消化，验证了λZAP粘性末端的连接和多克隆位点的制备。超声处理是产生适合表达文库构建的理想大小范围的随机染色体片段的快速方法。讨论了克隆到λZAP多克隆位点的Not I位点的优点。选择该位点无需对染色体DNA进行甲基化，而使用传统的Eco RI位点时则需要进行甲基化。该方法还便于对克隆插入片段进行限制性图谱分析。使用这种方法为集胞藻属PCC7942、聚球藻属PCC6803和荷兰原绿球藻构建了基因组表达文库。通过验证编码集胞藻属PCC7942细胞色素f的克隆的身份，证明了表达文库和体内切除的实用性，因为这些克隆插入片段的正确阅读框已明确确定。
