Lambda ZAP: improved strategies for expression library construction and use.
Lambda ZAP: improved strategies for expression library construction and use.
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Lambda ZAP：改进的表达库构建和使用策略。
DOI：
10.1089/dna.1.1989.8.69
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发表时间：
1989
期刊：
影响因子：
--
通讯作者：
Sherman,LA
中科院分区：
文献类型：
--
作者：
Webb,R;Reddy,KJ;Sherman,LA
提出了一种高效构建λ ZAP基因组表达文库的策略。描述了在文库构建的每个阶段评估载体DNA状态的程序，以便于故障排除。用限制性内切酶酶切载体DNA，验证λ ZAP粘性末端的连接和多克隆位点的制备。超声波处理是一种快速产生大小范围理想的表达文库构建的随机染色体片段的方法。讨论了在λ ZAP多接头的NotI位点克隆的优点。这个位点的选择消除了进行染色体DNA甲基化的需要，而这在使用常规EcoRI位点时是必需的。该方法还有助于克隆插入片段的限制性酶切图谱。基因组表达文库的构建使用这种方法为集球藻。PCC7942，集胞藻属Synechocystissp. PCC6803和荷兰原绿丝菌（Prochlorothrixhollandica.）表达文库和体内切除的实用性通过验证编码聚球藻属的克隆的身份来证明。PCC7942细胞色素f，因为这些克隆插入片段的正确阅读框被明确确定。
A strategy is presented for the efficient construction of λ 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 λ 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 theNotI site of the λ ZAP polylinker are discussed. The choice of this site eliminated the need to perform the methylation of chromosomal DNA, which is required when the conventionalEcoRI site is used. This method also facilitates restriction mapping of cloned inserts. Genomic expression libraries were constructed using this approach forSynechococcussp. PCC7942,Synechocystissp. PCC6803, andProchlorothrix hollandica. The utility of expression libraries andin vivoexcision was demonstrated by verifying the identity of clones coding forSynechococcussp. PCC7942 cytochrome f, since the correct reading frames of these cloned inserts were determined unambiguously.