Plasmid-based lacZalpha assay for DNA polymerase fidelity: application to archaeal family-B DNA polymerase.
Plasmid-based lacZalpha assay for DNA polymerase fidelity: application to archaeal family-B DNA polymerase.
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DOI：
10.1093/nar/gkp494
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发表时间：
2009-08
影响因子：
14.9
通讯作者：
Connolly BA
中科院分区：
文献类型：
--
作者：
Jozwiakowski SK;Connolly BA
本文报道了在单链区含有lacZα报告基因的缺口pUC 18衍生物的制备。通过在lacZα基因两侧插入两个相关切口核酸内切酶位点实现缺口，从而能够切除编码链或非编码链。然而，切除的链通过非共价的沃森-克里克碱基配对保持与质粒退火;因此，它的去除需要在完全互补的竞争DNA存在下的加热-冷却循环。缺口质粒可用于使用体外复制评估DNA聚合酶保真度，随后转化到大肠杆菌中并对蓝色/白色菌落比率进行评分。在背景（两种情况下均为± 0.08%）和用许多DNA聚合酶观察到的突变频率方面，用质粒发现的结果与基于缺口M13的成熟方法相似，为这种简单且技术上不复杂的方法提供了验证。已经研究了来自激烈火球菌的古细菌家族-B DNA聚合酶的几个容易出错的变体，阐明了该方法的潜力。
The preparation of a gapped pUC18 derivative, containing the lacZα reporter gene in the single-stranded region, is described. Gapping is achieved by flanking the lacZα gene with sites for two related nicking endonucleases, enabling the excision of either the coding or non-coding strand. However, the excised strand remains annealed to the plasmid through non-covalent Watson–Crick base-pairing; its removal, therefore, requires a heat–cool cycle in the presence of an exactly complementary competitor DNA. The gapped plasmids can be used to assess DNA polymerase fidelity using in vitro replication, followed by transformation into Escherichia coli and scoring the blue/white colony ratio. Results found with plasmids are similar to the well established method based on gapped M13, in terms of background (∼0.08% in both cases) and the mutation frequencies observed with a number of DNA polymerases, providing validation for this straightforward and technically uncomplicated approach. Several error prone variants of the archaeal family-B DNA polymerase from Pyrococcus furiosus have been investigated, illuminating the potential of the method.
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