科研之友
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摘要
A significantly improved DNA polymerase fidelity assay, based on a gapped plasmid containing the lacZ alpha reporter gene in a single-stranded region, is described. Nicking at two sites flanking lacZ alpha, and removing the excised strand by thermocycling in the presence of complementary competitor DNA, is used to generate the gap. Simple methods are presented for preparing the single-stranded competitor. The gapped plasmid can be purified, in high amounts and in a very pure state, using benzoylated-naphthoylated DEAE-cellulose, resulting in a low background mutation frequency (similar to 1 x 10(-4)). Two key parameters, the number of detectable sites and the expression frequency, necessary for measuring polymerase error rates have been determined. DNA polymerase fidelity is measured by gap filling in vitro, followed by transformation into Escherichia coli and scoring of blue/white colonies and converting the ratio to error rate. Several DNA polymerases have been used to fully validate this straightforward and highly sensitive system.
- 出版日期2013-2-15
