A high-throughput, restriction-free cloning and screening strategy based on ccd B-gene replacement

[摘要] BackgroundIn high-throughput demanding fields, such as biotechnology and structural biology, molecular cloning is an essential tool in obtaining high yields of recombinant protein. Here, we address recently developed restriction-free methods in cloning, and present a more cost-efficient protocol that has been optimized to improve both cloning and clone screening.ResultsIn our case study, three homologous β-lactamase genes were successfully cloned using these restriction-free protocols. To clone the genes, we chose a gene replacement strategy, where the recombinant genes contained overhangs that targeted a region of the expression vector including a cytotoxin-encoding ccd B-gene.ConclusionWe provide further evidence that gene replacement can be applied with high-throughput cloning protocols. Targeting a replacement of the ccd B- gene was found to be very successful for counterselection using these protocols. This eliminated the need for treatment with the restriction enzyme Dpn I that has so far been the preferred clone selection approach. We thus present an optimized cloning protocol using a restriction-free ccd B-gene replacement strategy, which allows for parallel cloning at a high-throughput level.

[发布日期] 2014-03-10

[关键词] Restriction-free cloning;Exponential megapriming PCR;High-throughput;Counterselection;Recombinant DNA technology;Parallel cloning