Abstract
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 ccdB-gene.ConclusionWe provide further evidence that gene replacement can be applied with high-throughput cloning protocols. Targeting a replacement of the ccdB-gene was found to be very successful for counterselection using these protocols. This eliminated the need for treatment with the restriction enzyme DpnI that has so far been the preferred clone selection approach. We thus present an optimized cloning protocol using a restriction-free ccdB-gene replacement strategy, which allows for parallel cloning at a high-throughput level.
Highlights
In high-throughput demanding fields, such as biotechnology and structural biology, molecular cloning is an essential tool in obtaining high yields of recombinant protein
We show that RF- and Exponential megapriming PCR (EMP)-cloning protocols can successfully be used in gene-replacement applications
The method holds a great potential in biotechnology, structural biology and other high-throughput demanding fields
Summary
In high-throughput demanding fields, such as biotechnology and structural biology, molecular cloning is an essential tool in obtaining high yields of recombinant protein. To meet the demands in biotechnology and structural biology, high-throughput molecular cloning methods have been developed to obtaining sufficient amounts of recombinant protein in a parallel manner For this purpose, restriction-free (RF) cloning is a simple PCR-based approach for inserting any DNA fragment into any position of a vector, independently of restriction-sites, ligation, without elaborate pretreatments of the vector and without sequence constraints [1,2,3]. Exponential megapriming PCR (EMP) cloning addresses the major shortcoming of the published RF-cloning protocol, the product- and size-limiting linear plasmid amplification, by introducing a reverse vector-specific primer making the amplification exponential [4] This protocol, requires two additional, successive steps of phosphorylation and ligation to close the plasmid. This makes these protocols beneficial for many high-throughput applications, such as structural genomics and biotechnology
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