Recombination-assisted megaprimer (RAM) cloning

Abstract

No molecular cloning technique is considered universally reliable, and many suffer from being too laborious, complex, or expensive. Restriction-free cloning is among the simplest, most rapid, and cost-effective methods, but does not always provide successful results. We modified this method to enhance its success rate through the use of exponential amplification coupled with homologous end-joining. This new method, recombination-assisted megaprimer (RAM) cloning, significantly extends the application of restriction-free cloning, and allows efficient vector construction with much less time and effort when restriction-free cloning fails to provide satisfactory results. The following modifications were made to the protocol: • Limited number of PCR cycles for both megaprimer synthesis and the cloning reaction to reduce error propagation. • Elimination of phosphorylation and ligation steps previously reported for cloning methods that used exponential amplification, through the inclusion of a reverse primer in the cloning reaction with a 20 base pair region of homology to the forward primer. • The inclusion of 1 M betaine to enhance both reaction specificity and yield.

Keywords

• Megaprimer
• Restriction-free cloning
• PCR cloning
• Exponential amplification
• Homologous end-joining