Recombination-assisted megaprimer (RAM) cloning
Mathieu, Jacques; Alvarez, Emilia; Alvarez, Pedro J.J.
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: 1) Limited number of PCR cycles for both megaprimer synthesis and the cloning reaction to reduce error propagation; 2) 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; 3) The inclusion of 1 M betaine to enhance both reaction specificity and yield.
megaprimer; restriction-free cloning; PCR cloning; exponential amplification; homologous end- joining