USER fusion: a rapid and efficient method for simultaneous fusion and cloning of multiple PCR products

TLDR

The authors introduce a rapid, efficient method for simultaneously fusing and cloning multiple PCR products. The technique uses primers bearing a single 5′ deoxyuridine, enzymatic excision to generate complementary 3′ overhangs, and the improved USER cloning system to assemble multiple fragments into a vector in a single step. Applying the method to three PCR fragments yielded ~90 % positive clones within about one hour, and the authors anticipate it will replace overlapping PCR and type IIS restriction enzyme strategies in many applications.

Abstract

We present a method that allows simultaneous fusion and cloning of multiple PCR products in a rapid and efficient manner. The procedure is based on the use of PCR primers that contain a single deoxyuridine residue near their 5′ end. Treatment of the PCR products with a commercial deoxyuridine-excision reagent generates long 3′ overhangs designed to specifically complement each other. The combination of this principle with the improved USER cloning technique provides a simple, fast and very efficient method to simultaneously fuse and clone multiple PCR fragments into a vector of interest. Around 90% positive clones were obtained when three different PCR products were fused and cloned into a USER-compatible vector in a simple procedure that, apart from the single PCR amplification step and the bacterial transformation, took approximately one hour. We expect this method to replace overlapping PCR and the use of type IIS restriction enzymes in many of their applications.