A novel rapid seamless cloning method: vector-free seamless cloning

At present, overlap-PCR and seamless cloning remain the main methods for cloning of gene segments. However, overlap-PCR is unable to reassemble too many fragments at a time, so there is a need to establish a fast multi-fragment recombination method with a guaranteed success rate. The vector-free seamless cloning method established in this study uses seamless cloning reagents to recombine multiple gene fragments and employs PCR to amplify the full length of the recombinant gene by using the recombinant product as the template. Through recombination of gene segments with different lengths in the Newcastle disease virus genome, the optimal conditions for vector-free seamless cloning were deduced. The genomes of DNA viruses (porcine circovirus 3), bacteria (Escherichia coli), fungi (Saccharomyces yeasts), parasites (Trichomonas trichomonas) and human (beta-actin) were tested to verify the universality of this method. Experimental verification showed that the vector-free seamless cloning method can recombine up to 4 gene fragments simultaneously, with the recombination success rate decreasing as the number and length of fragments increase, and it can recombine gene fragments of up to 4000 bp. Moreover, this method is applicable to genes of most species, proving to be a new and faster approach for gene recombination experiments.