One-step DNA fragment assembly and circularization for gene cloning.

Abstract

This article describes a one-step procedure based on Taq polymerase for the precise assembly of DNA fragments into circular constructs as long as 6 kb. The only prior step needed was the amplification of the gene to be cloned and the linear vector backbone, and the whole process up to assembly and circularization lasted only 2 days, compared with the conventional method's 2 weeks. Furthermore, the final DNA construct was used to transform Escherichia coli directly without any further treatment. By circumventing the need for DNA ligase, our "Quick Assemble" method offers an improvement over the combination of long PCR and overlap extension PCR, and is expected to facilitate various kinds of complex genetic engineering projects that require precise in-frame assembly of multiple fragments, such as multiple site-directed mutagenesis and whole-DNA library gene shuffling, as well as the construction of new plasmids with any promoter, resistance gene marker, restriction site, or any DNA tag.