In-Fusion® Cloning with Vaccinia Virus DNA Polymerase

Abstract

Vaccinia virus DNA polymerase (VVpol) encodes a 3'-to-5' proofreading exonuclease that can degrade the ends of duplex DNA and expose single-stranded DNA tails. The reaction plays a critical role in promoting virus recombination in vivo because single-strand annealing reactions can then fuse molecules sharing complementary tails into recombinant precursors called joint molecules. We have shown that this reaction can also occur in vitro, providing a simple method for the directional cloning of PCR products into any vector of interest. A commercial form of this recombineering technology called In-Fusion(®) that facilitates high-throughput directional cloning of PCR products has been commercialized by Clontech. To effect the in vitro cloning reaction, PCR products are prepared using primers that add 16-18 bp of sequence to each end of the PCR amplicon that are homologous to the two ends of a linearized vector. The linearized vector and PCR products are coincubated with VVpol, which exposes the complementary ends and promotes joint molecule formation. Vaccinia virus single-stranded DNA binding protein can be added to enhance this reaction, although it is not an essential component. The resulting joint molecules are used to transform E. coli, which convert these noncovalently joined molecules into stable recombinants. We illustrate how this technology works by using, as an example, the cloning of the vaccinia N2L gene into the vector pETBlue-2.

High-throughput recombinant protein expression in Escherichia coli : current status and future perspectives

“…However, LIC still requires enzymes such as T4 DNA polymerase and T5 exonuclease, depending on the protocols used, to generate single-stranded complementary ends in target genes and vector sequences (figure 2 c ). Several effective and convenient methods based on the LIC principle have been developed, including Gibson Assembly from NEB (Ipswich, MA, USA) [43], In-Fusion from Clontech [44], polymerase incomplete primer extension cloning [45], sequence and LIC [46], and overlap extension cloning [47,48]. The Gibson Assembly method [39] uses T5 exonuclease to remove portions of the 5′ ends to generate single-stranded complementary overhangs, which are joined together covalently by fusion DNA polymerase and Taq DNA ligase.…”

Generation of Plasmid Vectors Expressing FLAG-tagged Proteins Under the Regulation of Human Elongation Factor-1α Promoter Using Gibson Assembly

“…However, some of these systems still use restriction enzymes (i.e., Golden Gate cloning 18 ). Others use proprietary blends of enzymes based on vaccinia virus DNA polymerase and single-strand DNA binding protein from the same biological source 19 . Both systems are limited in comparison to GA by the shorter length of overlapping sequences.…”

Directional cloning of DNA fragments using deoxyinosine-containing oligonucleotides and endonuclease V

“…In-Fusion cloning) [37], cohesive terminal sequences are created via primer overhangs only four nucleotides in length. As a result, the PCR-primers used for insert DNA generation remain short, minimizing the chance for secondary structure and primer-dimer formation as well as synthesis errors to occur.…”

“…via T4 DNA polymerase or incomplete PCR [27-29], hybridization of PCR products [30], ribonucleotide-containing primers [31], terminal transferase [32], abasic sites [33], chemical or enzymatic cleavage of phosphorothioated DNA [34,35], or λ exonuclease [36]. Elegant enzyme-based in vitro systems have been developed, such as In-Fusion cloning [37], for which the polymerase is known but not the exact composition, as well as the combined isothermal usage of a DNA polymerase, a 5′ exonuclease and DNA ligase, named Gibson assembly cloning [38]. Although several of the described cloning systems with individual advantages and disadvantages are commercially available, many present costly alternatives or demand complex planning.…”