The USER-friendly DNA engineering method allows combining multiple PCR fragment assembly, nucleotide sequence alteration and directional cloning. In this approach, target DNA molecules and cloning vector are generated by PCR with 6-10 bases of homology between the neighboring fragments. PCR primers contain a single deoxyuracil residue (dU) flanking the 3’ end of the homology region, and can be designed to accommodate nucleotide substitutions, insertions and/or deletions. The primers are then used to amplify the vector and target DNA in discrete overlapping fragments that incorporate a dU at each end. The subsequent treatment of PCR fragments with USER Enzyme creates a single nucleotide gap at each location of dU resulting in PCR fragments flanked with single-stranded extensions that allow seamless and directional assembly of customized DNA molecules into a linearized vector. In this way, multi-fragment assemblies, as well as various mutagenic changes, can all be accomplished in a single-format experiment.
Figure 1: A) Design strategy of the pNEB206A vector compatible primers. An optional ATG should be included if protein expression is desired. B) PCR product cloning method using the USER Friendly Cloning Kit.
Figure 2: Effect of PCR product concentration on the cloning efficiency.
Bitinaite J. et al. (2007) USER friendly DNA engineering and cloning method by uracil excision. Nucl.Acids Res.35,1992-2002.
Bitinaite, J. and Nichols, N.M. (2009) DNA cloning and Engineering by uracil excision. Curr Protoc Mol Biol., Chapter 3, Unit 3.21.1-3.21.16
Vaisvila, R. and Bitinaite, J. (2013) Gene synthesis by assembly of deoxyuridine-containing oligonucleotides. Methods Mol.Biol. 978, 165-171.