Integration of PCR fragments at any specific site within cloning vectors without the use of restriction enzymes and DNA ligase.
Here, we describe a method that offers a unique way to engineer plasmids with precision but without digestion using restriction enzymes for the insertion of DNA. The method allows the insertion of PCR fragments in between any two nucleotides within a target plasmid. The only requirement is that the amplified fragments must be embedded between DNA sequences homologous to the site in which the integration is planned. This method is an adaptation of the QuikChange Site-Directed Mutagenesis protocol. It is simpler than the existing cloning strategies and is suitable for multiparallel constructions of new plasmids. We have demonstrated its utility by constructing plasmids in which we have successfully integrated PCR fragments up to 1117 bp.
Geiser
M
M
Novartis Pharma, CTA/PSU, Building WSJ 506.3.14, CH-4000 Basel, Switzerland. martin.geiser@pharma.novartis.com
Cèbe
R
R
Drewello
D
D
Schmitz
R
R
D003001:Cloning, Molecular / Q000379:methods*; D011088:DNA Ligases; D017931:DNA Primers; D004262:DNA Restriction Enzymes; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000235:genetics*; D016133:Polymerase Chain Reaction
D017931:DNA Primers; D004262:DNA Restriction Enzymes; D011088:DNA Ligases