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 wi th precision but without digestion using restriction enzymes for the insert ion of DNA. The method allows the insertion of PCR fragments in between any two nucleotides within a target plasmid. The only requirement that the amp lified fragments must be embedded between DNA sequences homologous to the s ite in which the integration is planned. This method is an adaptation of th e QuikChange (TM) Site-Directed Mutagenesis protocol. It is simpler than th e existing cloning strategies and is suitable for multi-parallel constructi ons of new plasmids. We have demonstrated its utility by constructing plasm ids in which we have successfully integrated PCR fragments up to 1117 bp.