Overlap extension cloning of PCR products into a Gateway-compatible plasmid vector

A PCR cloning method that combines a dual selection pGATE-1 plasmid vector and an improved overlap extension cloning was developed. This efficient and cost-effective method allows for the introduction of DNA fragments into the Gateway cloning pipeline. The cloning efficiency is facilitated by a dual selection that includes the ccdB gene and gentamicin resistance. For users of the Gateway cloning system, substantial cost saving comes from eliminating BP recombination and ligation reactions to introduce DNA fragments into pDONR or pENTR vectors. Beyond the Gateway technology, this recombination-based cloning system can be used to efficiently clone PCR amplicons by adding 24-base pair adaptor sequences that are utilized by bacterial homologous recombination mechanism. METHOD SUMMARYFor overlap extension cloning of PCR products into the pGATE-1 vector, a DNA fragment of interest is amplified using primers with adaptor oligonucleotide sequences complementary to the pGATE-1 dual-selection plasmid. The resulting PCR product is used as a megaprimer in the optimized overlap extension reaction to replace the ccdB negative selection gene in the Gateway cassette of pGATE-1. The product of the overlap extension reaction is used directly for transformation into bacteria.