title: An alternative approach in Gateway(®) cloning when the bacterial antibiotic selection cassettes of the entry clone and destination vector are the same.
abstract: The Gateway(®) recombination technology has revolutionized the method of gene cloning for functional analyses and high-throughput ORFeome projects. In general, Gateway cloning is highly efficient because after LR recombination and bacterial transformation, only cells containing the recombinant destination clone are selected on an antibiotic selection plate. However, when the antibiotic resistance gene for bacterial selection is the same in the entry and destination vectors, the direct selection of recombinant destination clones on an antibiotic plate is difficult. Here, we demonstrate an efficient and comprehensive approach to obtain positive destination clones directly on an antibiotic selection plate in this situation. The strategy involves polymerase chain reaction (PCR)-mediated amplification of the entry clone using entry vector-specific primers that bind outside the attL sequences and the subsequent use of this purified PCR product for LR recombination with the destination vector. Our results suggest that cloning of linear DNA fragments into circular destination vectors through LR recombination is an efficient method for inserts up to 7 kb in size. Using this approach, the yield of colony PCR positive destination clones was 100 % for genes of various sizes tested in our experiments.
authors.affiliation: National Institute of Plant Genome Research, New Delhi, India. kamalkumarinder@yahoo.co.in
authors.forename: Kamal
authors.initials: K
authors.lastname: Kumar
authors.forename: Saurabh
authors.initials: S
authors.lastname: Yadav
authors.forename: Savithri
authors.initials: S
authors.lastname: Purayannur
authors.forename: Praveen Kumar
authors.initials: PK
authors.lastname: Verma
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000235:genetics; D024881:Drug Resistance, Bacterial; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics*; D011995:Recombination, Genetic; D014169:Transformation, Bacterial / Q000235:genetics
chemical_list: D000900:Anti-Bacterial Agents; D004247:DNA