title: Genome-scale cloning and expression of individual open reading frames using topoisomerase I-mediated ligation.
abstract: The in vitro cloning of DNA molecules traditionally uses PCR amplification or site-specific restriction endonucleases to generate linear DNA inserts with defined termini and requires DNA ligase to covalently join those inserts to vectors with the corresponding ends. We have used the properties of Vaccinia DNA topoisomerase I to develop a ligase-free technology for the covalent joining of DNA fragments to suitable plasmid vectors. This system is much more efficient than cloning methods that require ligase because the rapid DNA rejoining activity of Vaccinia topoisomerase I allows ligation in only 5 min at room temperature, whereas the enzyme's high substrate specificity ensures a low rate of vector-alone transformants. We have used this topoisomerase I-mediated cloning technology to develop a process for accelerated cloning and expression of individual ORFs. Its suitability for genome-scale molecular cloning and expression is demonstrated in this report.
authors: Invitrogen Corporation, Carlsbad, California 92008 USA. john_heyman@invitrogen.com
authors: J A
authors: JA
authors: Heyman
authors: J
authors: J
authors: Cornthwaite
authors: L
authors: L
authors: Foncerrada
authors: J R
authors: JR
authors: Gilmore
authors: E
authors: E
authors: Gontang
authors: K J
authors: KJ
authors: Hartman
authors: C L
authors: CL
authors: Hernandez
authors: R
authors: R
authors: Hood
authors: H M
authors: HM
authors: Hull
authors: W Y
authors: WY
authors: Lee
authors: R
authors: R
authors: Marcil
authors: E J
authors: EJ
authors: Marsh
authors: K M
authors: KM
authors: Mudd
authors: M J
authors: MJ
authors: Patino
authors: T J
authors: TJ
authors: Purcell
authors: J J
authors: JJ
authors: Rowland
authors: M L
authors: ML
authors: Sindici
authors: J P
authors: JP
authors: Hoeffler
mesh_terms: D000818:Animals; D016466:CHO Cells / Q000502:physiology; D003001:Cloning, Molecular / Q000379:methods*; D006224:Cricetinae; D004264:DNA Topoisomerases, Type I / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005784:Gene Amplification; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D016678:Genome; D006801:Humans; D008322:Mammals; D016366:Open Reading Frames; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D011494:Protein Kinases / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics
chemical_list: D011494:Protein Kinases; D004264:DNA Topoisomerases, Type I