TRANSCRIPTOME 2002: From Functional Genomics to Systems
Full-length cDNA Library Construction Using Lambda Recombination (GatewayTM Technology)
Larissa G. Karnaoukhova, Mark R. Smith, Chris E. Gruber, and Martin Gleeson, Research and Development, Invitrogen Corporation, Carlsbad, CA
The Invitrogen GatewayTM Cloning Technology is a versatile and efficient system for cloning and subcloning DNA. It is based on a lambda phage recombinase that can be applied in vitro to stimulate site specific (att site) recombination. One application of this technology is the construction of directional cDNA libraries as Entry Clones. Once in Entry Clone format, the library can be recombined with any of the Destination Vectors creating an expression ready library. To construct Entry Clones, the cDNA is synthesized so that attB sites flank it. The attB-adapted cDNA is then recombined with a Donor Vector (attP sites) to create the attL Entry Clone library. Libraries constructed using this technology show that such recombination cloning of cDNA produces a higher quality library exceeding the standards seen using traditional cDNA library construction methods. Unlike standard cDNA cloning protocols, this approach requires no restriction enzyme digestion of the cDNA to create unique ends suitable for directional cloning. The recombination cloning reaction produces on average 1.3 million cfu/ng cDNA with average insert size of 2.3 kb and clone size ranging from 0.6-12 kb. Recombination cloning combined with the GeneRacerTM technology has been applied to clone cap-selected cDNA and create full-length enriched cDNA libraries.
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