TRANSCRIPTOME 2002: From Functional Genomics to Systems Biology
March 10-13, 2002
Seattle, Washington, USA


A Novel Approach to Eliminate Vector Background and Increase Sequencing Efficiency of cDNAs

Uday Matrubutham1, Jody Mirchandani1, Jian Liu1, Martin A. Gleeson1, K. MacDonald2, J. Asano2, Y. Butterfield2, N. Girn2, S. Lee2, T. Olson2, P. Pandoh2, U. Skalska2, D. Smailus2, L. Spence2, Jeff Stott2, G. Yang2, J. Schein2 and M. Marra2, 1Invitrogen Corporation, Carlsbad, CA, 2Genome Sequence Centre, BC Cancer Agency, Vancouver, CANADA

We, at Invitrogen Corporation, have developed a novel and highly efficient transposon-mediated method for generating cDNA sequences with low quantities of vector background.  The cDNA sequencing templates are generated in two consecutive in vitro reactions utilizing Mu transposition (GeneJumperTM) and att/clonase recombination (GatewayTM, Invitrogen Corporation).  First, the GeneJumperTM transposon is randomly inserted into the cDNA clone.  Second, the cDNA insert is transferred to a compatible GatewayTM vector by recombination. Transposon-containing cDNA recom-binants are identified using antibiotic selection and sequenced bi-directionally with transposon-specific primers.  This process has been applied to cDNAs cloned into pCMVSport and GatewayTM clones that have att recombination sites flanking the cloned insert DNA. This methodology was successfully evaluated for high-throughput process adaptation as part of the Mammalian Gene Collection initiative.  The approach reduces the number of reads required to complete cDNA sequences through reduction of reads initiated from within the cDNA vector.  In addition, we have adapted the approach to the pooled clone strategy currently in use at the BC Cancer Agency Genome Sequence Centre.  We anticipate that the increased efficiency provided by the method will reduce dramatically the cost of sequencing cDNA clones or other similarly sized clones.

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