Plant & Animal Genomes XI Conference

January 11-15, 2003
Town & Country Convention Center
San Diego, CA

Poster: Large Insert Libraries
            

AN ALTERNATIVE TO SHOTGUN LIBRARY CONSTRUCTION: IN-VITRO TRANSPOSITION OF LOW COPY BAC USING TN5 BASED TRANSPOSON

¹ EPICENTRE Technologies, 726 Post Road, Madison, WI53713

² Arizona Genomics Institute, 303 Forbes Building, University of Arizona, Tucson, AZ 85721

Traditionally, low copy BACs are sequenced using a shotgun library strategy. Library construction involves mechanical shearing of BAC DNA into smaller fragments and then sub-cloning into a high copy vector for high yield of DNA. We introduce here, a Tn5 transposon based system known as the EZTNInsertion Kit which can be used in place of shotgun library cloning, as an alternative strategy for sequencing low copy BACs. The In-vitro insertion reaction involves a 2 hour reaction resulting in a large population of subclones, each clone harboring a single randomly inserted transposon containing the inducible oriV and sequencing primer binding sites. The presence of oriV in the inserted clones allows an amplification of the BAC DNA into multiple copies when treated with an inducer, resulting in high yield of purer DNA. Primer binding sites allows bidirectional sequencing of the insert from each end of the transposon by using transposon specific primers. The number of subclones generated from one reaction can be used to obtain more than 100X sequence coverage of a BAC insert. In addition, repetitive sequences should be more readily assembled using transposon based sequencing as compared with the shotgun library strategy, since the integrity of the original sequence is maintained. We conclude that, sequencing of BAC DNA by using in-vitro insertion of the EZTNTransposon is a time and cost-effective method which can be easily adopted for high throughput sequencing of BACs.