Plant & Animal Genome IX Conference

Poster: Large Insert Libraries, Gene Isolation, Etc.
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PURIFICATION AND DIRECT SEQUENCING OF BACTERIAL ARTIFICIAL CHROMOSOMES

Princeton Separations, Inc. P.O. Box 300, Adelphia, NJ 07710

Current methods for the complete sequencing large molecules such as BACs and cosmids include shotgun cloning (Boysen et al. 1997) and primer walking using the current sequence information to design the next primer. In order to sequence an entire BAC clone (generally >100 kb) a system to sequence small segments at random is needed. Methods used to complete sequences in plasmids (e.g. Nested deletion sets, mapping and subcloning of segments (Sambrook et al. 1989) become very labor intensive when applied to large DNAs like BACs. A more direct method using the advantages of both shotgun cloning and primer walking is transposon mediated sequencing (TMS). This allows the direct sequencing of the single BAC molecule in a random fashion using one or two primers. Avoiding subcloning and multiple primer design reduces the initial labor and cost involved in generating a ôsequence libraryö of random clones. TMS coupled with a single and reliable BAC/cosmid miniprep method allows for rapid sequencing of large molecules.