Plant & Animal Genomes XI Conference

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

Poster: Genome Sequencing & ESTs
          

HIGH STABILITY VECTORS FOR CLONING UNSTABLE DNA

¹ Lucigen Corporation, 2120 W. Greenview Dr. #9, Middleton, WI 53562 USA

² University of Cincinnati College of Medicine, Cincinnati, OH

³ University of North Carolina, Chapel Hill, NC

Large sections of eukaryotic, microbial, and viral genomes are refractory to cloning with conventional host-vector systems. Most plasmid vectors induce transcription and translation of inserted fragments, leading to instability of certain classes of DNA sequences or gene products. This apparently unclonable DNA results in sequence "stacking", clone gaps, or a complete inability to create genomic libraries, especially from AT-rich DNA. The pSMART series of transcription free cloning vectors were therefore developed to generate random shotgun libraries from difficult-to-clone DNAs (www.lucigen.com). For example, telomeric repeats and other AT-rich fragments from Pneumocystis carinii were 100% stable in pSMART but were very unstable in pUC19. Likewise, using pSMART rather than pUC19 to clone the AT-rich genome of Lactobacillus helvecticus was 25-fold more efficient and significantly less biased. Toxic regions of the mouse hepatitis virus genome were readily cloned and stable in pSMART, but they were deleted, rearranged, and slow-growing in TOPO and pGEM vectors. A low background of empty pSMART vector (less than 1%) eliminated the need to screen for recombinants and facilitated cloning nanogram quantities of DNA. These and other features are currently being integrated into a high-stability, low-bias BAC vector. Finally, a new system for multiplex cloning was developed to allow two independent DNA fragments to be cloned simultaneously into separate sites of a transcription free vector.