Plant & Animal Genomes XIV Conference

January 14-18, 2006
Town & Country Convention Center
San Diego, CA

Novel Linear Vector Imparts High Stability To Large, Unstable Genes

David Mead¹ , Vinay Dhodda¹ , Nikolai Ravin² , Ronald Godiska¹

¹  Lucigen Corporation, Middleton, WI 53562

²  Centre

We have developed a novel, linear E. coli cloning vector incorporating transcription-free cloning capabilities. This vector shows unprecedented stability in maintaining large inserts (>10-20 kb) from AT-rich genomes. These same AT-rich inserts were cloneable in conventional plasmids. A transcription-free, linear vector has numerous important advantages in cloning, such as a significantly lower bias in size of fragments cloned, a low background of non-recombinants, simplified construction of large insert libraries, reduction of “sequence gaps” caused by secondary structures, and the ability to clone inverted repeats. Examples of these difficult-to-clone sequences are the large tracts of viral, microbial, and eukaryotic genomes that are refractory to cloning with conventional host-vector systems. Torsional strain inherent to supercoiled plasmids can induce localized melting and generate secondary structures, which are substrates for deletion or rearrangement by resolvases and replication enzymes. For example, tandem repeats and palindromic sequences are highly unstable, presumably due to cleavage of hairpin structures or to replication slippage across the secondary structures. Most plasmid vectors also induce strong transcription and translation of inserted fragments, and they allow transcription from cloned promoters to interfere with plasmid stability. As a result, certain DNA sequences are deleterious or highly unstable, leading to sequence “stacking”, clone gaps, or a complete inability to construct libraries, especially from AT-rich genomes or toxic cDNAs. These problems appear to be greatly diminished with the new linear vector.