pDUAL: a transposon-based cosmid cloning vector for generating nested deletions and DNA sequencing templates in vivo

Abstract

We describe a transposon gamma delta-containing cosmid cloning vector, pDUAL (previously called pJANUS), and demonstrate an efficient strategy for isolating nested deletions in both large-scale and small-scale DNA sequencing efforts. This "deletion factory" strategy takes advantage of the ability of gamma delta (Tn1000) to generate deletions that extend from an end of the transposon into adjacent DNA when gamma delta transposes to new sites in the same DNA molecule. pDUAL contains the contraselectable (conditional lethal) sacB+ (sucrose sensitivity) and strA+ (streptomycin sensitivity) genes just outside each end of an engineered gamma delta and selectable kan+ (Kanr) and tet+ (Tetr) genes between the cloning site and sacB and strA, respectively. Selection on sucrose tetracycline medium yields deletions that extend from one gamma delta end for various distances into the cloned DNA, while selection on streptomycin kanamycin medium yields comparable deletions in the other direction. Both types of deletions are recoverable because the essential plasmid replication origin is embedded in the gamma delta component and is thereby retained in each deletion product. Pilot experiments with pDUAL clones showed that deletion end points can be mapped or selected by plasmid size and that both DNA strands of any single clone can be accessed for sequencing by using a pair of universal primers specific for sequences that are just interior to the gamma delta ends.