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

Journal Article · Sun Aug 15 04:00:00 EDT 1993 · Proceedings of the National Academy of Sciences of the United States of America; (United States)

DOI:https://doi.org/10.1073/pnas.90.16.7874· OSTI ID:5053322

Wang, Gan; Berg, C M ^[1]; Blakesley, R W ^[2]; Berg, D E ^[3]

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1. Univ. of Connecticut, Storrs, CT (United States)
2. Life Technologies, Inc., Gaithersburg, MD (United States)
3. Washington Univ. Medical School, St. Louis, MO (United States)

The authors 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 [open quotes]deletion factory[close quotes] 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[sup +] (sucrose sensitivity) and strA[sup +] (streptomycin sensitivity) genes just outside each end of an engineered [gamma][delta] and selectable kan[sup +] (Kan[sup r]) and tet[sup +] (Tet[sup r]) 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 in to 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. 27 refs., 3 figs.

• OSTI ID: 5053322
• Journal Information: Proceedings of the National Academy of Sciences of the United States of America; (United States), Journal Name: Proceedings of the National Academy of Sciences of the United States of America; (United States) Vol. 90:16; ISSN PNASA6; ISSN 0027-8424
• Country of Publication: United States
• Language: English

Related Subjects

550400* -- Genetics
59 BASIC BIOLOGICAL SCIENCES
CLONING
DNA HYBRIDIZATION
DNA SEQUENCING
DNA-CLONING
GENES
GENETIC MAPPING
HYBRIDIZATION
LETHAL GENES
MAPPING
STRUCTURAL CHEMICAL ANALYSIS
TENSORS
VECTORS