DOE Human Genome Program Contractor-Grantee
10. pZIP: A Versatile Vector for Sequencing by Nested Deletions
John J. Dunn
Biology Department, Brookhaven National Laboratory, Upton, NY 11973
We have constructed a low-copy, amplifiable vector that should be particularly useful for cloning and sequencing full-length cDNAs and highly repeated DNAs. This pZIP vector is maintained in Escherichia coli at low copy number by the F replicon and can be amplified 300 fold from an IPTG-inducible phage P1 replicon (repL). A relatively small size of 4.4 kbp was achieved by removing the 2.5-kb sop (stability of plasmid genes) region of F, but the plasmid is stably maintained by selective growth in the presence of kanamycin. A multiple cloning region (MCR) is flanked by sites that allow the biochemical generation of unidirectional nested deletions crossing the cloned DNA. The resulting deletion clones can be ordered by size, and an ordered, overlapping set of sequences can be obtained by priming within the flanking vector sequence to produce the complete sequence of both strands. The correspondence of plasmid lengths with those predicted by the assembled sequence aids in and verifies the correctness of the assembly. The low copy number should allow the cloning of DNAs that might not be stable in higher copy vectors, and amplification provides ample DNA for generating the nested deletions.
Unidirectional nested deletions are produced by cutting the DNA specifically near one end of the cloned DNA to generate an end that is sensitive to digestion by E. coli exonuclease III (ExoIII) and an end that is resistant, or by specifically nicking the appropriate strand. The ends or nick are oriented so that ExoIII will digest one strand across the cloned DNA. The resulting single-strand gaps are converted to double-strand gaps by treatment with S1 nuclease, and the ends are repaired and ligated with T4 DNA polymerase and ligase. ExoIII digests quite synchronously, and treating pooled samples from several different ExoIII digestion times, followed by electroporation, produces a population of clones with a distribution of different deletion end points. ExoIII-resistant ends are produced by intron-encoded endonucleases that cut at very rare sites to produce 4-base 3' overhangs. I-CeuI and I-SceI flank the MCR on one side and I-PspI on the other. ExoIII-sensitive ends can be generated by cutting with a restriction endonuclease at any of several different 8-base or other rare cleavage sites located between the sites cut by the intron-encoded nucleases and the cloning sites in the MCR. The fd origin of replication is also located on the I-PspI side of the MCR, oriented so that the specific nick by the gene 2 protein can be extended across the cloned DNA by ExoIII.
In collaboration with the Joint Genome Institute, we are evaluating the capability of the pZIP vector and the nested-deletion sequencing strategy to close gaps that have resisted closure by standard sequencing strategies in several different regions of human chromosome 19. We hope to demonstrate cloning in the low-copy pZIP of regions that are difficult to clone in standard sequencing vectors, and to determine accurate sequences of highly repeated regions by the nested-deletion strategy.
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