Improved sequencing of cosmids using new primers and linearized DNA

Abstract

DNA sequencing is presently performed almost exclusively by primed DNA synthesis in the presence of the 2' ,3'-dideoxy chain terminators (1), with best results being obtained with singlestranded templates and using small plasmid clones. For largescale sequencing projects, direct sequencing of cosmids is a more desirable strategy, since the sequence of a cosmid (approx. 40 kb of insert DNA) could be determined from a single DNA preparation without the need for mapping or subcloning of smaller fragments. Targeted priming of the sequencing reactions along the 40 kb DNA would further reduce sequencing redundancy and thus minimize the amount of work required to determine the entire sequence of a cosmid. However, there are still technical difficulties associated with direct sequencing of cosmids, and most new sequence information presently generated comes from experiments on smaller DNA clones. Our initial efforts to sequence larger (20-40 kb) DNA, using sequence-specific primers and the Applied Biosystems DyeDeoxy Terminator cycle sequencing strategy gave satisfactory results with a phage clone XHAFP33 (2), but sequencing data obtained from cosmids were inferior to those obtained from X DNA. This led us to consider possible improvements and we decided to investigate whether certain parameters in the cycle sequencing reactions, such as temperature and topology of template DNA, might affect the quality of sequencing results.Our cosmid clones came from a library containing genomic DNA from human chromosome 4 cloned in the vector sCos-1 (3). The library was constructed at the Los Alamos National Laboratory (Dr Larry Deaven, personal communication). A unique BamHI site in sCos-l is the cloning site, which is flanked by T3-and T7-promoter sequences, and insert DNA can be excised by using NotI or EcoRI restriction endonucleases. The sequence surrounding the BamHI cloning site in sCos-1, including the position of our new primers, is shown in Figure 1. Individual clones from the cosmid library were grown overnight in Terrific Broth (4), and cosmid DNA was purified from the culture using Qiagen Tip columns according to the manufacturer's protocol. The isolated cosmid DNA is a mixture of two circular forms, the supercoiled and the relaxed nicked-circle.

A Method to Direct Sequence Cosmid LAWRIST16 Clones

“…Automated direct sequencing on cosmid DNA clones has proven to be difficult due to the complexity of the cosmid DNA template. One method of direct sequencing cosmid DNA clones using a dye terminator chemistry has been described (Dugaiczyk et al, 1992). We used AmpliTaqO FS DNA Polymerase , (Reichert ef al., 1995), commercially available from Perkin Elmer Corporation (Foster City, CA, USA), and ThermosequenaseTM (Reeve and Fuller, 1995), (Tabor and Richardson, 1995), available from Amersham International plc.…”

“…/-zollo/ DnaSequence /Cosmid.Results / dyeterm/liS.l aw 16P.lden.AmpliT.gif). In comparing this method with those previously described (Dugaiczyk et al, 1992), we did not find it necessary to digest the cosmid DNA template before starting the sequencing reaction. The same amount of DNA templates obtained from the same cosmid DNA preparation (cosmid 58), one linearized by using SfiI restriction enzyme (data available on our web page), and one not linearized (Fig.…”