Dave Burbee, Chris Davies, Michelle Gilbert, Kim Burzynski, Emilee Strunk, Jeff Schageman, James McFarland, Ken Kupfer[1], Skip Garner, and Glen A. Evans.
Recent developments in DNA sequencing technology allow the rapid analysis of multiple DNA samples. We have recently shown that with special template preparation and with the use of dye terminator chemistry, large DNA molecules such as intact cosmids can be readily analyzed. We are able to collect 400 to 600 base runs with > 98% accuracy of cosmid templates. For random sets of oligonucleotide primers, greater than 90% efficiently prime the Taq FS-catalyzed sequencing reaction. Also, recent developments in oligonucleotide synthesis have resulted in the decreased cost of custom-designed oligonucleotides. We are able to rapidly and inexpensively sequence cosmids by standard walk procedures: T7 and T3 promoter primers are used as entry points to sequence the insert, and each successive round of sequencing is used to design the next oligo that primes the sequencing reaction farther within the interior of the insert.
Over the past few years, a large number of strategies have been designed for the sequencing of large genomes, including standard shotgun sequencing, sequencing of nested deletions, and transposon-mediated primer insertion (dog-tagging). We propose a new strategy that extends our current development and use of GSS (genome sequence-sampled) maps, and is based on our success with cosmid template sequencing. We have completed a 100 kb resolution map of human chromosome 11 based on standard STS's as well as STS's designed from the sequencing of 17000 ends of a chromosome 11-specific cosmid library. Many of these cosmids have been associated by mapping of overlapping restriction fragments and/or binned within specific YACs by IRE-bubble PCR hybridization. This database was generated to identify start points for the cosmid-oriented walking (COW) DNA sequencing strategy. COW sequencing is initiated by the complete sequencing of a cosmid or cosmid contig by primer-mediated walking or else shotgunning. Extension of the initial contig is carried out by searching the GSS database for other identified cosmid ends, and so extend the sequence with the sequencing of the contiguous overlapping cosmid. Using this technique, cosmid template sets extending over several megabases may be completed.
Though ultimately limited by the cost of oligonucleotide primers, primer walking has several advantages over the current strategies. Little redundancy of sequence template preparation and analysis is required; specific regions of interest may be identified (by GSS) and then analyzed rapidly and economically. Furthermore, coupling of automated DNA sequencing instrumentation to DNA sequence analysis programs and multichannel oligonucleotide synthesizers will allow almost complete automation of the cosmid oriented walking strategy, allowing a small group of researchers to produce (for a 96 channel oligosynthesizer) 40 to 60 kb of DNA sequence per round of sequencing.
Supported by a grant from the Office of Health and Environmental Research, Department of Energy.
[1]SERCA Fellow of the National Center for Human Genome Research. Supported in part by the National Center for Human Genome Research, the Whitaker Foundation and the Eugene McDermott Foundation.