Harold R. Garner, David Burbee, Stafford Brignac, Kim Burzynski, Christopher Davies, Michelle Gilbert, Ken Kupfer,[1] Ping Li, Shane Probst, Simon Rayner, Emilee Strunk and Glen A. Evans,
McDermott Center for Human Growth and Development, University of Texas Southwestern Medical Center at Dallas, 6000 Harry Hines Boulevard, Dallas Texas, 75235-8591.
Cosmid oriented walking ("COW") is a novel and potentially powerful approach for determining the sequencing of human chromosomes based on sampled maps at any required level of accuracy. COW sequencing depends on the simultaneous sequencing of cosmid clones and the construction of contigs based on sequence matching to a large cosmid-end-sequence database. One advantage of COW sequencing is the potential for complete, unattended automation. A COW DNA sequencer prototype is being constructed which will carry out dye terminator DNA sequencing from 96 cosmid templates simultaneously, processing of 96 sequences, prediction of oligonucleotide "walking" primers for extending the sequence of each fragment, programming a network attached 96-channel oligonucleotide synthesizer, synthesis of the primers and placement of the primers into the cosmid template set to initiate a second round of sequencing. Using a set of nested cosmids covering 800 kb at 5X redundancy, COW sequencing should allow completion of 800 kb in 8 to 16 cycles. Such an instrument for unattended DNA sequencing is currently under construction.
The enabling technologies for COW sequencing are high quality dye terminator chemistry based sequencing on cosmids, the implementation of a high-throughput, low cost automatic oligo synthesizer and development of automated control and sequence assembly software. An oligo-synthesizer based on a 96 channel upgrade to the LBL 12-channel synthesizer, but using Macintosh hardware and Labview control software is being assembled. Network software based on Applescript and script-aware software for parallel assembly of 96+ independent walks is being written using a combination of commercial and in-house developed software packages.
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.