A long term effort is underway to map and sequence the human genome. Currently several projects to evaluate sequencing techniques and strategies are being scaled-up and automated. Most of these are based on highly redundant sequencing of random M13 clones. While having many advantages, this approach results in low efficiency due to the high redundancy and potentially serious difficulties in assembly and gap-filling. Recent advances have demonstrated that primer walking, an alternate strategy that avoids the pitfalls of a random strategy, could be carried out using sets of adjacent hexamers rather than a single longer primer (Kieleczawa, et al., Science 258:1787-91 (1992)). There are four specific aims of this project;
Specific aim 1) Optimize the use of hexamer strings to prime sequencing reactions in automated sequencers. Previous work on hexamer strings has used manual sequencing with radioistopes. In order to be scaled-up, this technology needs to be developed on automated sequencers to allow acquisition of computer readable data. Once this is done the impact of the hexamer string strategy will be assessed as part of a pilot project.
Specific aim 2) Test the large-scale utilization of double- stranded template preparation procedures. Optimize double-stranded template purification and compare the problems resulting from this strategy due to template failures to the benefits in assembly and gap filling this approach will provide. This will result in data indicating the likelihood of success in scaling-up to the megabase level.
Specific aim 3) Set up a database to store information that can be used to evaluate genome sequencing projects and make project management decisions. Design and implement a prototype database and system of software tools to support sequence assembly and project management.
Specific aim 4) Sequence approximately 600 kb from chromosome 2 of the fission yeast Schizoaccharomyces pombe. This sequence will have a major impact on the understanding of gene and genome structure in eukaryotes when compared to the sequence being obtained from S. cerevisiae.
| Lodhi, M A; McCombie, W R (1996) High-quality automated DNA sequencing primed with hexamer strings. Genome Res 6:10-8 |
| Johnson, A F; Lodhi, M A; McCombie, W R (1996) Fluorescence-based sequencing of double-stranded DNA by hexamer string priming. Anal Biochem 241:228-37 |
