A major problem in sequencing the genome of any organism is the difficulty in obtaining a complete set of contiguous DNA clones for the analysis. Although these clones may be chosen randomly at first, one will eventually be compelled to fill many large gaps in the clone collection in order to complete such a project. This task may be approached by chromosome walking. However, this will require an extraordinary effort in the case of genomes of higher animals due to the abundance of repeated DNA sequences. An alternative to chromosome walking would be to physically dissect specific regions of the chromosome and then isolate unique restriction fragment clones from the dissected chromosomal DNA. These clones might then be used as probes in order to isolate much larger genomic clones from libraries. This idea is not new, but a substantially improved method for obtaining clones from dissected chromosome fragments has been invented and is presented here. This method relies upon the ability to synthesize DNA in vitro using multiple cycles of DNA polymerase reactions where restriction fragments of dissected chromosomal DNA are the substrate. The amplified DNA is then either cloned or labelled for use as a probe of DNA libraries in order to obtain genomic clones from a specific region of a chromosome. I propose to use this method in collaboration with Dr. Daniel Hartl at Washington University (see letter in the Appendix) in order to identify a complete, ordered set of yeast-artificial- chromosome (YAC) clones of the euchromatic portion of the Drosophila melanogaster genome. I also propose to refine this method so that it may be used to isolate DNA from specific regions of vertebrate metaphase chromosomes and, if possible, make my laboratory a resource to investigators who are interested in the application of this new method.
| Overbeck, L D; Weston, M D; Kimberling, W J et al. (1993) Dinucleotide (CT)n (CA)n repeat polymorphism for D11S787 on chromosome 11q13.4. Hum Mol Genet 2:611 |
| Johnson, D H; Kroisel, P M; Klapper, H J et al. (1992) Microdissection of a human marker chromosome reveals its origin and a new family of centromeric repetitive DNA. Hum Mol Genet 1:741-7 |
| Overbeck, L D; Weston, M D; Kimberling, W J et al. (1992) A new highly polymorphic dinucleotide (CT)n repeat polymorphism D1S158 on chromosome 1q isolated by microdissection. Hum Mol Genet 1:141 |
