MATS; A novel, rapid method for the development of microsatellite markers from YACs
- Harvard Medical School, Boston, MA (United States)
The availability of high resolution genetic maps has enabled the rapid mapping of a large number of disease loci. In absence of good candidate genes, the eventual identification of the disease locus requires effective strategies for completing the positional cloning {open_quotes}end game{close_quotes}. An essential element of this exercise is the development of local high resolution genetic maps, enabling the precise localization of the minimal interval containing the targeted gene. In addition, one also requires an accurate clone map of this minimal interval. Currently, clone coverage can be obtained using YACs, but rapid development of new genetic markers or additional STSs for walking or confirmation of clone order is a tedious process. Here we describe the successful application of a novel, rapid and efficient procedure, based on subtractive hybridization and PCR amplification for generating microsatellite markers on non-polymorphic STSs directly from the YACs or other large insert cloning vectors. After a single round of subtraction, the target sequences (YAC) are amplified by PCR and cloned into plasmid vectors. Several key steps have been designed to achieve efficient subtractive hybridization and to obtain preferential amplification of the target sequences. These technical steps include the design of novel adapters, PCR primers, efficient development of small insert libraries and non-radioactive screening methods. For example, using a 600 kb YAC as a target, we developed 14 new microsatellite markers. These new markers greatly facilitated genetic localization of a disease locus and allowed the accurate ordering by STS content mapping of a cloned contig spanning the interval. In addition to the utility of this approach in positional cloning, this strategy may provide an approach for filling gaps in the emerging genetic maps.
- OSTI ID:
- 134391
- Report Number(s):
- CONF-941009--
- Journal Information:
- American Journal of Human Genetics, Journal Name: American Journal of Human Genetics Journal Issue: Suppl.3 Vol. 55; ISSN AJHGAG; ISSN 0002-9297
- Country of Publication:
- United States
- Language:
- English
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