In any large-scale mutagenesis effort, some phenotypes emanate from new alleles of known loci. Presently? such mutations are found only after fixation, mapping, and positional cloning. This greatly increases the? overall cost and limits the return of a forward genetic endeavor. Recent advances in resequencing? technology have led to a rapid, reliable, and cost-effective means of identifying mutations within a curated? population of coding exons and splice junctions. The Resequencing Core is intended to maintain? surveillance over all coding exons of known and likely candidate genes, within which mutations are known or? would be suspected to produce susceptibility to MCMV (the phenotype studied in Project 1) or faulty? signaling in response to Drosophila C virus, Sindbis virus, or Flockhouse virus (the phenotypes studied in? Project 2). In this process, which we term """"""""anticipatory resequencing,"""""""" the target sequences will be tiled? onto Affymetrix resequencing arrays. As transmissible phenotypes are identified, genomic DMA from the? index mouse or fly will be amplified robotically at all candidate loci and used as a probe to detect mutations? by hybridization. In this way, all animals with mutations in genes that are already known or believed to? participate in defense will be identified prior to initiation of the expensive and labor-intensive process of? positional cloning. The target sequences to be examined will be derived both from prior knowledge (for? example, all known or suspected MCMV susceptibility loci will be included) and from information deposited in? the Osaka, La Jolla, and Strasbourg databases. This will assure a steady flow of new information from? positional cloning. The resequencing arrays will be updated annually to incorporate all new candidates that? are acquired in our joint effort, as well as others that come to light from work elsewhere in the scientific? community. The Resequencing Core will also apply conventional sequencing to the confirmation of? mutations detected using resequencing arrays and to the analysis of critical regions developed after all array? candidates are excluded. Moreover, as the technology develops so that arrays become a resource shared? by all members of the mouse and Drosophila communities and as annotation is perfected, resequencing will? be utilized directly to explore critical regions.
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