Bacterial artificial chromosome (BAC) libraries have become pivotal reagents for modern molecular biology research, and are particularly important for comparative and functional genomics. However, due to their extreme cost, the expertise required for their construction, and the specialized equipment necessary for their generation and distribution, very few laboratories have been capable of routinely generating these resources. This RFA to establish resource laboratories to specifically generate BAC libraries from eukaryotic species of medical relevance is thus an imperative initiative, and one for which my laboratory is especially well-suited. Our research interests in molecular evolution and developmental genomics have dictated our need for generating BAC libraries, and consequently, we have constructed several libraries from the various chordate species that we study. My research group has recently moved to the Virginia Mason Research Center (Seattle) and our new laboratory has been set up with the necessary infrastructure, space, and robotics instruments expressly for performing the type of work prescribed by the RFA. Our main goal in this submission is to assure that high quality BAC libraries are generated for the eukaryotic species of interest and that these libraries are widely accessible to the scientific community. We propose to serve as a center for the generation of high quality 10X coverage BAC (equal to or > 150 kb average insert size) from six mammalian-sized genomes (species) per year. We will array these libraries and make requisite replicas for distribution to major users of the library and other entities that will serve as central resource distributors. Because of the heavy biological focus in my laboratory and because our strength is BAC library construction and not distribution, we thus adopt a model whereby we primarily serve as resource-generators. We advocate that the resources generated in this RFA program be as easily accessible (widely distributed) to the scientific community as possible, and to the extent that we are able, we will produce a limited number of high-density filters for the community and serve as an archival site for the libraries that we generate. Insofar as library generation per se, we have improved on the protocols involved in BAC construction, including high molecular weight DNA isolation, partial restriction digestion, preparative pulsed field gel electrophoresis (PFGE), and elution of partial digests from gels. We have also adopted logistical measures and procedures that streamline the entire BAC cloning process considerably. In keeping with the technology development component of this RFA, preliminary experiments have been performed in order to clone large DNA fragments from mechanically sheared samples and for specifically capturing large genomic fragments from heterogeneous populations of molecules. Finally, we wish to establish a good working relationship with the BAC Scientific Advisory Panel and to establish critical and cooperative crosstalk with the other BAC library resource centers.
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| Wang, Zhenshan; Miyake, Tsutomu; Edwards, Scott V et al. (2006) Tuatara (Sphenodon) genomics: BAC library construction, sequence survey, and application to the DMRT gene family. J Hered 97:541-8 |
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