The overall objective of this proposal is to develop new methods for the direct physical isolation of specific fragments of human DNA, ranging in size from 100-2000 kilobasepairs (Kbp), which are too large to be cloned into conventional phage or cosmid vectors and to apply these methods to the purification, mapping and cloning of NotI and SfiI DNA fragments from selected genetic loci in the human genome. Two distinct affinity enrichment strategies will be explored, both of which exploit biotin-labeled nucleic acids as probes. Novel """"""""hybridization"""""""" reactions that do not require mass denaturation of the DNA will be done using either probe-recA protein nucleofilaments to form triple-stranded D-loop complexes or controlled exonucleolytic resection of terminal fragment sequences to render them accessible to probes unique to the end of a single fragment. Affinity resins, such as avidin-agarose, suitable for the selective capture and subsequent recovery of specific biotinylated hybrid molecules, either from solution or from solid gel matrices, will be used for DNA purification. Primary DNA targets for purification and molecular characterization are the 170 Kbp SfiI fragment that contains the entire beta-globin gene cluster including distal flanking nuclease hypersensitive sites, a 1090 Kbp NotI fragment from within the major histocompatibility complex on which only a paucity of genes have yet been identified, and the complete complement of NotI fragments that span 5000 Kbp of sequence surrounding the Cystic fibrosis gene. Finally, additional unique sequence probe sets from chromosomes 6 and 7 will be prepared and used to test global mapping strategies that could be used in conjunction with affinity purified DNA for the coordinated construction of both a physical map and an ordered clone bank of these chromosomes.
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| Warren, Rene L; Varabei, Dmitry; Platt, Darren et al. (2006) Physical map-assisted whole-genome shotgun sequence assemblies. Genome Res 16:768-75 |
