The unifying goal of this center is the construction of a high resolution human genetic map. At the end of the five year period of proposed research, the map will consist of 3500 index quality markers providing an average marker density of 1 cM. The sex averaged resolution of this map, prior to gap filling, will be < 3 cM for 95% of the intervals. The goal of this project will be to develop a large set of STRPs that are uniformly distributed, readily amplified and easily interpreted. Markers will be obtained by use of a novel strategy termed """"""""marker selection"""""""" that allows the construction of libraries highly enriched for the STR marker of choice. Briefly, a genomic library with an average insert size of less than 500 base pairs was constructed in a phagemid vector. Amplification of this library in a dut ung strain of E. coli allows the recovery of the library as closed circular single-stranded DNA with uracil frequently incorporated in place of thymidine. This DNA was used as a template for second-strand DNA synthesis, primed with the desired simple sequence repeat oligonucleotides (e.g. [CA]20 or [TG]20) at elevated temperatures using a thermostable DNA polymerase. Transformation of this mixture into wild type E. coli strains resulted in the recovery of primer-extended products as a consequence of the strong genetic selection against single-stranded uracil- containing DNA molecules. In this manner, a library highly enriched for the targeted micro satellite-containing clones was recovered. This approach is widely applicable, and can be used to generate marker-selected libraries bearing any STR from cDNAs, whole genomes, single chromosomes, or more restricted chromosomal regions of interest. The use of this protocol, combined with high throughput sequencing strategies, will lead to the rapid accumulation of genetically useful STRPs and fuel the development of a robust genetic maps.
The specific aims of this project include: 1) Construction of small insert genomic DNA libraries in phagemid or M13 vectors. these libraries will be used to develop marker selected libraries highly enriched for di-, tri- and tetranucleotide repeats by the """"""""marker- selection"""""""" technique; 2) Construction of small insert oligo dT primed cDNA libraries as well as random primed cDNA libraries in phagemid or M13 vectors. These libraries will be used to develop marker selected libraries highly enriched for microsatellite repeats in the 3 untranslated region of expressed sequences and coding regions; 3) Complete a pilot study of markers derived from the diverse sources discussed above to establish the utility of various marker types and sources for the construction of high resolution genetic maps. The parameters to be studied include ease of development of PCR based assays, heterozygosity frequency, genomic distribution and ease of interpretation; 4) Construction of marker selected small insert genomic DNA libraries in phagemid or M13 vectors from flow sorted chromosome specific large insert libraries or directly from flow sorted material in order to facilitate the filling of gaps in the genetic map; 5) Development of methods for rapidly recovering STR sequences from physically mapped large insert clones to aid in filling gaps within the genetic map; 6) Continued development of the marker selection strategy. Specific goals include construction of improved bacterial host strains to optimize the representation of sequences in libraries, optimization of the enrichment protocol and explore the potential for improving the preferential selection of longer repeats.
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