Research Project
3333959
The long-term objective of the proposed research is to evaluate, develop and improve upon applications of the polymerase chain reaction (PCR) for genomic mapping and sequencing. The specific aims can be summarized into four general areas of technology development: (i) evaluation of bacteriophage P1 as a cloning system which is capable of carrying large DNA inserts, and which is amenable to all proposed PCR-based mapping and sequencing technologies; (ii) development of interspersed-repetitive sequence directed PCR (IRS PCR), to generate additional DNA markers throughout a targeted genomic region, to efficiently develop sequence tagged sites (STSs) and to fingerprint individual P1 clones carrying large fragments of cloned DNA; (iii) development of transposon based PCR, in which transposons will be used to integrate primer binding sites into targeted regions of DNA for application to a number of mapping purposes, including the generation of probes from regions recalcitrant to IRS PCR, and sequencing of targeted DNA fragments; and (iv) evaluation of novel thermophilic enzymes and/or conditions for performing PCR, to enhance the utility of PCR in large scale mapping and sequencing endeavors by enabling the amplification of longer DNA templates, and sequences which are currently difficult to amplify. All of the proposed research will be performed using a well characterized model system which includes irradiation-reduced Goss Harris hybrids containing overlapping segments of chromosome 11p13 as their sole human component, and the complete long-range restriction map of 11p13 which was constructed using these somatic cell hybrids as mapping reagents (Rose,et al.,1990). The resulting physical map contains DNA markers spaced at approximately 100 kb intervals throughout 11p13, and identifies the positions of several genes involved in both normal development and genetic disease. One of these genes has been isolated and characterized, and is believed to represent the gene on 11p13 which is responsible for predisposition to Wilms' tumor (Rose,et al,1990; Call, et al.,1990). The proposed research is expected to result in the development of new technologies which will have general applicability to the analysis of any targeted genomic region. These studies will also provide refined map and sequence information concerning the central portion of chromosome 11p13, additional DNA markers and STSs, and important information concerning the function of genes encoded within this region. The proposed research will thus benefit the Human Genome Initiative by providing new methodologies for genome analysis, while simultaneously enhancing our understanding of the basic biological functioning of the various genes within 11p13, and the relationship between genome structure and function.
