The general goal of the research being carried out in the Molecular Probe Resource Project is development of reagents and technologies which allow rapid, accurate appraisal of large scale (10's of kilobases) changes in the human genome which coincide with the cancer disease process. Currently, elucidation of causative relationships between genetic changes and cancer is hindered at least in part, by the difficulties of accurately identifying these changes on a case by case basis, and by the difficulty of surveying large numbers of cases for those specific changes which have already been observed and are suspected to be correlated with a given disease. In this initial year of the project, research aimed at improving the speed and accuracy of detection of these changes has been initiated and is ongoing in three areas. 1. Develop methods to characterize, stabilize, and effectively utilize the collections of region specific DNA segments which result from the process of micro-dissection and degenerate PCR amplification of normal and aberrant human chromosomes. DNA probes produced by micro-dissection have general utility as in-situ hybridization probes to rapidly identify the components of the complex chromosomal rearrangements and as probes in standard molecular biology protocols to provide defined points of entry at any cytogenetically definable region on the genome. The large investment in capital equipment and dedicated manpower required to generate these libraries prohibits many labs which would like to use these reagents from generating them themselves. In order to make the micro-dissected reagents generated at NCHGR available to the research community at large, methods for cloning the PCR products to allow widespread dissemination are being developed. A survey of the methods available for converting the PCR products to a cloned library is currently ongoing with model systems, the entire human chromosome 8, and a portion of chromosome 8 near the c-myc locus (8q24) which is frequently amplified in a variety of tumors. As a part of this research project, an assortment of molecular methods for examining the extent to which the micro-dissected materials contain all of the sequences in the original material, and the extent to which this level of representation is retained in the cloned material are being tested. To allow for efficient use of the cloned products as in-situ hybridization probes, an integrated protocol, which can be carried out with typical laboratory equipment, for the production of cloned DNA by cell culture, isolation of highly purified DNA from cultured cells by chromatography, fragmentation of the isolated DNA by depurination, chemical addition of amine groups to the DNA by trans-amination, and covalent addition of fluorescent dyes to the amine substituted DNA has been developed. 2. Develop extensions of the technology developed in the micro-dissection efforts which will allow highly localized samples taken from histopathologic samples to be subjected to analysis by the new technique of comparative genome hybridization (CGH). CGH analysis has been shown to be a very potent method for characterizing tumor materials for specific gains and losses at chromosomal loci. One of the weaknesses of the technique as currently practiced is that it requires a fairly large sample of tumor cells in order to extract sufficient DNA to carry out the test. Both the unavailability of large amounts of DNA from histopathology samples, especially archival materials, and the tendency of large samples to be more contaminated with normal cells limit the usefulness of CGH in evaluating tumors such as prostate and melanoma. Experimentation on the feasibility of preparing CGH samples by applying degenerate oligonucleotide priming methods (DOP PCR) to amplify DNA from very limited tissue sources is underway. Model experiments with limited amounts of template (1-10 ng) from cultured cells with known aberrations have been carried out and demonstrate that DOP PCR can produce comparable CGH results to those obtained with abundant extracted DNA. Experiments to allow determination of the best sources of cells for DOP PCR template extraction and of the best methods for extracting, purifying and labeling the sample DNA are ongoing. 3. Test the feasibility of analyzing genomic DNA by using hybridization arrays composed of region specific DNA targets immobilized on glass surfaces. The current practical limits in terms of the size of the regional DNA gain or loss which can be detected, and the capability to localize the gain or loss to precise genomic regions in the application of CGH are set by the use of metaphase chromosomes as the hybridization target. Greater sensitivity to changes involving small regions and higher precision of regional specificity could be obtained if a synthetic hybridization target containing more copies of fewer genes in somewhat larger areas could be produced to replace metaphase chromosomes. Experimentation to determine how micro-dissection products could be covalently linked to glass surfaces to provide such arrays has been initiated. Results to date indicate that very efficient loading of these products onto glass can be carried out by providing a 5' amino group on the PCR primers used to amplify the micro-dissected DNA and then attaching the denatured, amino terminated products to highly porous glass derivatized with isothiocyanate groups.
