) We applied an unbiased DNA fingerprinting technique, the Arbitrarily-Primed PCR (AP-PCR) to study tumor- specific genetic changes. AP-PCR is a PCR-based DNA fingerprinting method, which uses a single oligonucleotide primer of arbitrary sequence, and generates a profile of quantitative and qualitative differences between the fingerprints of tumor and matched-normal tissues. Our efforts to both automate the AP-PCR technique and make it more robust, gave us the idea of combining AP-PCR fingerprinting with DNA array hybridization technology. Developing this new technique, which we call Comparative Hybridization of AP- PCR Arrays (CHAPA), is the goal of this grant application. We propose to clone individual DNA fragments amplified by AP-PCR from human genomic DNA and array them on a solid base. The AP-PCR product can be viewed as a low- complexity representation of the genome. The array will be hybridized with AP-PCR products that are amplified from normal and tumor tissue DNAs, which have been labeled with green and red fluorescent dyes, respectively. The intensity ratio of the two colors at each hybridization spot will reflect tumor-specific losses, gains, or no change of corresponding genomic loci. For the Phase I application, we will develop a small array of 300 AP-PCR fragments and test the new technique to see if it is reproducible, sensitive, and reliable. Chromosomal and subchromosomal origins of the arrayed AP-PCR fragments will be determined by hybridizing the array with AP-PCR products from individual clones of radiation hybrid mapping panels. We will also test different arbitrary primers to obtain collections of AP-PCR products (representations) of the human genome with a degree of complexity that is optimal for a large scale array of AP-PCR fragments. We will also test AP- PCR's ability to produce quantitative fingerprints of genomic DNA isolated from minute amounts of fixed microdissected tissues. For the Phase II experiments, we propose to scale up the array to at least 5,000 nonredundant AP-PCR fragments. In its final form, the technique will allow the state of the tumor genome to be quickly and automatically analyzed with less than 1 Mbp of resolution. This high-density unbiased molecular karyotyping will facilitate the discovery of novel cancer genes and open new horizons for the diagnostic and prognostic analysis of cancer development.
