) The goal of this application is to develop two innovative mutation detection systems that may become powerful clinical tools for the diagnosis and staging of human cancers. Cancer results from the accumulation of mutations in multiple genes, however no single gene is always mutated in adult cancers. Thus, to identify an index mutation in primary tumors, there is an urgent need to develop a method that can rapidly survey fir mutations in many genes. A specific mutation identified in the primary tumor can be used as a clonal marker for tumor cells; however, screening for micrometastases poses the additional problem of detecting the mutation in a vast background of normal cells. We have assembled a team of investigators whose expertise will be directed toward an integrated solution to these two problems. We have focused on two genes, K-ras and p53, which are among the most commonly mutated in colon and breast cancers. In colon cancers, mutations in these two genes are highly clustered to specific codons, and thus they are an ideal model system in which to demonstrate """"""""proof of principle"""""""" for our cancer associated mutation detection methods. Our strategic approach is to: (i) Develop a multiplex polymerase chain reaction/ligase detection reaction (PCR/LDR) system for the detection of K-ras and p-53 mutations in tumors. (ii) Develop the capability to identify mutations at a sensitivity of 1 cancer cell in 100,000 - 1,000,000 normal cells using our PCR/restriction enzyme/LDR (PCR/RE/LDR) method. (iii) Design and synthesize nucleotide analogues to convert specific DNA sequences into restriction endonuclease sites, and to increase the fidelity of Tth DNA ligase. (iv) Develop the capability to detect multiple mutations at a sensitivity of 1 cancer cell in 100 - 1,000 normal cells using mutant Tth DNA ligases, primers containing nucleotide analogues, or other Thermus DNA ligases. (v) Develop methods for the simultaneous detection of large amounts of LDR, products using addressable oligonucleotide or peptide nucleic acid arrays. (vi) Explore the ability to multiplex PCR/LDR to screen for mutations in primary colon tumors, stool and colonic lavage samples. (vii) Explore the ability of PCR/RE/LDR to identify known mutations as clonal markers of occult tumor dissemination to colon cancer (lymph nodes, stool, and pelvic washings) and in breast cancer (lymph nodes, bone marrow).
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