) This project aims to develop two new technologies for detection of point mutations associated with colorectal cancer and breast cancer. We will combine the polymerase chain reaction (PCR), restriction endonuclease digestion reaction (RE), and the ligase detection reaction (LDR) to detect K-ras and p53 mutations in clinical samples from cancer patients.
Our specific aims are: (1) to develop a multiplex PCR/LDR method to screen rapidly and simultaneously for multiple mutant alleles at a sensitivity of 1 cancer cell in 100 - 1,000 normal cells, and (ii) to develop a highly sensitive PCR/RE/LDR method to identify specific mutant alleles at a sensitivity of 1 cancer cell in 100,000 - 1,000,000 normal cells. Ligation primers will be designed and tested to optimize ligation fidelity and compatibility for multiplexing. Mutant Tth DNA ligases and nucleotide analogue containing primers will be tested to improve PCR/LDR sensitivity and fidelity. For PCR/RE/LDR, a general method to selectively introduce restriction sites into the PCR product derived from the wild- type allele will be applied and tested using the K-ras and p53 genes. Multi-pairing and non-pairing nucleotide analogues as well as proofreading polymerases will be tested for their effect on the efficiency and specificity of the restriction site conversions. We intend to use clinical samples to demonstrate the feasibility and potential clinical relevance of mutation detection in K-ras and p53 using PCR/LDR and PCR/RE/LDR in colorectal and breast cancer. For colon cancer, we will evaluate multiplex PCR/LDR as a method for quantitative mutation detection in K-ras and p53 mutations as molecular markers of disseminated cancer cells in tissues at risk for occult micrometastases (lymph nodes, pelvic washings, and bone marrow aspirates). In addition, PCR/LDR and PCR/RE/LDR will be evaluated as diagnostic tests for colorecal cancer by screening stool and colonic lavage samples for K-ras mutations.
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