Lung cancer is the leading cause of cancer related deaths in American adults. Mutations in c-Kiras and p53 oncogenes occur early in tumor development and are retained as specific markers for lung cancer. Analysis of these genes in sputum samples revealed mutations in patients one year before clinical diagnosis.
We aim to develop a method to simultaneously detect approximately 200 specific point mutations in these genes as an early detection test for lung cancer. The method is based on ligation and selective amplification of a single oligonucleotide probe designed to give base discrimination and detection of 1 rare target in 105 normal molecules. Unlike allele specific PCR and LCR, this method analyzes mutations in parallel without loss of discrimination or sensitivity. Commercial plans are to adapt the genetic screen to microfabricated arrays for automated analysis. The objectives of Phase I will determine: i) optimal oligonucleotide probe sequence for target discrimination and selective amplification; ii) optimal ligation conditions for target discrimination and probe circularization; iii) if DNA polymerase fidelity is required for additional discrimination; and iv) signal amplification and compatibility with immobilized probe. In Phase II we will assemble the mutation panel, identify test format, and assess clinical utility.
Detection and screening of specific rare cell point mutations in a wild- type population can be used for earlier diagnosis of certain cancers. For example, with early screening of lung cancer, tumors can be identified at an earlier stage and removed surgically prior to metastasis. The proposed method can be automated for high volume screening at a cost below the current diagnostic method (chest X-ray).
| Thomas, D C; Nardone, G A; Randall, S K (1999) Amplification of padlock probes for DNA diagnostics by cascade rolling circle amplification or the polymerase chain reaction. Arch Pathol Lab Med 123:1170-6 |
