) This application is proposed to develop a mass spectrometric based technology for automated, multiplexed, high-throughput, sensitive, and specific detection of a small population of point mutation tumor cells in a large background of wild-type cells. The technology developed in this work consists of three major steps. First, the clinical DNA samples are amplified using the peptide nucleic analogues (PNA) directed PCR clamping reactions in which mutant DNA are preferentially amplified. Second, the PCR amplified DNA fragments are extended through mini-sequencing to generate diagnostic products. Third, diagnostic products are identified using matrix-assisted-laser- desorption-ionization time-of-flight (MALDI-TOF) mass spectrometry and therefore, the presence and nature of mutations are determined. Our preliminary results demonstrated that this approach could identify mutant alleles in the presence of 100,000-fold excess of normal alleles (at the 10 ppm level). Thus, the next logical step is to develop this method and to explore its potential in cancer research and detection. Two experimental goals will be achieved in this project. First, we will prove the feasibility of this new technology to identify various cancer-causing point mutations using both single and multiplex assays. Second, we will develop the proven assays for detection of the """"""""hotspot"""""""" point mutations in both k-ras and p53, two of the most important genes related to cancers. This application consists of two phases. Research in Year 1 is the R21 phase and work in Year 2-3 is the R33 phase. This technology has great potential to advance cancer research and diagnosis. Earlier detection of epithelial cancers is one of the most important application areas of this technology. Epithelium-derived cancers constitute a majority of all cancers including colon and lung cancers, where bronchoscopic biopsies, bronchoalveolar lavage, brush cytology, stool, and other specimens can be taken and one can look for abnormal cells in the normal background. This technology can also be used to determine the frequency of a particular point mutation in certain forms of cancer in a more accurate manner. This will provide insights into the correlation of cancer with gene mutations and the processing of genetic information in cellular function. Another important application is to seek the mutations that commonly occur in certain forms of cancer by screening a large number of patients. These mutations, in turn, can be used as cancer markers for earlier clinical diagnosis.
| Wang, Liming; Zhang, Jian; Banerjee, Sipra et al. (2010) Sumoylation of vimentin354 is associated with PIAS3 inhibition of glioma cell migration. Oncotarget 1:620-7 |
