Our long-term goal is to advance instrumentation and techniques for rapid, automated and sensitive variant analysis of nucleic acids. The LightCyclerTM has been our homogenous """"""""closed system"""""""" platform for genotyping. Fluorescent hybridization probes are included at the beginning of the reaction and are used for realtime PCR monitoring and genotyping. After rapid cycle PCR, the reaction is slowly heated (0.1 degree C/s) and products are identified by unique probe/target melting temperatures. The entire assay is performed from genomic DNA within 20 min and without any sample manipulation. Fluorescent melting curve analysis is a useful and popular method for repetitive genotyping of established mutations/polymorphisms. In phase I, we built and tested a new """"""""high-resolution"""""""" instrument for melting curve analysis. The high-resolution melting instrument has 10-fold less measurement error than the LightCyclerTM providing increased sensitivity for mutation detection and utility in mutation scanning. In phase II, we plan to integrate the """"""""low noise"""""""" components of the high-resolution instrument into a modified LightCyclerTM to allow both rapid amplification and smooth melting curve genotyping. Mathematical software algorithms will be written for sensitive variant detection and flagging unknown mutant sequences. The utility of the new instrument and algorithms will be demonstrated by homogenously scanning for mutations within critical exons of the p53 gene.
We propose developing instrumentation and analysis software that will allow homogenous mutation scanning for the first time. The system will be used for scanning the p53 tumor suppressor gene, which is mutated in almost all cancers at the time of invasive growth. The instrumentation and the assay will be commercially available for continued research on p53 and to provide a clinical test for the prognosis and treatment of cancer. The general method can be applied to other genes, as well.
| Erali, Maria; Wittwer, Carl T (2010) High resolution melting analysis for gene scanning. Methods 50:250-61 |
| Bennett, C D; Campbell, M N; Cook, C J et al. (2003) The LightTyper: high-throughput genotyping using fluorescent melting curve analysis. Biotechniques 34:1288-92, 1294-5 |
