The goal of this project is to develop ribonuclease protection/mismatch assays which will reliably and easily allow the detection of essentially all point mutations. Such assays will provide inexpensive and widely applicable methods for detecting mutations in oncogenes and tumor suppressor genes, screening for genetic diseases, typing viruses and a variety of other applications. Current methods, including SSCP do not achieve 100% detection efficiency and have other shortcomings as well. During Phase I we significantly improved reaction conditions which allowed us to detect point mutations in the p53 tumor suppressor gene which were refractory to RNase cleavage under standard conditions. In addition we developed a PCR/in vitro transcription strategy which allowed us to increase the efficiency of mutation detection. During Phase II we propose to further improve reaction conditions, develop specific reagents for mutation detection in p53, APC, NFI, and other genes, to work out techniques which maximize the size of the region which can be screened in a single assay, and to adapt these assays to non-radioisotopic formats. Collectively these improvements will make RNase protection/mismatch assays the most attractive technique for screening for single base mutations.
|Goldrick, M M; Kimball, G R; Liu, Q et al. (1996) NIRCA: a rapid robust method for screening for unknown point mutations. Biotechniques 21:106-12|