CAPILLARY HYBRIDIZATION FOR IDENTIFICATION OF MUTATIONS

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DESCRIPTION: (Applicant's Description) The long-term objective is to measure mutational spectra in humans in order to determine the causes and mechanisms leading to mutations, cancer and aging, as well as to apply the technology developed for mutational spectrometry to immediate clinical applications. It is proposed to develop a rapid method for unambiguous identification and measurement of mutants comprising mutational profiles. The approach will not require isolation and sequencing of individual mutants and will work in conjunction with the fast, sensitive and efficient method for separation of mutants called constant denaturant capillary electrophoresis (CDCE). Identification is based on DNA hybridization of a sample with a panel of previously sequenced mutants inside the capillary, Nonidentity is indicated by a change in the mobility associated with resultant heteroduplexes. The power of this approach is that once a representative set of mutants in a particular sequence is determined, interpretation of spectra in subsequent samples will be very efficient. Preliminary studies have demonstrated the ability to identify individual mutants in complex mutational spectra of human tissues. The aims of the proposal include application to cancer-associated mutations in such genes as N-ras and p53. The method will be further developed for identification and measurement of double stranded DNA species as separated by non-denaturing electrophoresis.