Denaturing gradient gel electrophoretic analysis of the human cHa-ras1 proto-oncogene

Activation of ras proto-oncogenes frequently involves point mutations at different sites in the gene. Here we describe the application of denaturing gradient gel electrophoresis to identify and characterize such mutations in the cHa-ras1 proto-oncogene. We calculated a melting map of the cHa-ras1 gene and designed optimal conditions to separate mutant from wild type sequences in the first exon. As an example we examined the T24 guanosine to thymidine point mutation in the first exon which has been found in T24 bladder carcinoma cells. Denaturing gradient gel analysis of both homoduplex as well as heteroduplex molecules resulted in separation of the wild type sequence from the mutant sequence. On the basis of the melting map we present a general scheme for screening the cHa-ras1 proto-oncogene sequence for the occurrence of point mutations.