Search for Oncogene Mutations in X-ray-transformed Mouse 10T½ Cells by Denaturing Gradient Gel Electrophoresis Blotting

Abstract

We have sought evidence for possible mutations within the c-myc, c-Ha-ras and c-Ki-ras loci of X-ray-transformed mouse C3H10T½ cell clones using the denaturing gradient gel electrophoresis (DGGE) blot technique. This highly sensitive method was developed to detect any mutations (e.g. single base changes, small deletions) in genomic DNA, by measuring differences in the melting behaviour of short DNA fragments (50–800 bp) obtained by digestion of genomic DNA with several specific 4 bp recognition site restriction enzymes. In this study, genomic DNAs derived from 23 X-ray-transformed clones were digested with several restriction enzymes, electrophorezed on denaturing gradient gel and hybridized to c-myc, c-Ha-ras and c-Ki-ras cDNA probes. No alterations in melting patterns were observed for any of these oncogenes as compared with DNA from 18 control, non-irradiated wild-type 10T½ cell clones, suggesting that transformation was not associated with mutation of these genes nor with changes in their patterns of methylation. However, our screening of the large portion of exons 2 and 3 of c-myc as well as of exons 1 and 2 of c-Ha-ras gene cannot exclude the possibility that some sequence differences in the high melting domains of examined fragments were not detected by this assay.