Separation of transforming N-ras mutations in codons 12, 13 and 61 by denaturing gradient gel electrophoresis: investigation based on a set of phagemid constructs.

In the present study we have introduced 19 activating base pair substitutions into N-ras cDNA by use of an in vitro site-directed mutagenesis system. Six mutants were constructed for N-ras codon 12 (exon 1), six for codon 13 (exon 1), and seven for codon 61 (exon 2). Fifteen out of 19 PCR-amplified mutation sequences showed a clear separation from the wild type on denaturing gradient gel electrophoresis runs as homoduplex band, and the rest could be separated after heteroduplex formation with wild-type DNA. These constructs can be used as controls in many screening systems for analyzing activating point mutations of the N-ras gene.