Separation of transforming amino acid-substituting mutations in codons 12, 13 and 61 of the N-ras gene by constant denaturant capillary electrophoresis (CDCE)

We used high fidelity PCR and constant denaturant capillaryelectrophoresis (CDCE) [Khrapko et al. (1994) Nucleic AcidsRes., 22, 364–369] to separate wild type and differentmutant N-ras exon 1 and 2 sequences. The set of plasmids containingN-ras cDNA, wild type or mutant sequences representing all transformingamino acid-substituting single base pair changes in codons 12,13 (exon 1) and 61 (exon 2), were amplified using Pfu polymerasein a limited cycle polymerase chain reaction. One of the primersused for the amplification of each exon included a 40 nucleotideGC rich sequence that created high and low melting domains.The amplified fragments 151 bp (exon 1) and 150 bp (exon 2)were run on the CDCE with the ‘denaturant zone’temperature of the capillary corresponding to the melting temperatureof 111 bp (exon 1) and 110 bp (exon 2) low melting domains.The separation was achieved between wild type and mutant sequencesas homoduplexes in 15 out of 19 cases, as a single base substitutionalters the electrophoretic mobility of a partially melted doublestranded fragment. The denaturation and reannealing of wildtype and mutant fragments together created wild type/ mutantheteroduplexes. All the heteroduplexes were well resolved fromwild type homoduplex. In the current form mutant sequences weredetected at a frequency of 10^–3 in the presence of wildtype. This study has resulted in obtaining electrophoretic spectrumof different N-ras mutants on CDCE as homoduplexes as well asheteroduplexes.