SEPARATION OF TRANSFORMING AMINO ACID-SUBSTITUTING MUTATIONS IN CODON-12, CODON-13 AND CODON-61 OF THE N-RAS GENE BY CONSTANT DENATURANT CAPILLARY ELECTROPHORESIS (CDCE)

We used high fidelity PCR and constant denaturant capillary electropho resis (CDCE) [Khrapko et al. (1994) Nucleic Acids Res., 22, 364-369] t o separate wild type and different mutant N-ras exon 1 and 2 sequences , The set of plasmids containing N-ras cDNA, wild type or mutant seque nces representing all transforming amino acid-substituting single base pair changes in codons 12, 13 (exon 1) and 61 (exon 2), were amplifie d using Pfu polymerase in a limited cycle polymerase chain reaction, O ne of the primers used for the amplification of each exon included a 4 0 nucleotide GC rich sequence that created high and low melting domain s, The amplified fragments 151 bp (exon 1) and 150 bp (exon 2) were ru n on the CDCE with the 'denaturant zone' temperature of the capillary corresponding to the melting temperature of 111 bp (exon 1) and 110 bp (exon 2) low melting domains. The separation was achieved between wil d type and mutant sequences as homoduplexes in 15 out of 19 cases, as a single base substitution alters the electrophoretic mobility of a pa rtially melted double stranded fragment, The denaturation and reanneal ing of wild type and mutant fragments together created wild type/mutan t heteroduplexes. All the heteroduplexes were web resolved from wild t ype homoduplex. In the current form mutant sequences were detected at a frequency of 10(-3) in the presence of wild type. This study has res ulted in obtaining electrophoretic spectrum of different N-ras mutants on CDCE as homoduplexes as well as heteroduplexes.