Automated constant denaturant capillary electrophoresis applied for detection of KRAS exon 1 mutations

In this study, we have applied automated constant denaturant capillary elec trophoresis (ACDCE) for the detection of KRAS exon 1 mutations. Samples fro m 191 sporadic colon carcinomas previously analyzed for KRAS mutations with allele-specific PCR (ASPCR), temporal temperature gradient electrophoresis (TTGE), and constant denaturant capillary electrophoresis (CDCE) were anal yzed. In ACDCE, an unmodified ABI PRISM(TM) 310 genetic analyzer with const ant denaturant conditions separated fluorescein-labeled PCR products. Tempe rature in combination with a chemical denaturant was used for separation. T he optimal separation conditions for PCR-amplified KRAS exon 1 fragments we re determined by adjusting the temperature before electrophoresis. In the A CDCE analysis, the sequence of a mutant was determined by comparing the ele ctropherogram of the fragment to that of known mutations followed by mixing the sample with control mutations before reanalysis. In a titration experi ment mixing mutant and wild-type alleles, the sensitivity for mutation dete ction was shown to be 0.6% in this automated CDCE technique. The automation of CDCE allowed rapid analysis of a large number of test samples over as s hort period of time and with a commercially available apparatus.