Automated constant denaturant capillary electrophoresis applied for detection of KRAS exon 1 mutations.
In this study, we have applied automated constant denaturant capillary electrophoresis (ACDCE) for the detection of KRAS exon 1 mutations. Samples from 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 analyzed. In ACDCE, an unmodified ABI Prism 310 genetic analyzer with constant denaturant conditions separated fluorescein-labeled PCR products. Temperature in combination with a chemical denaturant was used for separation. The optimal separation conditions for PCR-amplified KRAS exon 1 fragments were determined by adjusting the temperature before electrophoresis. In the ACDCE analysis, the sequence of a mutant was determined by comparing the electropherogram of the fragment to that of known mutations followed by mixing the sample with control mutations before reanalysis. In a titration experiment mixing mutant and wild-type alleles, the sensitivity for mutation detection 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 short period of time and with a commercially available apparatus.
Bjørheim
J
J
Department of Immunology, Section for Immunotherapy, Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, 0310 Oslo, Norway. j@bjorheim.net
Ekstrøm
P O
PO
Fossberg
E
E
Børresen-Dale
A L
AL
Gaudernack
G
G
D001322:Autoanalysis; D003062:Codon; D015179:Colorectal Neoplasms / Q000235:genetics*; D015925:Cryopreservation; D004252:DNA Mutational Analysis; D019075:Electrophoresis, Capillary / Q000379:methods*; D005091:Exons; D011905:Genes, ras / Q000235:genetics*; D006358:Hot Temperature; D006801:Humans; D009154:Mutation; D016133:Polymerase Chain Reaction; D011489:Protein Denaturation; D012680:Sensitivity and Specificity; D012967:Sodium Dodecyl Sulfate; D014407:Tumor Cells, Cultured
D003062:Codon; D012967:Sodium Dodecyl Sulfate