Mutation analysis of TP53 exons 5-8 by automated constant denaturant capillary electrophoresis

Abstract

DNA fragments melt characteristically according to their nucleotide sequence and length, when exposed to denaturants such as temperature, urea or formamide. Small differences within a defined sequence, like a base mutation, will result in a slightly different melting behavior of the aberrant DNA fragment compared to that of the wild type sequence. This feature has previously been exploited for mutation detection by constant denaturant capillary electrophoresis (CDCE). In this report, we describe an automated approach (ACDCE) using a commercially available apparatus (ABI 310 Genetic Analyzer) to analyze mutations in exons 5-8 of TP53. The running conditions were determined by temperature titration of the fragments on the apparatus, and an operating sensitivity showed that 0.1% mutated alleles could be detected against a background of wild-type alleles. Up to 48 samples can be analyzed by ACDCE without any need for operator intervention. The apparatus is commercially available, and there is no need for instrument modification. To our knowledge this is the first report on the analysis of TP53 exons 5-8 by ACDCE.