Evaluation of denaturing conditions in analysis of DNA variants applied to multi‐capillary electrophoresis instruments

Abstract

AbstractDenaturant slab gel techniques (DGGE and variants thereof) and denaturing CE (DCE) have been used for the analysis of mutations and single nucleotide polymorphisms by several research groups. Recently, DCE applied to commercially available capillary electrophoresis instruments has been demonstrated to be sensitive, specific, and robust for mutation and SNP analysis. However, instruments currently available on the market are not designed for DCE in their present forms. In both single and multi‐capillary instruments inaccurate temperature control units lead to irreproducible denaturing conditions, both between runs and from capillary to capillary within an electrophoresis run. We have previously suggested that cycling a temperature gradient several times around a theoretically defined optimum for a given DNA fragment results in more reproducible and controllable denaturing conditions compared to a constant denaturant or single sweep temperature gradient. In this paper, we demonstrate that with the temperature differences observed, the common technique of applying a temperature gradient is not sufficient for optimal separation conditions in all capillaries. Furthermore, we demonstrate that cycling of the temperature around a fragment's theoretically calculated melting temperature during electrophoresis leads to reproducible results in all capillaries. These results clearly show the potential of standard commercial capillary instruments in automated cycling temperature CE applications.