High resolution for single-strand conformation polymorphism analysis by capillary electrophoresis.

Abstract

Since the successful completion of the Human Genome Project, increasing concern is being directed toward the polymorphic aspect of the genome and its clinical relevance. A form of single-strand DNA-conformation polymorphism analysis (SSCP) employing nondenaturing slab-gel electrophoresis (SGE) is applicable to the genetic diagnosis of bladder cancer from urine samples. To bring this technique into routine clinical practice, the use of capillary electrophoresis (CE) is naturally favorable in terms of speed and automation. However, the resolving power of SSCP, a prerequisite basis for reliability required in diagnostics, remains as a challenge for CE systems. We thus focused on this topic and conducted studies on CE instruments equipped with a single capillary or an array of multiple capillaries, using the resolution (Rs) as a quantitative scale for the resolving power. Polymer concentration and buffer are shown to be the decisive parameters. High Rs values of >2.5 are achieved for representative SNPs markers under the optimized conditions, without sacrificing such intrinsic advantages of CE over SGE as the 10-fold quicker migration time and operation that is reproducible, continuous, and automatic. The strategies presented broaden the limits of CE in both the current and related applications.