Conformation-sensitive gel electrophoresis for rapid detection of single-base differences in double-stranded PCR products and DNA fragments: evidence for solvent-induced bends in DNA heteroduplexes.

TLDR

Recent methods exist to detect single‑base mismatches in DNA heteroduplexes composed of wild‑type and mutant strands. The study tested whether mildly denaturing solvents amplify mismatch‑induced bends to enhance heteroduplex–homoduplex separation by gel electrophoresis. The method employs a 6 % polyacrylamide gel with 10 % ethylene glycol/15 % formamide/Tris‑taurine buffer, requires no special equipment, and applies to 200–800 bp PCR products. Optimized conditions detected 60 of 68 mismatches across 59 contexts, while mismatches within 50 bp of ends or in high‑melting domains showed reduced separation, confirming solvent‑induced bends improve detection.

Abstract

Several techniques have recently been developed to detect single-base mismatches in DNA heteroduplexes that contain one strand of wild-type and one strand of mutated DNA. Here we tested the hypothesis that an appropriate system of mildly denaturing solvents can amplify the tendency of single-base mismatches to produce conformational changes, such as bends in the double helix, and thereby increase the differential migration of DNA heteroduplexes and homoduplexes during gel electrophoresis. The best separations of heteroduplexes and homoduplexes were obtained with a standard 6% polyacrylamide gel polymerized in 10% ethylene glycol/15% formamide/Tris-taurine buffer. As predicted by the hypothesis of solvent-induced bends, when the concentration of either ethylene glycol or formamide was increased, the differential migration decreased. Also, single-base mismatches within 50 bp of one end of a heteroduplex did not produce differential migration. Sixty of 68 single-base mismatches in a series of PCR products were detected in some 59 different sequence contexts. The eight mismatches not detected were either within 50 bp of the nearest end of the PCR product or in isolated high-melting-temperature domains. Therefore, it was possible to predict in advance the end regions and sequence contexts in which mismatches may be difficult to detect. The procedure can be applied to any PCR products of 200-800 bp and requires no special equipment or preparation of samples.