Mismatch Detection Using Heteroduplex Analysis

AbstractThis protocol describes a technique that can be used to identify point mutations or single‐base polymorphisms in heterozygous individuals. This technique takes advantage of the fact that heteroduplex molecules containing single‐base mismatches can be separated under particular conditions of gel electrophoresis from nearly identical molecules containing no mismatches. RNA or DNA from a potentially heterozygous individual is amplified by PCR, and the products are denatured and allowed to renature, forming heteroduplexes. Renatured PCR products are run on nondenaturing mutation‐detection‐enhancement polyacrylamide gels (HydroLink MDE gels). On this gel, hybrid molecules containing a mismatch migrate more slowly than their corresponding homoduplexes. This protocol describes the analysis of unlabeled PCR products; however, radiolabeled PCR products can also be analyzed by this method.