Mismatch detection using heteroduplex analysis

Curr Protoc Hum Genet. 2002 Aug:Chapter 7:Unit 7.3. doi: 10.1002/0471142905.hg0703s33.

Abstract

This 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.

MeSH terms

  • Base Pair Mismatch*
  • DNA Mutational Analysis / methods
  • Genetics, Medical
  • Heteroduplex Analysis / methods*
  • Heterozygote
  • Humans
  • Point Mutation
  • Polymorphism, Single Nucleotide