Mismatch Detection Using Heteroduplex Analysis

Anne‐Lise Børresen1

1 The Norwegian Radium Hospital, Oslo
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 7.3
DOI:  10.1002/0471142905.hg0703s33
Online Posting Date:  August, 2002
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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.

     
 
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Table of Contents

  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1:

  Materials
    For common stock solutions, see appendix 2D; for suppliers, see suppliers appendix.
  • Mutation detection enhancement gel solution (HydroLink MDE; AT Biochem) or 50% (w/v) acrylamide stock solution (unit 7.4)
  • 10× TBE buffer ( appendix 2D)
  • Glycerol
  • TEMED
  • 10% (w/v) ammonium persulfate (APS; prepare fresh weekly and store at 4°C)
  • RNA or DNA sample and control (unit 10.4 & appendix 3B)
  • 10× gel loading buffer ( appendix 2D)
  • 10 mg/ml ethidium bromide solution ( appendix 2D)
  • Sequencing gel apparatus with 40 × 20–cm glass plates and 0.8‐mm spacers and comb
  • 20°C water bath
  • Additional reagents and equipment for nondenaturing PAGE (unit 7.4) and PCR amplification of sequences from affected individuals (unit 7.1)
CAUTION: TEMED and ethidium bromide are hazardous; see appendix 2A for guidelines on handling, storage, and disposal.
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Figures

Videos

Literature Cited

Literature Cited
   Cotton, R. G. H. 1992. Detection of mutations in DNA. Curr. Opin. Biotechnol. 3:24‐30.
   Keen, J., Lester, D., Ingelhearn, C., Curtis, A., and Bhattacharya, S. 1991. Rapid detection of single base mismatches as heteroduplexes on hydrolink gels. Trends Genet. 7:5.
   Soto, D. and Sukumar, S. 1992. Improved detection of mutations in the p53 gene in human tumors as single‐stranded conformation polymorphs and double‐stranded heteroduplex DNA. PCR Meth. Appl. 2:96‐98.
   White, M.B., Carvalho, M., Derse, D., O'Brien, S.J., and Dean, M. 1992. Detecting single base substitutions as heteroduplexes polymorphisms. Genomics 12:301‐306.
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