Detection of Mutations by Denaturing Gradient Gel Electrophoresis

Anne‐Lise Børresen‐Dale1, Eivind Hovig1, Birgitte Smith‐Sørensen1

1 The Norwegian Radium Hospital, Oslo
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 7.5
DOI:  10.1002/0471142905.hg0705s17
Online Posting Date:  May, 2001
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Abstract

This unit describes the procedure for determining the melting profile for a given PCR‐amplified sequence by perpendicular denaturing gradient gel electrophoresis (DGGE) and, using that information, for developing a screening assay based on either parallel DGGE, CDGE (Constant Denaturant Gel Electrophoresis), or TTGE (Temporal /Temperature Gradient Electrophoresis). Four support protocols describe techniques for pouring perpendicular and parallel denaturing gradient gels, constant denaturant gels, and temporal temperature gradient gels.

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Mutational Analysis Using Denaturing Gradient Gel Electrophoresis
  • Support Protocol 1: Casting a Perpendicular Denaturing Gradient Gel (0% to 100% Denaturant)
  • Support Protocol 2: Casting a Parallel Denaturing Gradient Gel
  • Support Protocol 3: Casting a Constant Denaturant Gel
  • Support Protocol 4: Casting and Running a Temporal Temperature Gradient Gel
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Mutational Analysis Using Denaturing Gradient Gel Electrophoresis

  Materials
  • Sample DNA
  • protocol 2Perpendicular denaturing gradient gel (see protocol 2)
  • 50× TAE buffer ( appendix 2D)
  • recipeGel loading buffer (see recipe)
  • 10 mg/ml ethidium bromide ( appendix 2D)
  • protocol 3Parallel denaturing gradient gel, protocol 4constant denaturant gel, or protocol 5temporal temperature gradient gel (see Support Protocols protocol 32, protocol 43, and protocol 54)
  • Vertical minigel electrophoresis unit (Bio‐Rad Mini‐Protean II modified as in Fig. , or equivalent)
  • Water bath, accurate and adjustable for 45° to 60°C
  • 80‐V constant‐voltage power supply
  • Additional reagents and equipment for PCR amplification of sequences from affected individuals (unit 7.1)

Support Protocol 1: Casting a Perpendicular Denaturing Gradient Gel (0% to 100% Denaturant)

  Materials
  • Acetone
  • recipe7.5% (w/v) acrylamide/0% denaturant gel solution (see recipe)
  • recipe7.5% (w/v) acrylamide/100% denaturant gel solution (see recipe)
  • TEMED
  • 20% (w/v) ammonium persulfate (APS; prepare fresh)
  • 0.1% (w/v) SDS
  • Vertical minigel electrophoresis unit (Bio‐Rad Mini‐Protean II or equivalent)
  • Gradient maker, 10 ml per chamber (Bio‐Rad)
  • Tygon tubing with micropipet tip
  • Peristaltic pump (optional; Markson minipump or equivalent)

Support Protocol 2: Casting a Parallel Denaturing Gradient Gel

  • Minigel casting tray (Bio‐Rad or Hoefer)
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Figures

Videos

Literature Cited

Literature Cited
   Børresen, A.‐L. 1996. Constant denaturant gel electrophoresis (CDGE) in mutation screening. In Technologies for Detection of DNA Damage and Mutation (G.P. Pfeifer, ed.) pp. 267‐279. Plenum, New York.
   Børresen, A.‐L., Hovig, E., and Brøgger, A. 1988. Detection of base mutations in genomic DNA using denaturing gradient gel electrophoresis (DGGE) followed by transfer and hybridization with gene‐specific probes. Mutat. Res. 202:77‐83.
   Børresen, A.‐L., Hovig, E., Smith‐Sørensen, B., Malkin, D., Lystad, S., Andersen, T.I., Nesland, J.M., Isselbacher, K.J., and Friend, S.H. 1991. Constant denaturant gel electrophoresis as a rapid screening technique for p53 mutations. Proc. Natl. Acad. Sci. U.S.A. 88:8405‐8409.
   Børresen‐Dale, A.‐L., Lystad, S., and Langerød, A. 1997. Temporal temperature gradient gel electrophoresis (TTGE) compared with denaturing gradient gel electrophoresis (DGGE) and constant denaturant gel electrophoresis (CDGE) in mutation screening. BioRadiations 99:12‐113.
   Condie, A., Børresen, A.‐L., Eeles, R., Coles, C., Cooper, C., and Prosser, J. 1993. Point‐mutation detection: Comparison of SSCP, CDGE and HOT. Hum. Mut. 2:58‐66.
   Fischer, S.G. and Lerman, L.S. 1979. Two‐dimensional electrophoretic separation of restriction enzyme fragments of DNA. Methods Enzymol. 68:183‐191.
   Fischer, S.G. and Lerman, L.S. 1983. DNA fragments differing by single base‐pair substitutions are separated in denaturing gradient gels: Correspondence with melting theory. Proc. Natl. Acad. Sci. U.S.A. 80:1579‐1583.
   Gray, M.R. 1992. Detection of DNA sequence polymorphisms in human genomic DNA by using denaturing gradient gel blots. Am. J. Hum. Genet. 50:331‐346.
   Hovig, E., Smith‐Sørensen, B., Brøgger, A., and Børresen, A.‐L. 1991. Constant denaturant gel electrophoresis, a modification of denaturing gradient gel electrophoresis, in mutation detection. Mutat. Res. 262:63‐71.
   Lerman, L.S., Fischer, S.G., Hurley, I., Silverstein, K., and Lumelsky, N. 1984. Sequence‐determined DNA separations. Annu. Rev. Biophys. Bioeng. 13:399‐423.
   Lerman, L.S. and Silverstein, K. 1987. Computational simulation of DNA melting and its application to denaturing gradient gel electrophoresis. Methods Enzymol. 155:482‐501.
   Myers, R.M., Lumelsky, N., Lerman, L.S., and Maniatis, T. 1985. Detection of single base substitutions in total genomic DNA. Nature 313:495‐498.
   Sheffield, V.C., Cox, D.R., Lerman, L.S., and Myers, R.M. 1989. Attachment of a 40‐base‐pair G+C rich sequence (GC‐clamp) to genomic DNA fragments by the polymerase chain reaction results in improved detection of single‐base changes. Proc. Natl. Acad. Sci. U.S.A. 86:232‐236.
   Smith‐Sørensen, B., Hovig, E., Andersson, B., and Børresen, A.‐L. 1992. Screening for mutations in the human HPRT cDNA using the polymerase chain reaction (PCR) in combination with constant denaturant gel electrophoresis (CDGE). Mutat. Res. 269:41‐53.
   Smith‐Sørensen, B., Gebhardt, M.C., Kloen, P., Aquilar, F., Cerutti, P., Børresen, A.‐L. 1993. Screening for TP53 mutations in osteosarcomas using constant denaturant gel electrophoresis (CDGE). Hum. Mut. 2:274‐285.
   Uitterlinden, A.G., Slagboom, P.E., Knook, D.L., and Vijg, J. 1989. Two‐dimensional DNA fingerprinting of human individuals. Proc. Natl. Acad. Sci. U.S.A. 86:2742‐2746.
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