Denaturing Gel Electrophoresis for Sequencing

Barton E. Slatko1, Lisa M. Albright2

1 New England Biolabs, Beverly, Massachusetts, 2 null, Reading, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 7.6
DOI:  10.1002/0471142727.mb0706s16
Online Posting Date:  May, 2001
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Abstract

The accuracy of DNA sequence determination depends largely upon resolution of the sequencing products in denaturing polyacrylamide gels. This unit provides a detailed description of the setup, electrophoresis, and processing of such gels. In general, the gels required for DNA sequencing are 40‐cm long, of uniform thickness, and contain 4% to 8% acrylamide and 7 M urea. Modifications of this protocol increase the length of readable sequence information which can be obtained from a single gel (i.e., forming the gel with wedge‐shaped spacers to create a field gradient, or incorporating a buffer gradient, an electrolyte gradient, or an acrylamide step gradient into the gel). A modification to the ‐‐inclusion of formamide in the sequencing gel‐‐is designed to overcome gel compressions arising from secondary structure in the sequencing products during gel electrophoresis. A discussion of acrylamide concentrations and electrophoresis conditions is included in the Commentary.

     
 
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Table of Contents

  • Basic Protocol 1: Pouring, Running, and Processing Sequencing Gels
  • Alternate Protocol 1: Buffer‐Gradient Sequencing Gels
  • Alternate Protocol 2: Electrolyte‐Gradient Sequencing Gels
  • Alternate Protocol 3: Formamide‐Containing Sequencing Gels
  • Reagents and Solutions
  • Commentary
  • Tables
     
 
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Materials

Basic Protocol 1: Pouring, Running, and Processing Sequencing Gels

  Materials
  • 70% ethanol or isopropanol in squirt bottle
  • 5% dimethyldichlorosilane (diluted in CHCl 3; Sigma #D‐3879)
  • recipeDenaturing acrylamide gel solution
  • TEMED
  • 10% (w/v) ammonium persulfate (made fresh weekly and stored at 4°C)
  • 1× TBE buffer, pH 8.3‐8.9 ( appendix 22)
  • Sequencing samples in formamide/dye solution (units 7.4 or 7.5)
  • 5% acetic acid/5% methanol (vol/vol) fixer solution
  • 30 × 40–cm front and back gel plates
  • 0.2‐ to 0.4‐mm uniform‐thickness spacers
  • Large book‐binder clamps
  • 60‐ml syringe
  • Pipet tip rack or stopper
  • 0.2‐ to 0.4‐mm sharkstooth or preformed‐well combs
  • Sequencing gel electrophoresis apparatus
  • Pasteur pipet or Beral thin stem
  • Power supply with leads
  • Sequencing pipet tip
  • Gel dryer
  • Shallow fixer tray
  • 46 × 57–cm gel blotting paper (e.g., Whatman 3MM)
  • Kodak XAR‐5 X‐ray film
NOTE: Many companies provide equipment needed for sequencing experiments; a list of suppliers is provided in Table 7.6.1.
Table 7.6.1   Materials   Suppliers of Sequencing Gel Electrophoresis Equipment c   Suppliers of Sequencing Gel Electrophoresis Equipment

Equipment Supplier d
Sequencing gel apparatus, including necessary clamps, combs and spacers AAP, ABA, BR, GB, HO, IBI, JS, OSP, PH, SS
Gel tape ABA, GB, HS, IBI, OSP
Power supplies ABA, ACS, BR, EC, FD, GB, HO, IBI, IS, OSP, PH, SS, ST
Beral thin stem BE
Ultrathin pipet tips for loading gels ABA, BR, CO, DR, DY, HO, IBI, IS, MB, PH, SS, ST
Gel thermometer BR
Shallow fixer tray OSP
Transfer paper ABA, SS, WH
Gel dryers ATR, BR, HO, SV

 cModified from Slatko, .
 dAbbreviations: AAP, Ann Arbor Plastics; ABA, American Bioanalytical; ACS, Accurate Chemical and Scientific; ATR, ATR; BE, Beral Enterprises; BR, Bio‐Rad; CO, Costar; DR, Drummond; DY, Dynalab; EC, EC Apparatus; FD, Fotodyne; GB, Gibco/BRL; HO, Hoefer; IBI, International Biotechnologies; IS, Integrated Separation Systems; JS, Jordan Scientific; MB, Marsh Biomedical; OSP, Owl Scientific Plastics; PH, Pharmacia LKB; SS, Schleicher & Schuell; ST, Stratagene; SV, Savant; WH, Whatman. See appendix 44 for addresses of suppliers.

Alternate Protocol 1: Buffer‐Gradient Sequencing Gels

  Additional Materials
  • recipeBuffer‐gradient gel solutions (containing 0.5× and 2.5× TBE; see reagents and solutions)
  • 25‐ml pipet equipped with a rubber pipet‐filler bulb

Alternate Protocol 2: Electrolyte‐Gradient Sequencing Gels

  Additional Materials
  • 0.5× and 1× TBE buffer ( appendix 22)
  • 3 M sodium acetate unbuffered

Alternate Protocol 3: Formamide‐Containing Sequencing Gels

  Additional Materials
  • recipeFormamide gel solution
  • 5% acetic acid/20% methanol (v/v) fixer solution
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Figures

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Literature Cited

Literature Cited
   Ansorge, W. and Labeit, S. 1984. Field gradients improve resolution in DNA sequencing gels. J. Biochem. Biophys. Methods 10:237‐243.
   Biggin, M.D., Gibson, T.J., and Hong, G.F. 1983. Buffer gradient gels and 35S‐label as an aid to rapid DNA sequence determination. Proc. Natl. Acad. Sci. U.S.A. 80:3963‐3965.
   Brown, N.L. 1984. DNA sequencing. Methods Microbiol. 17:259‐13.
   Isfort, R. and Ihle, J. 1988. The 4‐6‐8 method of sequence analysis. BioTechniques 6:138‐141.
   Mayeda, A. and Krainer, A.R. 1991. Long‐term storage of concentrated Tris borate‐EDTA electrophoresis buffers without precipitation. BioTechniques 10:182.
   Olsson, A., Moks, T., Muhlen, J., and Gaal, A.B. 1984. Uniformly spaced banding patterns in DNA sequencing gels by the use of a field‐strength gradient. J. Biochem. Biophys. Methods 10:83‐90.
   Sheen, J.‐Y. and Seed, B. 1988. Electrolyte gradient gels for DNA sequencing. BioTechniques 6:942‐944.
   Slatko, B. 1991a. Sources of reagents and supplies for dideoxy DNA sequencing and other applications. In Methods in Nucleic Acids Research (J. Karam, L. Chao, and G. Warr, eds.) pp. 379‐392. CRC Press, Boca Raton, Fla.
   Tabor, S. and Richardson, C.C. 1987. DNA sequence analysis with a modified bacteriophage T7 DNA polymerase. Proc. Natl. Acad. Sci. U.S.A. 84:4767‐4771.
   U.S. Biochemical. 1990. Formamide gels (40%) for sequencing DNA. Comments 17(1):31.
   Williams, S.A., Slatko, B.E., Moran, L.S., and DiSimone, S.M. 1986. Sequencing in the fast lane: A rapid protocol for [α‐35S]dATP dideoxy DNA sequencing. BioTechniques 4:138‐147.
Key Reference
   Slatko, B. 1991b. Protocols for manual dideoxy DNA sequencing. In Methods in Nucleic Acids Research (J. Karam, L. Chao, and G. Warr, eds.) pp. 83‐129. CRC Press, Boca Raton, Fla.
  Contains references for numerous modifications to the basic sequencing gel protocol.
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