Double‐Stranded DNA Sequence Analysis

Sherie L. Morrison1

1 University of California Los Angeles, Los Angeles, California
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 10.25
DOI:  10.1002/0471142735.im1025s14
Online Posting Date:  May, 2001
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Abstract

There are several different methods for analyzing the sequence of cloned DNA segments. For analysis of immunoglobulin variable (V) regions, doubleā€stranded DNA sequencing is routinely used. Although this method does not yield the longest DNA sequences, it is easy and provides sufficient information to determine the correct sequence. The protocols in this unit are for use with the Sequenase Version 2.0 kit from U.S. Biochemicals, which is based on dideoxy sequencing with a modified T7 DNA polymerase. The describes performing a sequencing reaction for cloned immunoglobulin V regions and running and analyzing a sequencing gel. A support protocol describes pouring and setting up the sequencing gel.

     
 
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Table of Contents

  • Basic Protocol 1: Double‐Stranded DNA Sequence Analysis of Immunoglobulin Variable Regions Cloned by PCR
  • Support Protocol 1: Preparing a DNA Sequencing Gel
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Double‐Stranded DNA Sequence Analysis of Immunoglobulin Variable Regions Cloned by PCR

  Materials
  • DNA template cloned in M13 sequencing vector, miniprep purified (unit 10.3) and resuspended to 0.5 µg/µl in TE buffer ( appendix 2A)
  • 5 mg/ml DNAse‐free RNase
  • 1:1 (v/v) phenol/chloroform
  • 2 M sodium hydroxide (NaOH)
  • 3 M sodium acetate ( appendix 2A)
  • 100% and 70% (v/v) ethanol, −20°C
  • Primers (30 to 60 ng, or 0.5 pmol)
  •  M13 reverse primer: 5′‐GTAAAACGACGGCCAGT‐3′
  •  Universal primer: 5′‐GTTTCCCAGTCACGAC‐3′
  • Sequenase Version 2.0 Kit (U.S. Biochemical) containing:
  •  5× annealing buffer
  •  5× labeling mix
  •  Enzyme dilution buffer
  •  Enzyme
  •  Termination mixes
  •  0.1 M DTT
  •  Stop solution
  • 70% and 100% ethanol, −20°C
  • 0.1 M dithiothreitol (DTT)
  • 12.5 µCi/µl [α‐35S]dATP (1415 Ci/mmol)
  • 6% polyacrylamide sequencing gel ( protocol 2)
  • Flexible plastic 96‐well microtiter dish (e.g., Falcon)
  • Positive‐displacement pipet (optional)
  • Power supply for sequencing gel apparatus
  • Whatman 3MM filter paper
  • Gel dryer
  • Additional reagents and equipment for autoradiography ( appendix 3J)
CAUTION: This procedure should be performed only by personnel trained in the proper use of 35S isotope and in NRC‐licensed sites. Standard precautions to prevent excessive exposure and radioactive contamination of personnel and equipment should be followed at all times.

Support Protocol 1: Preparing a DNA Sequencing Gel

  Materials
  • 70% (v/v) ethanol in squirt bottle
  • recipeDenaturing acrylamide gel solution (see recipe)
  • TEMED
  • 10% (w/v) ammonium persulfate (prepare fresh weekly and store in glass container at 4°C)
  • 1× TBE buffer (unit 10.4)
  • 30 × 40–cm sequencing gel plates
  • 0.2‐ or 0.4‐mm spacers and sharkstooth or preformed‐well comb
  • Book‐binder clamps, large
  • Yellow vinyl electrical tape (optional)
  • Rubber stoppers
  • Sequencing gel electrophoresis apparatus
  • 50‐ml syringe with 18‐gauge needle bent to 90°
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Figures

Videos

Literature Cited

Literature Cited
   Chen, E.Y. and Seeburg, P.H. 1985. Super‐coil sequencing. A fast and simple method for sequencing plasmid DNA. DNA 4:165‐170.
   Martin, C.S. 1994. Dideoxy DNA sequencing with chemiluminescent detection. In Current Protocols in Molecular Biology. (F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith and K. Struhl, eds.) pp. 7.4.1‐7.4.35. John Wiley & Sons, New York.
   Sanger, F., Nicklen, S., and Coulson, A.R. 1977. DNA sequencing with chain‐terminating inhibitors. Proc. Natl. Acad. Sci. U.S.A. 74:5463‐5467.
   Slatko, B.E. and Albright, L.M. 1992. Denaturing gel electrophoresis for sequencing. In Current Protocols in Molecular Biology. (F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 7.6.1‐7.6.13. John Wiley & Sons, New York.
   Slatko, B.E., Albright, L.M., and Tabor, S. 1994. DNA Sequencing by the dideoxy method. In Current Protocols in Molecular Biology. (F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 7.4.1‐7.4.35. John Wiley & Sons, New York.
   Wang, Y. 1988. Double‐stranded DNA sequencing with T7 polymerase. 1988. BioTechniques 6:843‐845.
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