DNA Sequencing by the Dideoxy Method

Barton E. Slatko1, Lisa M. Albright2, Stanley Tabor3, Jingyue Ju4

1 New England Biolabs, Beverly, Massachusetts, 2 null, Reading, Massachusetts, 3 Harvard Medical School, Boston, Massachusetts, 4 Incyte Pharmaceuticals, Palo Alto, California
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 7.4A
DOI:  10.1002/0471142727.mb0704as47
Online Posting Date:  May, 2001
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Abstract

In the basic dideoxy sequencing reaction, an oligonucleotide primer is annealed to a single‐stranded DNA template and extended by DNA polymerase in the presence of four deoxyribonucleoside triphosphates (dNTPs), one of which is 35S‐labeled. The reaction also contains one of four dideoxyribonucleoside triphosphates (ddNTPs), which terminate elongation when incorporated into the growing DNA chain. After completion of the sequencing reactions, the products are subjected to electrophoresis on a high‐resolution denaturing polyacrylamide gel and then autoradiographed to visualize the DNA sequence. Three variations of the dideoxy sequencing procedure are currently in use and are presented in this unit. In the “labeling/termination“ procedure, primer chains are initially extended and labeled in the absence of terminating ddNTPs, whereas in the traditional “Sanger” procedure, labeling and termination of primer chains occur in a single step. A recent variation of the dideoxy sequencing method is thermal cycle sequencing in which the reaction mixture, containing template DNA, primer, thermostable DNA polymerase, dNTPs, and ddNTPs, is subjected to repeated rounds of denaturation, annealing, and elongation steps. The resulting linear amplification of the sequencing products allows much less template DNA to be used and eliminates independent primer annealing and template denaturation steps, which are required for the labeling/termination or Sanger procedures. The use of automated fluorescent sequencers ofr four‐color dideoxy DNA sequencing is also described in detail.

     
 
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Table of Contents

  • Basic Protocol 1: Labeling/Termination Sequencing Reactions Using Sequenase
  • Alternate Protocol 1: Using Mn2+ in the Labeling/Termination Reactions
  • Using Other Polymerases in the Labeling/Termination Reactions
  • Alternate Protocol 2: Labeling/Termination Reactions Using Taq DNA Polymerase
  • Alternate Protocol 3: Labeling/Termination Reactions Using Klenow Fragment
  • Basic Protocol 2: Sequencing by the Sanger Procedure Using Klenow Fragment
  • Alternate Protocol 4: Using Taq DNA Polymerase in the Sanger Procedure
  • Alternate Protocol 5: One‐Step Sequencing Reactions Using 5′‐End‐Labeled Primers
  • Basic Protocol 3: Thermal Cycle Sequencing Reactions Using α‐Labeled Nucleotides
  • Alternate Protocol 6: Thermal Cycle Sequencing Reactions Using 5′‐End‐Labeled Primers
  • Cycle Sequencing Using Fluorescence Dye‐Labeled Primer (Dyeprimer) or Terminator (Dyeterminator)
  • Alternate Protocol 7: Sequencing with Energy Transfer Dye‐Labeled Primers (ET Primer)
  • Alternate Protocol 8: Sequencing with Energy Transfer Dye‐Labeled Terminators (BigDye Terminators)
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Labeling/Termination Sequencing Reactions Using Sequenase

  Materials
  • 0.5 pmol single‐stranded or denatured double‐stranded DNA template (unit 7.3)
  • 0.5 to 1 pmol/µl oligonucleotide primer in water (store at −20°C; unit 2.11)
  • recipe10× Sequenase buffer (see recipe)
  • recipeSequenase termination mixes (see recipe)
  • recipeSequenase/pyrophosphatase mix (see recipe)
  • recipeSequenase diluent (see recipe)
  • recipeLabeling mixes (see recipe)
  • 10 mCi/ml [α‐35S]dATP (500 to 1200 Ci/mmol)
  • recipeStop/loading dye (see recipe)
  • 65° and 95°C water baths
  • 0.5‐ml microcentrifuge tubes
  • Heat‐resistant microtiter plates (optional; Table 7.4.1)

Alternate Protocol 1: Using Mn2+ in the Labeling/Termination Reactions

  • Mn buffer: 0.15 M sodium isocitrate/0.1 M MnCl 2 (store up to 3 months at −20°C)
To sequence using Mn buffer, carry out the labeling/termination basic protocol, making the addition at the step indicated below.

Alternate Protocol 2: Labeling/Termination Reactions Using Taq DNA Polymerase

  • 10× Taq sequencing buffer: 500 mM Tris⋅Cl, pH 9.0 ( appendix 22)
  • recipeTaq termination mixes (see recipe)
  • Taq DNA polymerase
  • 45°C water bath
To use Taq DNA polymerase in the labeling/termination reactions, modify the steps in protocol 1 as indicated below.

Alternate Protocol 3: Labeling/Termination Reactions Using Klenow Fragment

  • recipe10× Klenow sequencing buffer (see recipe)
  • recipeKlenow termination mixes (see recipe)
  • Klenow fragment (unit 3.5)
  • 37° to 42°C water bath

Basic Protocol 2: Sequencing by the Sanger Procedure Using Klenow Fragment

  Materials
  • 0.5 to 1 pmol single‐stranded or denatured double‐stranded DNA (unit 7.3)
  • 0.5 to 1 pmol/µl oligonucleotide primer in water (store at −20°C; units 2.11 & 2.12)
  • 10× and 1× recipeKlenow sequencing buffer (see recipe)
  • recipeKlenow Sanger mixes (see recipe)
  • Klenow fragment (unit 3.5)
  • 10 mCi/ml [α‐35S]dATP (500 to 1200 Ci/mmol)
  • recipedNTP chase (see recipe)
  • recipeStop/loading dye (see recipe)
  • 0.5‐ml microcentrifuge tubes
  • 37° to 42°C water bath
  • Additional reagents and equipment for labeling/termination sequencing reactions (see protocol 1)

Alternate Protocol 4: Using Taq DNA Polymerase in the Sanger Procedure

  • 10× Taq sequencing buffer: 500 mM Tris⋅Cl, pH 9.0 ( appendix 22)
  • recipeTaq Sanger mixes (see recipe)
  • Taq DNA polymerase
  • 50° to 75°C water bath
To use Taq DNA polymerase in the Sanger procedure (see protocol 5), modify the steps in that protocol as indicated below.

Alternate Protocol 5: One‐Step Sequencing Reactions Using 5′‐End‐Labeled Primers

  • 32P]ATP (3000 Ci/mol) or35S]ATP (1300 Ci/mmol)
  • recipeSequenase termination mixes (see recipe), recipeTaq termination mixes (see recipe), or recipeKlenow one‐step sequencing mixes (see recipe)
  • DNA polymerase and appropriate 10× buffer: Sequenase (see protocol 1), Taq DNA polymerase (see protocol 3), or Klenow fragment (see protocol 4)
  • recipedNTP chase (for Klenow fragment; see recipe)
  • 37° to 42°C water bath (for Klenow fragment or 50° to 75°C water bath (for Taq polymerase)
  • Additional reagents and equipment for end labeling (unit 3.10)

Basic Protocol 3: Thermal Cycle Sequencing Reactions Using α‐Labeled Nucleotides

  Materials
  • recipeVent R (exo) sequencing mixes (see recipe)
  • 0.04 to 0.1 pmol single‐stranded or double‐stranded DNA template (unit 7.3)
  • 0.6 to 1.2 pmol oligonucleotide primer (unit 2.11)
  • recipe10× Vent R (exo) DNA sequencing buffer (see recipe)
  • 3% (v/v) Triton X‐100
  • 10 mCi/ml [α‐35S]dATP, [α‐32P]dATP, or [α‐33P]dATP (500 to 3000 Ci/mmol)
  • 2000 U/ml Vent R (exo) DNA polymerase
  • Mineral oil, sterile
  • recipeTCS stop/loading dye (see recipe)
  • Thermal cycling apparatus

Alternate Protocol 6: Thermal Cycle Sequencing Reactions Using 5′‐End‐Labeled Primers

  • 10 mCi/ml [γ‐32P]ATP or [γ‐33P]ATP (3000 Ci/mmol), or biotin or fluorescent conjugates (see )
  • Additional reagents and equipment for end‐labeling as described for one‐step sequencing reactions (see protocol 7, steps and )

Alternate Protocol 7: Sequencing with Energy Transfer Dye‐Labeled Primers (ET Primer)

  Materials
  • ThermoSequenase reaction buffer: 260 mM Tris⋅Cl/65 mM MgCl 2, pH 9.5
  • ThermoSequenase nucleotide mix: 750 µM each dNTP + 2.5 µM of specific ddNTP
  • ET primer: 0.5 µM dye‐labeled A or C primer; 1.0 µM dye‐labeled G or T primer
  • 1.5 U/µl ThermoSequenase (Amersham Pharmacia Biotech)
  • 0.25 µg/µl BAC DNA
  • recipeFormamide loading buffer (see recipe)
  • 0.2‐ml strip tubes
  • Thermal cycler
  • Automated DNA sequencer (e.g., Perkin‐Elmer)

Alternate Protocol 8: Sequencing with Energy Transfer Dye‐Labeled Terminators (BigDye Terminators)

  Materials
  • recipeTermination nucleotide mix (see recipe)
  • 5× sequencing buffer: 400 mM Tris⋅Cl, pH 9.0/10 mM MgCl 2
  • AmpliTaq FS for terminators (Perkin‐Elmer)
  • Primer
  • 0.25 µg/µl BAC DNA
  • Centri‐Sep spin columns (Princeton Separations)
  • Thermal cycler
  • Automated DNA sequencer (e.g., Perkin‐Elmer)
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Figures

Videos

Literature Cited

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Key References
   Tabor and Richardson, 1987b, 1990. See above.
  Describes the procedure for DNA sequence analysis with Sequenase.
   Sanger, et al., 1977. See above.
  Describes the Sanger protocol using Klenow fragment.
   Sears, et al., 1992. See above.
  Describes the thermal cycle reaction.
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