Preparation of Templates for DNA Sequencing

Barton E. Slatko1, Peter Heinrich2, B. Tracy Nixon3, Richard L. Eckert4

1 New England Biolabs, Beverly, Massachusetts, 2 Consortium für Elektrochemische Industrie, Munich, Federal Republic of Germany, 3 Pennsylvania State University, University Park, Pennsylvania, 4 Case Western Reserve University, Cleveland, Ohio
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 7.3
DOI:  10.1002/0471142727.mb0703s21
Online Posting Date:  May, 2001
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Abstract

This unit contains protocols for preparing DNA suitable for use as dideoxy sequencing templates and as material for end labeling and chemical sequencing. In all protocols, the starting material contains the recombinant molecule to be sequenced. DNA from M13mp‐derived phage is easily prepared and is currently the most reliable source of template for large‐scale dideoxy sequencing projects. Because it is occasionally necessary or convenient to use a λ‐derived phage as a source of DNA, a protocol for preparing λ phage DNA from plate lysates is provided. Two protocols for minipreps of plasmid DNA are provided, one intended for dideoxy sequencing, the other for end labeling and chemical sequencing; they differ primarily in the way in which cellular RNA is removed. Alkali denaturation of double‐stranded DNA (necessary prior to annealing) is described, and a final protocol describes the preparation of template for thermal cycle sequencing from a single phage plaque or bacterial colony.

     
 
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Table of Contents

  • Basic Protocol 1: Preparation of Single‐Stranded M13 Phage DNA
  • Basic Protocol 2: Preparation of λ DNA from Small‐Scale Lysates
  • Basic Protocol 3: Miniprep of Recombinant pSP64CS or pSP65CS Plasmid DNA for Chemical Sequencing
  • Basic Protocol 4: Miniprep of Double‐Stranded Plasmid DNA for Dideoxy Sequencing
  • Basic Protocol 5: Alkali Denaturation of Double‐Stranded Plasmid DNA for Dideoxy Sequencing
  • Basic Protocol 6: Preparation of Plasmid DNA from an E. coli Colony or Phage DNA from a Plaque for Thermal Cycle Sequencing
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Preparation of Single‐Stranded M13 Phage DNA

  Materials
  • E. coli DH5αF′ (or equivalent; Table 97.80.4711), competent (unit 1.8) and overnight cultures in LB medium (units 1.1 & 1.2)
  • Recombinant M13mp RF DNA or M13mp single‐stranded DNA (unit 1.15)
  • H top agar (unit 1.1)
  • LB plates (unit 1.1), 37°C
  • 2× TY medium (unit 1.1)
  • recipeM13 polyethylene glycol (PEG) solution
  • TE buffer ( appendix 22)
  • Buffered phenol (unit 2.1)
  • 3 M sodium acetate, pH 5.2 ( appendix 22)
  • 100% ethanol and 70% ethanol, ice‐cold
  • Pasteur pipets, regular and drawn‐out (sterile)
  • 18 × 150–mm tubes
  • Additional reagents and equipment for transformation (unit 1.8), cell growth (unit 1.2), and agarose gel electrophoresis (unit 2.5)

Basic Protocol 2: Preparation of λ DNA from Small‐Scale Lysates

  Materials
  • Recombinant λ phage
  • E. coli strain appropriate for λ phage of interest
  • Lambda top agarose and agarose plates (i.e., substitute agarose for agar; unit 1.1), freshly prepared on the day of use
  • 1 mg/ml DNase I (unit 3.12)
  • recipe1 mg/ml RNase A, heat‐inactivated (see )
  • recipeLambda polyethylene glycol (PEG) solution
  • SM medium (unit 1.11)
  • 10% sodium dodecyl sulfate (SDS)
  • Buffered phenol (unit 2.1)
  • 0.5 M EDTA
  • 1:1 phenol/chloroform
  • Chloroform
  • 3 M sodium acetate, pH 7.0
  • Isopropanol
  • TE buffer, pH 8.0 ( appendix 22)
  • Pasteur pipets, drawn out (sterile)
  • Additional reagents and equipment for preparing and titering a λ phage plate lysate (units 1.11 & 1.12)

Basic Protocol 3: Miniprep of Recombinant pSP64CS or pSP65CS Plasmid DNA for Chemical Sequencing

  Materials
  • LB medium containing 40 µg/ml ampicillin (unit 1.1)
  • E. coli strain carrying recombinant pSP64CS or pSP65CS
  • recipeTris/EDTA/glucose buffer
  • 2 N NaOH
  • 4% SDS
  • 3 M sodium acetate, pH 6.0
  • 10 µg/ml RNase A, heat‐inactivated (see )
  • 25:24:1 phenol/chloroform/isoamyl alcohol (unit 2.1)
  • Isopropanol
  • 70% ethanol
  • 18 × 150–mm tubes

Basic Protocol 4: Miniprep of Double‐Stranded Plasmid DNA for Dideoxy Sequencing

  Materials
  • LB medium containing appropriate antibiotic (unit 1.1)
  • E. coli strain carrying recombinant plasmid
  • recipeTris/EDTA/glucose buffer
  • 1.0% SDS/0.2 N NaOH
  • 3 M sodium acetate, pH 4.8 ( appendix 22)
  • Isopropanol
  • TE buffer, pH 8.0 ( appendix 22)
  • 4 M LiCl
  • Buffered phenol (unit 2.1)
  • Chloroform
  • Isopropanol
  • 70% ethanol, ice‐cold
  • TE buffer, pH 8.0 ( appendix 22)
  • Additional reagents and equipment for agarose gel electrophoresis (unit 2.5)

Basic Protocol 5: Alkali Denaturation of Double‐Stranded Plasmid DNA for Dideoxy Sequencing

  Materials
  • Recombinant plasmid DNA (third protocol 3basic protocol and/or fourth protocol 4basic protocol)
  • 2 M NaOH/ 2 mM EDTA
  • 3 M sodium acetate, pH 6.0
  • 95% and 70% ethanol
  • 0.5‐ml microcentrifuge tubes

Basic Protocol 6: Preparation of Plasmid DNA from an E. coli Colony or Phage DNA from a Plaque for Thermal Cycle Sequencing

  Materials
  • Agar plate containing E. coli colonies carrying a recombinant plasmid or λ or M13 recombinant phage plaques
  • recipeTris/EDTA/proteinase K
  • Sterile toothpick or glass rod
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Figures

Videos

Literature Cited

Literature Cited
   Adams, S. and Blakesley, R. 1991. Linear amplification sequencing. Focus (BRL) 13:56‐57.
   Chen, E.Y. and Seeburg, P.H. 1985. Supercoil sequencing: A fast and simple method for sequencing plasmid DNA. DNA (N.Y.) 4:165‐170.
   Hattori, M. and Sakaki, Y. 1986. Dideoxy DNA sequencing method using denatured plasmid templates. Anal. Biochem. 152:232‐238.
   Hultman, T., Stahl, S., Hornes, E., and Uhlen, M. 1989. Direct solid phase sequencing of genomic and plasmid DNA using magnetic beads as solid supports. Nucl. Acids Res. 17:4937‐4946.
   Kaneoka, H., Lee, D.R., Hsu, K.‐C., Sharp, G.C., and Hoffman, R.W. 1991. Solid phase DNA sequencing of allele specific polymerase chain reaction amplified HLA‐DR genes. BioTechniques 10:30‐40.
   Krishnan, B.R., Blakesley, R.W., and Berg, D.E. 1991. Linear amplification DNA sequencing directly from single phage plaques and bacterial colonies. Nucl. Acids Res. 19:1153.
   Sears, L.B., Moran, L.S., Kissinger, C., Creasey, T., Perry‐O'Keefe, H., Roskey, M., Sutherland, E., and Slatko, B.E. 1992. CircumVent thermal cycle sequencing and alternative manual and automated DNA sequencing protocols using the highly thermostable VentR (exo−) DNA polymerase. BioTechniques. 13:626‐633.
   Silhavy, T., Berman, M., and Enquist, L. 1984. Experiments with Gene Fusions. Cold Spring Harbor Laboratory Press. Cold Spring Harbor, N.Y.
   Wang, B. and Sodja, A. 1991. Alternate approach to sequencing double‐stranded template DNAs. BioTechniques 10:198‐201.
   Zagursky, R.J., Baumeister, K., Lomax, N., and Berman, M.L. 1985. Rapid and easy sequencing of large double‐stranded DNA and supercoiled plasmid DNA. Gene Anal. Tech. 2:89‐94.
   Zhang, H., Scholl, R., Browse, J., and Sommerville, C. 1988. Double‐stranded DNA sequencing as a choice for DNA sequencing. Nucl. Acids Res. 16:1220.
   Zimmerman, J., Dietrich, T., Voss, H., Erfle, H., Schwager, C., Stegemann, J., Hewitt, N., and Ansorge, W. 1992. Fully automated sequencing portocol for double‐stranded DNA. Methods Mol. Cell. Biol. 3:39‐42.
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