Direct DNA Sequencing of PCR Products

Robert L. Dorit1, Osamu Ohara2, Charles B‐C. Hwang3, Jae Bum Kim3, Seth Blackshaw3

1 Yale University, New Haven, Connecticut, 2 Shionogi Research Laboratories, Osaka, Japan, 3 Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 15.2
DOI:  10.1002/0471142727.mb1502s56
Online Posting Date:  November, 2001
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Abstract

PCR products can be sequenced using either the dideoxy (Sanger) or chemical (Maxam‐Gilbert) approaches. In the dideoxy methods presented here, the target sequence is amplified and an excess of one strand of the target sequence (relative to its complement) is then generated by “asymmetric PCR,” where one primer is present in vast excess over the other. This single‐stranded product serves as the template for conventional dideoxy sequencing methods. Another procedure prepares PCR products for use as templates fes for characterizing unlabeled product by genomic sequencing and chemical sequencing of end‐labeled products are also presented.

     
 
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Table of Contents

  • Basic Protocol 1: Generating Single‐Stranded Products for Dideoxy Sequencing by Asymmetric PCR
  • Alternate Protocol 1: Generating Single‐Stranded Template for Dideoxy Sequencing by Single‐Primer Reamplification
  • Alternate Protocol 2: Preparing Double‐Stranded PCR Products for Dideoxy Sequencing
  • Alternate Protocol 3: Generating Single‐Stranded Template for Dideoxy Sequencing by λ Exonuclease Digestion of Double‐Stranded PCR Products
  • Alternate Protocol 4: One‐Step Enzymatic Purification of PCR Products for Direct Sequencing
  • Basic Protocol 2: Labeling PCR Products for Chemical Sequencing
  • Alternate Protocol 5: Genomic Sequencing of PCR Products
  • Reagents and Solutions
  • Commentary
     
 
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Materials

Basic Protocol 1: Generating Single‐Stranded Products for Dideoxy Sequencing by Asymmetric PCR

  Materials
  • Oligonucleotide primers 1 and 2
  • 32P‐labeled dNTPs (optional; unit 3.4)
  • 10 M ammonium acetate ( appendix 22)
  • 100% and 70% ethanol, room temperature
  • 0.1× TE buffer, pH 8.0 ( appendix 22)
  • Centricon 30 or 100 column (optional; Amicon)
  • Additional reagents and equipment for PCR (unit 15.1), electrophoresis using agarose gels (unit 2.5) or nondenaturing polyacrylamide gels (unit 2.7), Southern blotting and hybridization (units 2.9 & 6.4), ethanol precipitation (unit 2.1), and dideoxy sequencing (unit 7.4)

Alternate Protocol 1: Generating Single‐Stranded Template for Dideoxy Sequencing by Single‐Primer Reamplification

  • recipePEG/NaCl solution (see recipe)
  • 70% ethanol, cold
  • 1 pmol (2 to 5 ng) sequencing primer
  • recipe5× annealing buffer (see recipe)
  • Additional reagents and equipment for phenol/chloroform extractions (unit 2.1)

Alternate Protocol 2: Preparing Double‐Stranded PCR Products for Dideoxy Sequencing

  • T4 polynucleotide kinase (unit 3.10) and 1× buffer (unit 3.4)
  • 3 M sodium acetate, pH 5.2 ( appendix 22)
  • λ exonuclease (unit 3.11) and 1× buffer (unit 3.4)

Alternate Protocol 3: Generating Single‐Stranded Template for Dideoxy Sequencing by λ Exonuclease Digestion of Double‐Stranded PCR Products

  Materials
  • PCR product to be sequenced (unit 15.1), 25 to 250 ng/µl
  • 1 U/µl shrimp alkaline phosphatase (ShrAP; e.g., USB)
  • 10 U/µl exonuclease I (Exo I; e.g., USB)
  • 50 mM Tris⋅Cl, pH 8.0 ( appendix 22)
  • PCR tubes or 96‐well PCR plate
  • Thermal cycler to accommodate tubes or 96‐well plates
  • Additional reagents and equipment for agarose gel electrophoresis (unit 2.5)

Alternate Protocol 4: One‐Step Enzymatic Purification of PCR Products for Direct Sequencing

  Materials
  • 5 to 10 pmol oligonucleotide primer 1 (to be end‐labeled)
  • T4 polynucleotide kinase (unit 3.10) and 1× buffer (unit 3.4)
  • [γ‐32P]ATP, specific activity 6000 Ci/mmol
  • 20 to 40 pmol oligonucleotide primer 2
  • 10× PCR amplification buffer (unit 15.1)
  • 2 mM 4dNTP mix (unit 15.1)
  • DNA template (20 to 1000 ng for eukaryotic DNA; 10 to 100 ng for bacterial DNA; 1 to 20 ng for cloned DNA inserts)
  • Taq DNA polymerase (unit 15.1)
  • Mineral oil
  • Disposable columns or dialysis cartridges for DNA purification (optional; NACS prepack cartridge, Life Technologies; Centricon 30 column, Amicon; or Select 6L column, 5 Prime→3 Prime)
  • Additional reagents and equipment for PCR (unit 15.1), end‐labeling with T4 polynucleotide kinase ( 97.80.4711Table 3.4.1 & unit 3.10), electrophoresis using agarose gels (unit 2.5) or nondenaturing polyacrylamide gels (unit 2.7), autoradiography ( appendix 3A), electroelution from agarose gels (unit 2.6), phenol extraction and ethanol precipitation (unit 2.1), sequencing by the chemical method (unit 7.5), and sequencing gels ( )

Basic Protocol 2: Labeling PCR Products for Chemical Sequencing

  Materials
  • Filter paper (Schleicher & Schuell #410), precut to gel size
  • Additional reagents and equipment for phenol extraction and ethanol precipitation (unit 2.1); transfer by electroblotting, UV cross‐linking, and hybridization (units 2.9, 6.3, & 6.4); and labeling with terminal deoxynucleotidyltransferase (unit 3.6)
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Figures

Videos

Literature Cited

Literature Cited
   Church, G.M. and Gilbert, W. 1984. Genomic sequencing. Proc. Natl. Acad. Sci. U.S.A. 81:1991‐1995.
   Church, G.M. and Kieffer‐Higgins, S. 1988. Multiplex DNA sequencing. Science 240:185‐188.
   DiMarzo, R., Dowling, C.E., Wong, C., Maggio, A., and Kazazian, H.H. 1988. The spectrum of β‐thalassaemia mutations in Sicily. Br. J. Haematol. 69:393‐397.
   Gyllensten, U.B. 1989. Direct sequencing of in vitro amplified DNA. In PCR Technology (H.A. Erlich, ed.) pp. 45‐60. Stockton Press, New York.
   Gyllensten, U.B. and Erlich, H.A. 1988. Generation of single‐stranded DNA by the polymerase chain reaction and its application to the direct sequencing of the HLA‐DQA locus. Proc. Natl. Acad. Sci. U.S.A. 85:7652‐7656.
   Hanke, M. and Wink, M. 1994. Direct DNA sequencing of PCR‐amplified vector inserts following enzymatic degradation of primer and dNTPs. Biotechniques 17:858‐860.
   Higuchi, R.G. and Ochman, H. 1989. Production of single‐stranded DNA templates by exonuclease digestion following the polymerase chain reaction. Nucl. Acids Res. 17:5865.
   Innis, T.M.A., Myambo, K.B., Gelfand, D.H., and Brow, M.A. 1988. DNA sequencing with Thermus aquaticus DNA polymerase and direct sequencing of polymerase chain reaction‐amplified DNA. Proc. Natl. Acad. Sci. U.S.A. 85:9436‐9440.
   Kreitman, M. and Landweber, L. 1989. A strategy for producing single‐stranded DNA in the polymerase chain reaction. Gene Anal. Technol. 6:84‐88.
   Myers, R.M., Sheffield, V., and Cox, D.R. 1988. Detection of single‐base changes in DNA: Ribonuclease cleavage and denaturing gel electrophoresis. In Genomic Analysis: A Practical Approach (K. Davies, ed.) pp. 95‐139. IRL Press, Oxford.
   Ohara, O., Dorit, R.L., and Gilbert, W. 1989. One‐sided polymerase chain reaction: The amplification of cDNA. Proc. Natl. Acad. Sci. U.S.A. 86:5673‐5677.
   Werle, E., Schneider, C., Renner, M., Volker, M., and Fiehn, W. 1994. Convenient single‐step, one tube purification of PCR products for direct sequencing. Nucl. Acids Res. 22:4354‐4355.
   Wrischnik, L.A., Higuchi, R.G., Stoneking, M., Erlich, H.A., Arnheim, N., and Wilson, A.C. 1987. Length mutations in human mitochondrial DNA: Direct sequencing of enzymatically amplified DNA. Nucl. Acids Res. 15:529‐542.
Key References
   Kusukawa, N., Uemori, T., Asada, K., and Kato, I. 1990. Rapid and reliable protocol for direct sequencing of material amplified by polymerase chain reaction. BioTechniques 9:66‐72.
  Outlines the method for double‐stranded sequencing of PCR products.
   Maxam, A.M. and Gilbert, W. 1980. Sequencing end‐labeled DNA with base‐specific chemical cleavages. Methods Enzymol. 65:499‐559.
  These two papers outline the basic principles and techniques of chemical and dideoxy sequencing.
   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.
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