Ligation‐Mediated PCR for Genomic Sequencing and Footprinting

Paul R. Mueller1, Barbara Wold1, Paul A. Garrity2

1 California Institute of Technology, Pasadena, California, 2 University of California, Los Angeles, Los Angeles, California
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 15.3
DOI:  10.1002/0471142727.mb1503s56
Online Posting Date:  November, 2001
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Abstract

This unit describes how PCR can be used to exponentially amplify segments of DNA located between two specified primer hybridization sites. A single-sided PCR method is used that initially requires specification of only one primer hybridization site; the second is defined by the ligation-based addition of a unique DNA linker. This linker, together with the flanking gene-specific primer, allows exponential amplification of any fragment of DNA. Because a defined, discrete-length sequence is added to every fragment, complex populations of DNA such as sequence ladders can be amplified intact with retention of single-base resolution. The ligation-based protocol was specifically designed for genomic footprinting and direct sequencing reactions, and is described in this context; it can, however, be used for other applications.

     
 
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Table of Contents

  • Unit Introduction
  • Basic Protocol: Ligation-Mediated Single-Sided PCR
  • Support Protocol 1: Preparation of Genomic DNA from Monolayer Cells for DMS Footprinting
  • Support Protocol 2: Preparation of Genomic DNA from Suspension Cells for DMS Footprinting
  • Support Protocol 3: Preparation of Genomic DNA for Chemical Sequencing
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol: Ligation-Mediated Single-Sided PCR

 Materials
  • 0.4 µg/µl cleaved genomic DNA in TE buffer, pH 7.5 (Support Protocol 1, 2, or 3)
  • First-strand synthesis mix (see recipe), containing oligonucleotide primer 1 (Figs. 15.3.1 and 15.3.2; also see Critical Parameters)
     FigureFigure 15.3.2 Two possible arrangements of gene-specific primers (see Critical Parameters).
  • 20 µM unidirectional linker mix (see recipe)
  • Ligase dilution solution (see recipe)
  • Ligase mix (see recipe)
  • 2000 to 3000 “Weiss” U/ml T4 DNA ligase (UNIT 3.14; Promega or Pharmacia Biotech)
  • Precipitation salt mix (see recipe)
  • 100% ethanol, ice-cold and room temperature
  • 75% ethanol, room temperature
  • Amplification mix (see recipe), containing linker primer and primer 2
  • 2 U/µl Vent DNA polymerase mix (see recipe; also see UNIT 7.4A)
  • Mineral oil
  • End-labeling mix (see recipe), containing end-labeled primer 3 (Fig. 15.3.3)
     FigureFigure 15.3.3 Staggered linker used in ligation-mediated PCR (LMPCR). This staggered linker is made by annealing the oligonucleotides LMPCR.1 and LMPCR.2 to each other. LMPCR.1 has 25 bases and a GC content of 60%, whereas LMPCR.2 has 11 bases and a GC content of 36%. Other oligonucleotides could be used in place of these as long as they meet the criteria defined in text. Note the lack of 5¢ phosphates on oligonucleotides (i.e., 5¢ OH instead) and the unconventional orientation of LMPCR.2 (3¢ to 5¢) in the figure.
  • Vent DNA polymerase stop solution (see recipe)
  • 25:24:1 (v/v/v) phenol/chloroform/isoamyl alcohol (see recipe)
  • Loading buffer (see recipe)
  • 1.5-ml microcentrifuge tubes, silanized (APPENDIX 3A) and with Lid-Loks (optional; Intermountain Scientific)
  • 4° and 17°C water baths
  • Automated thermal cycler or water baths at 95°, 60°, 76°, and 60° to 70°C
  • Additional reagents and equipment for PCR (UNIT 15.1), denaturing gel electro- phoresis for DNA sequencing (UNIT 7.6), and autoradiography (APPENDIX 3A)

Support Protocol 1: Preparation of Genomic DNA from Monolayer Cells for DMS Footprinting

 Materials
  • Phosphate-buffered saline (PBS; APPENDIX 2)
  • Tissue culture medium appropriate for sample cells
  • Lysis solution (see recipe)
  • Duplicate 15-cm plates of cells in monolayer culture (see Critical Parameters)
  • 100% dimethyl sulfate (DMS; Aldrich)
  • Equilibrated buffered phenol (UNIT 2.1A)
  • 25:24:1 (v:v:v) phenol/chloroform/isoamyl alcohol (see recipe)
  • 24:1 (v/v) chloroform/isoamyl alcohol
  • Ethyl ether
  • Isopropanol
  • TE buffer, pH 7.5 (APPENDIX 2)
  • 3 M sodium acetate, pH 7.0
  • 100% ethanol, room temperature and chilled on dry ice
  • 75% ethanol, room temperature
  • DMS stop buffer (UNIT 7.5), ice-cold
  • Piperidine (Aldrich)
  • 8 M ammonium acetate
  • 50- and 15-ml disposable polypropylene screw-cap tubes (e.g., Corning)
  • Aspirator attached to waste flask
  • Disposable cell scraper
  • Tabletop centrifuge (e.g., IEC Centra-7R)
  • 1.5-ml microcentrifuge tubes, silanized (APPENDIX 3A) and with Lid-Loks (Intermountain Scientific)
  • Sealed Pasteur pipet or thin glass rod
  • Additional reagents and equipment for quantitation of DNA (APPENDIX 3A)

CAUTION: DMS and concentrated piperidine are extremely toxic. All manipulations that use DMS or piperidine should be performed in a properly functioning fume hood. Before starting this procedure, review precautions for working with and disposing of DMS and piperidine in UNIT 7.5.

Support Protocol 2: Preparation of Genomic DNA from Suspension Cells for DMS Footprinting

 Materials
  • Cells in suspension culture
  • Phosphate-buffered saline (PBS; APPENDIX 2)
  • Lysis solution (see recipe)
  • 100% dimethyl sulfate (DMS; Aldrich)
  • 100% ethanol, room-temperature
  • 50-ml polypropylene screw-cap tubes (e.g., Corning)
  • Tabletop centrifuge (IEC Centra-7R or equivalent)

CAUTION: DMS is extremely toxic. All manipulations that use DMS should be performed in a properly functioning fume hood. Before starting this procedure, review precautions for working with and disposing of DMS in UNIT 7.5.

Support Protocol 3: Preparation of Genomic DNA for Chemical Sequencing

 Materials
  • 0.5 to 1.0 µg/µl untreated genomic DNA in TE buffer, pH 7.5 (control DNA from steps to in Support Protocols 1 and 2)
  • TE buffer, pH 7.5 (APPENDIX 2)
  • 3 M sodium acetate, pH 7.0 (APPENDIX 2)
  • 100% ethanol, room temperature and chilled on dry ice
  • 75% ethanol, room temperature
  • 88% formic acid (Fisher Chemical)
  • G+A stop solution (see recipe)
  • Hydrazine, 98% anhydrous (Aldrich #21,515-5)
  • C+T/C stop solution (see recipe)
  • 5 M sodium chloride (APPENDIX 2)

CAUTION: DMS, formic acid, and hydrazine are toxic. All manipulations using these chemicals should be performed in a properly functioning fume hood. Before starting this procedure, review precautions for working with and disposing of DMS and hydrazine in UNIT 7.5.
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Figures

  •  FigureFigure 15.3.1 Flowchart of ligation-mediated PCR protocol (see text for details). The steps correspond to those listed in the Basic Protocol.
    View Image
  •  FigureFigure 15.3.2 Two possible arrangements of gene-specific primers (see Critical Parameters).
    View Image
  •  FigureFigure 15.3.3 Staggered linker used in ligation-mediated PCR (LMPCR). This staggered linker is made by annealing the oligonucleotides LMPCR.1 and LMPCR.2 to each other. LMPCR.1 has 25 bases and a GC content of 60%, whereas LMPCR.2 has 11 bases and a GC content of 36%. Other oligonucleotides could be used in place of these as long as they meet the criteria defined in text. Note the lack of 5¢ phosphates on oligonucleotides (i.e., 5¢ OH instead) and the unconventional orientation of LMPCR.2 (3¢ to 5¢) in the figure.
    View Image
  •  FigureFigure 15.3.4 Flow chart of preparation of DMS-footprinted genomic DNA for use in ligation-mediated PCR.
    View Image

Videos

Literature Cited

Literature Cited
    Becker, P.D. and Schutz, G. 1988. Genomic footprinting. In Genetic Engineering Principles and Methods, Vol. 10 (J.K. Setlow and A. Hollaender, eds.) pp. 1-19. Plenum, New York.
    Church, G.M. and Gilbert, W. 1984. Genomic sequencing. Proc. Natl. Acad. Sci. U.S.A. 81:1991-1995.
    Clark, J.M. 1988. Novel nontemplated nucleotide addition reactions catalyzed by prokaryotic and eukaryotic DNA polymerases. Nucl. Acids Res. 16:9677-9686.
    Fors, L., Saavedra, R.A., and Hood, L. 1990. Cloning of the shark Po promoter using genomic walking technique based on the polymerase chain reaction. Nucl. Acids Res. 18:2793-2799.
    Frohman, M.A., Dush, M.K., and Martin, G.R. 1988. Rapid production of full-length cDNAs from rare transcripts: Amplification using a single gene-specific oligonucleotide primer. Proc. Natl. Acad. Sci. U.S.A. 85:8998-9002.
    Garrity, P.A. and Wold, B.J. 1992. Effects of different DNA polymerases in ligation-mediated PCR: Enhanced genomic sequencing and in vivo footprinting. Proc. Natl. Acad. Sci. U.S.A. 89:1021-1025.
    Huibregtse, J.M. and Engelke, D.R. 1986. Direct identification of small sequence changes in chromosomal DNA. Gene 44:151-158.
    Jackson, P.D. and Felsenfeld, G. 1985. A method for mapping intranuclear protein-DNA interactions and its applications to a nuclease hypersensitive site. Proc. Natl. Acad. Sci. U.S.A. 82:2296-2300.
    Linz, U. 1990. Thermocycler temperature variation invalidates PCR results. BioTechniques 9:286-293.
    Loh, E.Y., Elliott, J.F., Cwirla, S., Lanier, L.L., and Davis, M.M. 1989. Polymerase chain reaction with single-sided specificity: Analysis of T cell receptor chain. Science 243:217-220.
    Mueller, P.R. and Wold, B. 1991. Ligation-mediated PCR: Applications to genomic footprinting. Methods 2:20-31.
    Mueller, P.R., Salser, S.J., and Wold, B. 1988. Constitutive and metal inducible protein: DNA interactions at the mouse metallothionein-I promoter examined by in vivo and in vitro footprinting. Genes & Dev. 2:412-427.
    Mueller, P.R. and Wold, B. 1989. In vivo footprinting of a muscle specific enhancer by ligation mediated PCR. Science 246:780-786.
    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.
    Pfeifer, G.P., Steigerwald, S.D., Mueller, P.R., Wold, B., and Riggs, A.D. 1989. Genomic sequencing and methylation analysis by ligation mediated PCR. Science 246:810-813.
    Pfeifer, G.P., Tanguay, R.L., Steigerwald, S.D., and Riggs, A.D. 1990. In vivo footprint and methylation analysis by PCR-aided genomic sequencing: Comparison of active and inactive X chromosomal DNA at the CpG island and promoter of human PGK-1. Genes & Dev. 4:1277-1287.
    Rideout III, W.M., Coetzee, G.A., Olumi, A.F., and Jones, P.A. 1990. 5-Methylcytosine as an endogenous mutagen in the human LDL receptor and p53 genes. Science 249:1288-1290.
    Rigaud, G., Roux, J., Pictet, R., and Grange, T. 1991. In vivo footprinting of Rat TAT Gene: Dynamic interplay between the glucocorticoid receptor and a liver-specific factor. Cell 67:977-986.
    Saiki, R.K., Gelfand, D.H., Stoffel, S., Scharf, S.J., Higuchi, R., Horn, G.T., Mullis, K.B., and Erich, H.A. 1988. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239:487-491.
    Saluz, H.P. and Jost, J.P. 1987. A Laboratory Guide to Genomic Sequencing: The Direct Sequencing of Native Uncloned DNA. Birkhauser, Boston.
    Tanguay, R.L., Pfeifer, G.P., and Riggs, A.D. 1990. PCR-aided DNase I footprinting of single copy gene sequences in permeabilized cells. Nucl. Acids Res. 18:5902.
    Wahl, G.M., Berger, S.L., and Kimmel, A.R. 1987. Molecular hybridization of immobilized nucleic acids: Theoretic concepts in practical considerations. Methods Enzymol. 152:399-407.
    White, T.J., Arnheim, N., and Erlich, H.A. 1989. The polymerase chain reaction. Trends Genet. 5:185-189.
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