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

  • Basic Protocol 1: 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 1: Ligation‐Mediated Single‐Sided PCR

  Materials
  • 0.4 µg/µl cleaved genomic DNA in TE buffer, pH 7.5 ( protocol 2, protocol 32, or protocol 43)
  • recipeFirst‐strand synthesis mix (see recipe), containing oligonucleotide primer 1 (Figs. and ; also see )
  • recipe20 µM unidirectional linker mix (see recipe)
  • recipeLigase dilution solution (see recipe)
  • recipeLigase mix (see recipe)
  • 2000 to 3000 “Weiss” U/ml T4 DNA ligase (unit 3.14; Promega or Pharmacia Biotech)
  • recipePrecipitation salt mix (see recipe)
  • 100% ethanol, ice‐cold and room temperature
  • 75% ethanol, room temperature
  • recipeAmplification mix (see recipe), containing linker primer and primer 2
  • recipe2 U/µl Vent DNA polymerase mix (see recipe; also see unit 7.4)
  • Mineral oil
  • recipeEnd‐labeling mix (see recipe), containing end‐labeled primer 3 (Fig. )
  • recipeVent DNA polymerase stop solution (see recipe)
  • recipe25:24:1 (v/v/v) phenol/chloroform/isoamyl alcohol (see recipe)
  • recipeLoading 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 22)
  • Tissue culture medium appropriate for sample cells
  • recipeLysis solution (see recipe)
  • Duplicate 15‐cm plates of cells in monolayer culture (see )
  • 100% dimethyl sulfate (DMS; Aldrich)
  • Equilibrated buffered phenol (unit 2.1)
  • recipe25:24:1 (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 22)
  • 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 22)
  • recipeLysis 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 protocol 21 and protocol 32)
  • TE buffer, pH 7.5 ( appendix 22)
  • 3 M sodium acetate, pH 7.0 ( appendix 22)
  • 100% ethanol, room temperature and chilled on dry ice
  • 75% ethanol, room temperature
  • 88% formic acid (Fisher Chemical)
  • recipeG+A stop solution (see recipe)
  • Hydrazine, 98% anhydrous (Aldrich #21,515‐5)
  • recipeC+T/C stop solution (see recipe)
  • 5 M sodium chloride ( appendix 22)
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

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.
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   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.
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   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.
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   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.
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   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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