Pulsed‐Field Gel Electrophoresis for Long‐Range Restriction Mapping

Robert M. Gemmill1, Richard Bolin2, Hans Albertsen (LiDS prep)3, Jeff P. Tomkins4, Rod A. Wing4

1 Eleanor Roosevelt Institute for Cancer Research, Denver, Colorado, 2 Nexagen, Boulder, Colorado, 3 Eccles Institute of Human Genetics, Salt Lake City, Utah, 4 Clemson University Genomics Institute, Clemson, South Carolina
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 5.1
DOI:  10.1002/0471142905.hg0501s31
Online Posting Date:  February, 2002
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Abstract

This unit describes procedures for generating long‐range restriction maps of genomic DNA and for analysis of large insert clones. The basic protocol details restriction digestion of agarose‐embedded DNA, PFGE separation, Southern transfer, and hybridization. Support protocols describe the preparation of high‐molecular‐weight genomic DNA samples in agarose blocks and in agarose microbeads, respectively. Additional support protocols describe the preparation of DNA size standards from l phage and two yeast species, Saccharomyces cerevisiae and Schizosaccharomyces pombe. An alternative method of preparing S. cerevisiae size standards using lithium dodecyl sulfate (LiDS) solubilization is provided. The final protocol details the preparation of BAC DNA suitable for digestion, mapping, and sequencing.

     
 
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Table of Contents

  • Basic Protocol 1: Constructing Long‐Range Restriction Maps of Genomic Regions
  • Support Protocol 1: Preparation of High‐Molecular‐Weight Mammalian Genomic DNA Embedded in Agarose Blocks
  • Support Protocol 2: Preparation of High‐Molecular‐Weight DNA Samples in Agarose Microbeads
  • Preparation of High‐Molecular‐Weight DNA Size Standards in Agarose Blocks
  • Support Protocol 3: Preparation of Lambda Phage DNA Concatemers
  • Support Protocol 4: Preparation of Chromosomes and YACs from S. cerevisiae Strains
  • Support Protocol 5: Preparation of Schizosaccharomyces Pombe Chromosomes
  • Support Protocol 6: Preparation of S. cerevisiae Chromosomes and YACs Using Lithium Dodecyl Sulfate
  • Support Protocol 7: Preparation of BAC DNA, Restriction Digestion, and CHEF Gel Analysis
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Constructing Long‐Range Restriction Maps of Genomic Regions

  Materials
  • Agarose‐embedded DNA samples (see protocol 2 or protocol 32)
  • Rare‐cutting restriction endonuclease (Table 5.1.1) and 10× buffer
  • 1 mg/ml BSA, acetylated and nuclease‐free
  • 100 mM spermidine trihydrochloride
  • recipeTE buffer ( appendix 2D)
  • Agarose (ultrapure; e.g., Life Technologies)
  • Electrophoresis buffer (e.g., recipe0.5× TBE; appendix 2D), 10°C
  • recipe6× loading dye ( appendix 2D)
  • DNA size standards (see Support Protocols protocol 43 to protocol 76)
  • recipe0.5 µg/ml ethidium bromide ( appendix 2D)
  • 100‐mm petri plates
  • Water bath at appropriate temperature for restriction digestion (Table 5.1.1)
  • Pulsed‐field gel electrophoresis apparatus
  • Constant‐voltage power supply
  • Pulse generator
  • Positively charged nylon membranes
  • Additional reagents and equipment for radiolabeling probe with DNA‐dependent polymerases ( appendix 3E), and Southern blotting and hybridization(unit 2.7)
CAUTION: Ethidium bromide and radiolabeled probes are hazardous; see appendix 2A for guidelines on handling, storage, and disposal.

Support Protocol 1: Preparation of High‐Molecular‐Weight Mammalian Genomic DNA Embedded in Agarose Blocks

  Materials
  • 1.0% (w/v) low gelling/melting temperature agarose (e.g., InCert; FMC Bioproducts) prepared in HBSS
  • 70% (v/v) ethanol
  • Mammalian cells
  • HBSS ( appendix 2D), 37°C
  • recipeCell lysis buffer (see recipe)
  • recipePMSF/TE wash buffer (make fresh; see recipe)
  • Plexiglas block mold (e.g., Bio‐Rad 10‐well sample‐plug mold)
  • Beckman GPR centrifuge with GH‐3.7 swinging‐bucket rotor (or equivalent)
  • 50° and 42°C water baths
  • Additional reagents and equipment for counting cells with a hemacytometer ( appendix 3G)
CAUTION: Human cells and PMSF/TE wash buffer are hazardous; see APPENDIX 2A for guidelines on handling, storage, and disposal.

Support Protocol 2: Preparation of High‐Molecular‐Weight DNA Samples in Agarose Microbeads

  • 1% (w/v) low‐gelling/melting temperature agarose (e.g., InCert; FMC Bioproducts) prepared in HBSS ( appendix 2D)
  • Light mineral oil, prewarmed
  • HBSS ( appendix 2D), ice‐cold
  • 45° and 65°C water baths
  • 50‐ml conical centrifuge tube
  • Beckman GPR centrifuge with GH‐3.7 swinging‐bucket rotor (or equivalent)
  • Additional reagents and equipment for quantitation of DNA by UV spectrophotometry ( appendix 3D)

Support Protocol 3: Preparation of Lambda Phage DNA Concatemers

  Materials
  • 70% (v/v) ethanol
  • 1% (w/v) low gelling/melting temperature agarose (e.g., InCert; FMC Bioproducts) in recipeλ concatemer buffer (see recipe)
  • CsCl‐banded and dialyzed preparation (unit 2.2 or CPMB UNIT ) of cI857Sam7 λ phage
  • recipeλ dilution buffer (see recipe)
  • recipeλ lysis buffer (see recipe)
  • Plexiglas block mold (e.g., Bio‐Rad 10‐well sample‐plug mold)
  • 42° and 50°C water baths

Support Protocol 4: Preparation of Chromosomes and YACs from S. cerevisiae Strains

  Materials
  • S. cerevisiae cells of desired strain
  • recipeYPD plates and liquid medium (unit 5.5)
  • recipe50 mM EDTA (pH 7.5), sterile
  • 70% (v/v) ethanol
  • Zymolyase solution: 100 U/ml Zymolase 100T (dry powder; ICN Biomedicals)/100 mM mercaptoethanol (2‐ME) in recipeSCE buffer
  • recipeSCE buffer (see recipe)
  • 1.4% (w/v) low gelling/melting temperature agarose prepared in SCE buffer
  • 2‐ME solution: 70 mM 2‐ME in recipeSCE buffer
  • recipeCell lysis buffer (see recipe)
  • 30°C incubator
  • 30°C shaking water bath
  • 250‐ml centrifuge bottles
  • Sorvall centrifuge with GSA rotor (or equivalent)
  • 50‐ml conical centrifuge tube, screw‐cap
  • Plexiglas block mold (Bio‐Rad)
  • Beckman GPR centrifuge with GH‐3.7 rotor (or equivalent)
  • 42° and 50°C water baths
Additional reagents and equipment for counting cells with a hemacytometer ( appendix 3G)CAUTION: 2‐ME is hazardous; see appendix 2A for guidelines on handling, storage, and disposal.

Support Protocol 5: Preparation of Schizosaccharomyces Pombe Chromosomes

  Materials
  • S. pombe cells
  • recipeYPD plates and liquid medium (unit 5.5)
  • recipeAcid wash buffer (see recipe)
  • recipeSpheroplasting buffer (see recipe)
  • 10% (w/v) SDS
  • recipeNeutralization buffer (see recipe)
  • 30°C shaking and stationary incubators
  • 250‐ml centrifuge bottles
  • Sorvall centrifuge with GSA rotor (or equivalent)
  • 50‐ml conical centrifuge tube, screw‐cap
  • Beckman GPR centrifuge with GH‐3.7 rotor (or equivalent)
Additional reagents and equipment for counting cells with a hemacytometer ( appendix 3G)

Support Protocol 6: Preparation of S. cerevisiae Chromosomes and YACs Using Lithium Dodecyl Sulfate

  • recipeSORB buffer (see recipe)
  • 10 U/µl yeast lytic enzyme (70 U/mg Arthrobacter luteus; ICN Biomedicals) in recipeSORB buffer
  • recipeLithium dodecyl sulfate (LiDS) buffer (see recipe)
  • ET buffer: 5 mM EDTA (pH 8.0)/10 mM Tris⋅Cl (pH 7.8)
  • 50‐ml conical centrifuge tube, screw‐cap

Support Protocol 7: Preparation of BAC DNA, Restriction Digestion, and CHEF Gel Analysis

  Materials
  • LB media with antibiotic (same concentration as solid media)
  • E. coli strain DH10B streaked cultures of desired BAC clones grown on solid LB/antibiotic/IPTG (25 µg/ml)/X‐gal (50 µg/ml). Typical antibiotic concentrations needed for the most common BAC vectors is 12.5 µg/ml chloramphenicol or 25 µg/ml kanamycin.
  • recipeGTE solution (see recipe), ice cold
  • NaOH/SDS solution: 4.65 ml sterile water, 100 µl of 10 N NaOH, 250 µl of recipe20% (w/v) SDS; prepare fresh
  • Potassium acetate solution: add 29.5 g potassium acetate in sterile water bringing volume to 88.5 ml, add 11.5 ml glacial acetic acid
  • Isopropanol
  • 70% (v/v) ethanol
  • TE buffer ( appendix 2D), pH 8.0
  • NotI buffer (NEB)
  • 100× BSA (NEB)
  • 10 U/µl Not I restriction enzyme (NEB)
  • 6× loading dye ( appendix 2D)
  • 1% (w/v) electrophoresis grade agarose gel prepared in recipe0.5× TBE
  • DNA size standards (see Support Protocols protocol 43 through protocol 76)
  • 0.5 µg/ml ethidium bromide
  • 15‐ml test tubes
  • 37°C incubator with shaker
  • Table‐top centrifuge with swinging bucket rotor
  • 1.5‐ml microcentrifuge tubes
  • Microcentrifuge
  • 37°C incubator or water bath
  • BIO‐RAD CHEF unit (DR‐II, DR‐III, or Mapper) with chiller and pump
  • UV light box
  • Additional reagents and equipment for Southern blotting and hybridization with probe (see protocol 1)
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Figures

Videos

Literature Cited

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   Bird, A.P. 1986. CpG‐rich islands and the function of DNA methylation. Nature 321:209‐213.
   Birren, B. and Lai, E. 1993. Pulsed field gel electrophoresis: A practical guide. Academic Press, San Diego.
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   Fan, J.B., Chikashige, Y., Smith, C.L., Niwa, O., Yanagida, M., and Cantor, C.R. 1988. Construction of a NotI restriction map of the fission yeast Schizosaccharomyces pombe genome. Nucl. Acids Res. 17:2801‐2818.
   Finney, M. 1992. Pulsed‐field gel electrophoresis. In Current Protocols in Molecular Biology (F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 2.5.9‐2.5.17. Greene Publishing Associates and John Wiley & Sons, New York.
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   Gemmill, R. 1990. Pulsed field gel electrophoresis. Adv. Electrophor. 4:1‐48.
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   Link, A.J. and Olson, M.V. 1991. Physical map of the Saccharomyces cerevisiae genome at 110‐kilobase resolution. Genetics 127:681‐698.
   Marra, M., Kkucaba, T., Dietrich, N., Green, E., and Brownstein, B. 1997. High throughput fingerprint analysis of large‐insert clones. Genome Res. 7:1072‐1084.
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   Shiyuza, H., Birren, B., Kim, U.J., Manicino, V., Slepak, T., Tashiiri, Y., and Simon, M. 1992. Cloning and stable maintenance of 300‐kilobase pair fragments of human DNA in Escherichi coli using an F‐factor based vector. Proc. Natl. Acad. Sci. U.S.A. 89:8794‐8797.
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Key References
   Schwartz and Cantor, 1984. See above.
  Original description of PFGE providing the key insights to development of large DNA separation methods.
   Olson, 1989. See above.
  Excellent review of all aspects of PFGE.
   Birren and Lai, 1993. See above.
  Examples of long‐range mapping in human genome using PFGE.
   Compton et al., 1988. See above.
   Drumm et al., 1988. See above.
   Gardiner et al., 1990. See above.
   Kenwrick et al., 1987. See above.
   Poustka et al., 1988. See above.
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