Analysis of Human Papillomavirus Genome Replication Using Two‐ and Three‐Dimensional Agarose Gel Electrophoresis

Liisi Henno1, Eva‐Maria Tombak1, Jelizaveta Geimanen1, Marit Orav1, Ene Ustav1, Mart Ustav2

1 University of Tartu, Institute of Technology, Tartu, 2 Academy of Sciences, Tallinn
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 14B.10
DOI:  10.1002/cpmc.28
Online Posting Date:  May, 2017
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This unit includes the necessary information to conduct neutral/neutral and neutral/alkaline two‐dimensional and neutral/neutral/alkaline three‐dimensional agarose gel electrophoresis. The methodology has been optimized over the years to gain a better outcome from the hard‐to‐interpret signals of human papilloma virus replication intermediates obtained from two‐ and three‐dimensional agarose gels. Examples of typical results and interpretation of replication intermediate patterns are included, and the outcomes of multiple‐dimension assays are assessed using previously published experimental data. © 2017 by John Wiley & Sons, Inc.

Keywords: two‐dimensional agarose gel electrophoresis; three‐dimensional agarose gel electrophoresis; HPV; DNA replication; minicircle production; southern blot

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Table of Contents

  • Introduction
  • Basic Protocol 1: Production of High‐Quality Recircularized ccc HPV DNA by Minicircle Technology
  • Basic Protocol 2: Transfection of Viral DNA Into Mammalian Cells and Isolation of Replication Intermediates and Products
  • Basic Protocol 3: 2D Neutral/Neutral Age
  • Basic Protocol 4: 2D Neutral/Alkaline Age
  • Basic Protocol 5: 3D Neutral/Neutral/Alkaline AGE
  • Basic Protocol 6: Southern Blotting
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Production of High‐Quality Recircularized ccc HPV DNA by Minicircle Technology

  • E. coli strain ZYCY10P3S2T (Kay et al., ; System Biosciences, cat. no. MN900A‐1)
  • LB medium (see recipe)
  • LB agar plates (see recipe) with and without kanamycin
  • Kanamycin
  • Inoue transformation buffer (see recipe), ice cold
  • Dimethyl sulfoxide (DMSO)
  • Liquid nitrogen
  • pMC.BESPX‐HPV18 DNA: HPV‐containing parental plasmid, prepared from vector pMC.BESPX (Kay et al., ; System Biosciences, cat. no. MN100A‐1), transferred into HPV18 between positions 7374/7474
  • Terrific broth with kanamycin (see recipe)
  • 20% (w/v) L‐arabinose (see recipe)
  • 1 N NaOH
  • 1 M HCl
  • Endotoxin‐free DNA extraction kit (e.g., NucleoBond Xtra Maxi EF, Macherey‐Nagel) including: resuspension buffer with RNase A, alkaline lysis buffer, neutralization buffer, equilibration buffer, wash buffers, elution buffer, endotoxin‐free TE‐EF buffer, columns with column filters
  • Isopropanol
  • 70% (v/v) endotoxin‐free ethanol
  • 100‐ml, 250‐ml, 1‐liter, and 2‐liter glass conical flasks
  • Shaking incubator (18°, 32°, and 37°C)
  • 37°C heating block
  • UV spectrophotometer and plastic cuvettes
  • 50‐ml conical centrifuge tubes
  • 1.5‐ml microcentrifuge tubes
  • pH indicator strips
  • 400‐ml centrifuge bottles
  • Centrifuge (e.g., Sorvall RC‐5B Plus)
Additional reagents and equipment for standard agarose gel electrophoresis

Basic Protocol 2: Transfection of Viral DNA Into Mammalian Cells and Isolation of Replication Intermediates and Products

  • U2OS cells (ATCC, no. HTB‐96)
  • Iscove's modified Dulbecco's medium (IMDM) with 10% fetal calf serum (FCS) (e.g., Naxo)
  • Penicillin
  • Streptomycin
  • Phosphate‐buffered saline (PBS, appendix 2A)
  • 0.05% trypsin/EDTA in PBS, tissue culture grade (e.g., Naxo)
  • Plasmid DNA to be tested for replication activity (HPV genome; see protocol 1)
  • Endotoxin‐free plasmid carrier DNA (e.g., pAuxoMCS plasmid, Icosagen)
  • Hirt lysis buffer (see recipe)
  • 5.0 M NaCl
  • CTAB extraction buffer (see recipe)
  • Isopropanol
  • Tris extraction buffer (see recipe)
  • 20 mg/ml proteinase K (Thermo Fisher Scientific)
  • Phenol (AppliChem, equilibrated, stabilized)
  • Chloroform (Sigma‐Aldrich)
  • 96% (v/v) ethanol
  • Endotoxin‐free TE‐EF buffer, pH 7.0 (Macherey‐Nagel)
  • 10 mg/ml RNase A (Thermo Fisher Scientific)
  • 100‐mm cell culture dishes
  • 37°C, 5% CO 2 incubator
  • 15‐ and 50‐ml conical tubes
  • Cell counter (e.g., Invitrogen Countess)
  • Electroporator and cuvettes (e.g., Bio‐Rad GenePulser Xcell with capacitance extender and 4‐mm cuvettes)
  • Natural‐rubber scraper (or equivalent)
  • 1.5‐ml microcentrifuge tubes
  • 37°C heating block

Basic Protocol 3: 2D Neutral/Neutral Age

  • Agarose (e.g., TopVision agarose, Thermo Fisher Scientific)
  • 0.5× TBE electrophoresis buffer (see recipe)
  • 6× loading dye (see recipe)
  • Markers and ladders:
    • Undigested minicircle DNA
    • Linearized minicircle DNA
    • Supercoiled DNA ladder (e.g., New England Biolabs)
    • High‐range DNA ladder (e.g., Thermo Fisher Scientific O'GeneRuler High Range DNA Ladder)
    • Linear 1‐kb DNA ladder (250‐10,000 bp; e.g., Naxo)
  • 10 mg/ml ethidium bromide (EtBr; appendix 2A)
  • Gel electrophoresis apparatus with gel trays, 1.5‐mm combs, and power supply
  • UV fluorescent ruler
  • UV transilluminator
  • Scalpel

Basic Protocol 4: 2D Neutral/Alkaline Age

  • Alkaline electrophoresis buffer (AEB; see recipe)
  • 0.25 M HCl
  • Additional reagents and equipment for 2D N/N AGE (see protocol 3)

Basic Protocol 5: 3D Neutral/Neutral/Alkaline AGE

  • Alkaline electrophoresis buffer (AEB; see recipe)
  • 0.25 M HCl
  • Additional reagents and equipment for 2D N/N AGE (see protocol 3)

Basic Protocol 6: Southern Blotting

  • Agarose gel containing separated DNA (see Basic Protocols protocol 33, protocol 44, and protocol 55)
  • Denaturation buffer (see recipe)
  • Neutralization buffer (see recipe)
  • 20× SSC ( appendix 2A)
  • Prehybridization buffer (see recipe), freshly prepared
  • DNA template for probe labeling
  • DecaLabel DNA Labeling kit (Thermo Fisher Scientific) including: decanucleotides in 5× reaction buffer, mix C, DNA polymerase I Klenow fragment exo (5 U/μl), and dNTPs
  • 10 mCi/ml [α‐32P]dCTP (specific activity, 111 TBq/mmol; Hartmann Analytics)
  • 10% (w/v) SDS ( appendix 2A)
  • Positively charged nylon transfer membrane, 0.45‐μm pore size (e.g., Naxo)
  • Whatman 3 MM blotting paper (Macherey‐Nagel, cat. no. MN 218B)
  • Glass bowl
  • Glass plates or slides
  • Plastic cling film (e.g., Saran Wrap)
  • Paper towels
  • UV cross‐linker (e.g., Stratagene Stratalinker)
  • Hybridization oven and tubes
  • 1.5‐ml microcentrifuge tube
  • Heating block (37° and 100°C)
  • 50‐ml conical tube
  • X‐ray cassette with intensifying screen
  • X‐ray film (e.g., Agfa)
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Literature Cited

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