Rapid Full‐Length Cloning of Nonpolyadenylated RNA Virus Genomes

Randy Beckett1, W. Allen Miller1

1 Iowa State University, Ames, Iowa
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 16F.3
DOI:  10.1002/9780471729259.mc16f03s4
Online Posting Date:  February, 2007
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Abstract

Access to a full‐length infectious clone of a viral genome is a virtual necessity for research aimed at understanding virus replication and gene expression mechanisms. While construction of a full‐length clone may be straightforward, obtaining one that is infectious is by no means routine. Here the authors describe methods to maximize the likelihood of obtaining a full‐length infectious clone. These include protocols to (1) sequence the ends of nonpolyadenylated RNA genomes, (2) obtain a full‐length PCR product in a single reaction directly from viral RNA, and (3) efficiently clone the PCR product into a vector that allows in vitro transcription of viral RNA containing perfect or near‐perfect termini. Given the traditional difficulty of obtaining infectious clones of RNA genomes (especially of nonpolyadenylated RNA viruses), this unit should be valuable to all virologists working with nonpolyadenylated as well as polyadenylated viruses of plants and animals.

Keywords: infectious clone; in vitro transcription; ligation‐anchored PCR; RT‐PCR; positive strand RNA virus

     
 
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Table of Contents

  • Basic Protocol 1: Cloning Full‐Length Viral Genomes of Known Sequence
  • Determining the 5′ and 3′ Terminal Sequences of Unknown or Partially Known Viral Genomes
  • Support Protocol 1: Determination of 3′ End Sequence
  • Support Protocol 2: Determination of 5′ End Sequence
  • Support Protocol 3: Gradient Block PCR for Optimization of Annealing Temperatures
  • Support Protocol 4: Gel Purification of PCR Products
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Cloning Full‐Length Viral Genomes of Known Sequence

  Materials
  • Downstream primer (25 pmol/µl in nuclease‐free water)
  • Purified viral RNA
  • Nuclease‐free water, sterile (Ambion)
  • 10 mM dNTP mixture: 10 mM each of dATP, dCTP, dGTP, dTTP (also see appendix 2A)
  • Zero Blunt TOPO PCR Cloning Kit (Invitrogen, no. K2800‐20) containing:
    • 12.5 mM dNTP mixture
    • Salt solution
    • TOPO Vector: pCR‐Blunt II‐TOPO
    • SOC medium (also see appendix 4A)
    • One Shot TOP10 Chemically Competent E. coli
  • 5× cDNA synthesis buffer (supplied by Invitrogen with Thermoscript reverse transcriptase)
  • 0.1 M dithiothreitol (DTT; appendix 2A)
  • RNase inhibitor: RNasin (Promega) or equivalent
  • Thermoscript reverse transcriptase (Invitrogen)
  • 10× PCR buffer (supplied by Invitrogen with Platinum Pfx DNA polymerase)
  • 50 mM MgSO 4
  • 2.5 mM dNTP mixture: 2.5 mM each of dATP, dCTP, dGTP, dTTP (also see appendix 2A)
  • Upstream primer (25 pmol/µl in nuclease‐free water)
  • Thermostable DNA polymerase: Platinum Pfx (see Table 16.3.1)
  • QIAquick Gel Extraction Kit (Qiagen)
  • LB plates containing selective antibiotics ( appendix 4A)
  • 0.2‐, 0.5‐, or 1.5‐ml microcentrifuge tubes, sterile and nuclease free
  • Water bath or thermal cycler
  • 0.5‐ml or 0.2‐ml thin‐walled PCR tubes
  • −80°C freezer
  • Thermal cycler
  • Additional reagents and equipment for agarose gel electrophoresis (Voytas, ) and isolating DNA from an agarose gel (Moore et al., , optional)
    Table 6.0.1   MaterialsCommercial Proof‐Reading DNA Polymerases

    Polymerase Comments a Supplier
    Vent polymerase Requires optimization for each template New England Biolabs
    Deep Vent polymerase New England Biolabs
    Pfu polymerase Promega
    Tli polymerase Promega
    Pfu polymerase Very high fidelity, but low yield Stratagene
    Pfu Turbo polymerase Stratagene
    rTth polymerase XL Very high fidelity and yield; setup is complicated Applied Biosystems
    Pwo polymerase Roche
    ExTaq polymerase Not as high fidelity as others Panvera
    LA Taq polymerase Panvera
    KOD HiFi polymerase Novagen
    KOD XL polymerase Novagen
    Platinum Pfx polymerase High fidelity and yield on diverse templates; built‐in hot‐start Invitrogen
    Platinum Taq polymerase High Fidelity Invitrogen
    Elongase Amplification System Complicated to use Invitrogen
    Pfu polymerase Fermentas

     aComments indicate the authors' experience with selected polymerases, under specific conditions. In some cases the authors did not attempt to optimize the reactions, thus the properties indicated may not apply in all conditions.

Support Protocol 1: Determination of 3′ End Sequence

  • 10× RNA ligation buffer (supplied by Ambion with T4 RNA Ligase)
  • Anchor oligo
  • T4 RNA Ligase (Ambion)
  • 17°C incubator

Support Protocol 2: Determination of 5′ End Sequence

  • 1.5 M sodium hydroxide
  • 10× RNA ligation buffer (supplied by Ambion with T4 RNA Ligase)
  • T4 RNA Ligase (Ambion)
  • Boiling water bath
  • YM‐30 column (Amicon)
  • 17°C incubator

Support Protocol 3: Gradient Block PCR for Optimization of Annealing Temperatures

  • 0.2‐ml tubes in a 12‐tube strip (USA Scientific)
  • Gradient PCR block and compatible PCR base unit (e.g., Bio‐Rad)

Support Protocol 4: Gel Purification of PCR Products

  • 0.8% low‐melting‐point agarose gel (Bio‐Rad; also see Voytas, )
  • Buffered phenol ( appendix 2A)
  • 25:24:1 (v:v:v) phenol/chloroform/isoamyl alcohol ( appendix 2A)
  • 3 M sodium acetate, pH 5.5
  • 70% and 95% ethanol, −20°C
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Figures

Videos

Literature Cited

Literature Cited
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