PCR‐Based Analysis of Mitochondrial DNA Copy Number, Mitochondrial DNA Damage, and Nuclear DNA Damage

Claudia P. Gonzalez‐Hunt1, John P. Rooney1, Ian T. Ryde1, Charumathi Anbalagan1, Rashmi Joglekar1, Joel N. Meyer1

1 Nicholas School of the Environment, Duke University, Durham, North Carolina
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 20.11
DOI:  10.1002/0471140856.tx2011s67
Online Posting Date:  February, 2016
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Abstract

Because of the role that DNA damage and depletion play in human disease, it is important to develop and improve tools to assess these endpoints. This unit describes PCR‐based methods to measure nuclear and mitochondrial DNA damage and copy number. Long amplicon quantitative polymerase chain reaction (LA‐QPCR) is used to detect DNA damage by measuring the number of polymerase‐inhibiting lesions present based on the amount of PCR amplification; real‐time PCR (RT‐PCR) is used to calculate genome content. In this unit, we provide step‐by‐step instructions to perform these assays in Homo sapiens, Mus musculus, Rattus norvegicus, Caenorhabditis elegans, Drosophila melanogaster, Danio rerio, Oryzias latipes, Fundulus grandis, and Fundulus heteroclitus, and discuss the advantages and disadvantages of these assays. © 2016 by John Wiley & Sons, Inc.

Keywords: qPCR; DNA damage; mitochondrial DNA copy number; mitochondrial DNA; C. elegans

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Analysis of Mitochondrial and Nuclear DNA Damage
  • Basic Protocol 2: Analysis of Mitchondrial and Nuclear Genome Copy Number
  • Support Protocol 1: DNA Template Extraction from C. elegans (Small Number of Worms)
  • Support Protocol 2: DNA Template Extraction from C. elegans (Large Number of Worms) or Animal Tissue
  • Support Protocol 3: DNA Quantification
  • Support Protocol 4: Linearization of Mitochondrial DNA Following Automated DNA Extraction
  • Support Protocol 5: Generating Standard Curve Plasmids for Mitchondrial and Nuclear Genome Copy Number Analysis
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Analysis of Mitochondrial and Nuclear DNA Damage

  Materials
  • Nuclease‐free H 2O
  • LongAmp Hot Start Taq 2× Master Mix (New England Biolabs)
  • 10 μM primers (see appendix 3C; Kramer and Coen, ), diluted in 0.1× TE buffer [1 mM Tris·Cl, pH 8 ( appendix 2A)/0.1 mM disodium EDTA]
  • Template DNA, purified
  • 0.2‐ml PCR tubes
  • Microcentrifuge and PCR‐tube minicentrifuge
  • 96‐well format thermal cycler
  • Dedicated workstation (e.g., PCR hood equipped with UV lamp for sterilization)
  • Additional reagents and equipment for the polymerase chain reaction ( appendix 3C; Kramer and Coen, ) and quantification of PCR products ( protocol 5)

Basic Protocol 2: Analysis of Mitchondrial and Nuclear Genome Copy Number

  Materials
  • Nuclease‐free H 2O
  • 100,000 copies/μl aliquots of pCR 2.1 plasmid containing cloned species‐specific nuclear or mitochondrial gene – if calculating with standard curve (refer to protocol 7)
  • Power SYBR Green PCR Master Mix (Life Technologies)10 μM primers (see appendix 3C; Kramer and Coen, ), diluted in 0.1× TE buffer [1 mM Tris·Cl, pH 8 ( appendix 2A)/0.1 mM disodium EDTA]
  • Template DNA, purified, or 40 μl young adult (24 hr post‐L4) glp‐1 worm lysate (20 worms; 1567 copies/μl; alternative to C. elegans nuclear plasmid for standard curve calculations; refer to protocol 3)
  • Sterile, aerosol filter tips and pipettors dedicated to LA‐QPCR
  • Optical 96‐well PCR plate and optical adhesive film
  • Plate vortexer
  • Centrifuge
  • Real‐time PCR system

Support Protocol 1: DNA Template Extraction from C. elegans (Small Number of Worms)

  Materials
  • Worm lysis buffer (see recipe)
  • Worms of interest
  • Platinum wire worm pick
  • 0.2‐ml PCR tubes
  • Ice or cryogenic 96‐well plate (PCR cooler)
  • −80°C freezer
  • 96‐well format thermal cycler or heat block

Support Protocol 2: DNA Template Extraction from C. elegans (Large Number of Worms) or Animal Tissue

  Materials
  • Worms or tissue of interest
  • K medium (see recipe)
  • 20% glycerol solution (for animal tissues)
  • Liquid nitrogen
  • Genomic‐tip 20/G kit (Qiagen) including:
    • Buffer G2
    • Buffer QBT
    • Buffer QC
    • Buffer QF
    • RNase A
    • Proteinase K
    • Isopropanol
    • 70% ethanol
  • 15‐ml screw‐cap conical tubes
  • Orbital shaker
  • Mortar and pestle
  • 1‐ to 2‐ml cryotubes
  • Handheld homogenizer (for softer animal tissue)
  • Spatula, sterile

Support Protocol 3: DNA Quantification

  Materials
  • λ DNA/Hind III Fragments (Invitrogen)
  • 1× TE buffer: 10 mM Tris·Cl, pH 8.0 ( appendix 2A)/1 mM EDTA
  • 0.1× TE buffer 1 mM Tris·Cl, pH 8.0 ( appendix 2A)/0.1 mM EDTA
  • Extracted DNA samples
  • 50‐μl aliquots (stored at −20°C) of Quant‐iT PicoGreen dsDNA reagent (Molecular Probes)
  • 96‐well white‐ or black‐bottomed plates
  • Fluorescence plate reader capable of measuring 485 nm excitation and 528 nm emission

Support Protocol 4: Linearization of Mitochondrial DNA Following Automated DNA Extraction

  Materials
  • 225 ng purified DNA
  • Nuclease‐free H 2O
  • Restriction enzyme (HaeII for human, PvuII for mouse, and XhoI for rat, and corresponding CutSmart or NEBuffer buffer; New England Biolabs)
  • 0.2‐ml PCR tubes
  • 96‐well format thermal cycler

Support Protocol 5: Generating Standard Curve Plasmids for Mitchondrial and Nuclear Genome Copy Number Analysis

  Materials
  • Genomic DNA ( protocol 4)
  • Nuclease‐free H 2O
  • Taq DNA polymerase with standard Taq buffer
  • dNTP mix (2.5 mM of each nucleotide)
  • Magnesium chloride (MgCl 2)
  • Forward and reverse primers (see Table 20.11.4; also see appendix 3C; Kramer and Coen, )
  • QIAquick PCR Purification Kit (Qiagen)
  • pCR2.1 plasmid DNA (bacterial stocks available from us upon request)
  • Restriction enzymes (HindIII and XhoI and associated NEBuffer; New England Biolabs)
  • Gel extraction kit (available from various molecular biology suppliers)
  • T4 DNA ligase and associated buffer (New England Biolabs)
  • Heat shock–competent E. coli (Any recAcloning strain)
  • SOC medium (see recipe)
  • LB agar plates containing 100 μg/ml ampicillin (see recipe)
  • LB broth containing 50 μg/ml ampicillin or 50 μg/ml kanamycin (see recipe)
  • Sterile, aerosol filter tips and pipets dedicated to PCR
  • NanoDrop2000 UV‐Vis Spectrophotometer
  • 0.2‐ml PCR tubes
  • Thermal cycler with heated lid
  • Heat block
  • Additional reagents and equipment for the polymerase chain reaction (PCR; appendix 3C; Kramer and Coen, ), agarose gel electrophoresis (Voytas, )
Table 0.1.4   MaterialsCloning PCR Primers and Conditions

Species Genome Target gene Forward primer seq. a Reverse primer seq. a Amplicon (bp) Annealing temp. (°C) Reference
C. elegans mt nd‐1 NR NR NR NR Bratic et al. ( )
nuc cox‐4 5′‐ATCT AAGCTT GCC GAC TGG AAG AAC TTG TC‐3′ 5′‐ATAG CTCGAG GCG GAG ATC ACC TTC CAG TA‐3′ 184 60 This unit
H. sapiens mt tRNA‐Leu(UUR) 5′‐ATCTAAGCTT GCC TTC CCC CGT AAA TGA TA‐3′ 5′‐ATAG CTCGAG AGG AAT GCC ATT GCG ATT AG‐3′ 215 55 This unit
nuc B2M 5′‐ATCT AAGCTT TGC TGT CTC CAT GTT TGA TGT ATC T‐3′ 5′‐ATAG CTCGAG TCT CTG CTC CCC ACC TCT AAG T‐3′ 106 62 This unit
F. heteroclitus mt 16S rRNA 5′‐ATAG AAGCTT AAA ATT AAC GGC CCC AAC CC‐3′ 5′‐ACTT CTCGAG CCG AGT TCC TTC TTC CCC TT‐3′ 131 64 This unit
nuc CFTR 5′‐ATAG AAGCTT GCC GCT GCC TTC ATT GCT GT‐3′ 5′‐AATT CTCGAG ATG AGC TGG GTG TGC GCT GA‐3′ 234 65 Hunter et al. ( )
D. rerio mt nd‐1 5′‐ATCT AAGCTT CGT TTA CCC CAG ATG CAC CT‐3′ 5′‐ATAG CTCGAG GTG CGA TTG GTA GGG CGA TA‐3′ 215 60 This unit
nuc vtg2 5′‐ATCT AAGCTT TGG ATA CCT GAC CGA GAG CT‐3′ 5′‐ATAG CTCGAG AGA CAA CTC TTA CGG CTG GC‐3′ 110 60 This unit

 aUnderlined regions denote restriction sites.
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Figures

Videos

Literature Cited

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