Analysis of Mitochondrial DNA Point Mutation Heteroplasmy by ARMS Quantitative PCR

Jing Wang1, Victor Venegas1, Fangyuan Li1, Lee‐Jun Wong1

1 Baylor College of Medicine, Houston, Texas
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 19.6
DOI:  10.1002/0471142905.hg1906s68
Online Posting Date:  January, 2011
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Abstract

Mitochondrial disorders are a group of complex and heterogeneous diseases that may be caused by molecular defects in both the nuclear and mitochondrial genomes. Pathogenic mitochondrial DNA (mtDNA) mutations are usually present in the heteroplasmic form. The degree of mtDNA mutation heteroplasmy (mutation load) varies among different tissues. Manifestation of clinical phenotypes depends on the mutation load in affected tissues. Thus, it is important to quantify the degree of mutation heteroplasmy in various tissues. This unit outlines the design of allele refractory mutation system (ARMS)‐based quantitative PCR (qPCR) analysis of three common mtDNA point mutations. This is a cost‐effective and sensitive single‐step method to simultaneously detect and quantify heteroplasmic mtDNA point mutations. It requires as little as 0.1 ng of total genomic DNA per reaction and can be used to quantify mutation heteroplasmy in various noninvasively obtained tissues such as hair follicles, buccal swab, and urine sediment. Detailed protocols for ARMS primer design and qPCR set up, validation procedures, and cautions in the interpretation of results, as well as advantages and limitations are discussed. ARMS qPCR is an important tool for addressing some of the diagnostic challenges of mitochondrial disease. Curr. Protoc. Hum. Genet. 68:19.6.1‐19.6.16 © 2011 by John Wiley & Sons, Inc.

Keywords: mitochondrial DNA point mutations; mtDNA; ARMS qPCR; mtDNA heteroplasmy; mtDNA mutations; quantitative PCR

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: ARMS Real‐Time qPCR for the Quantification of mtDNA Point Mutation Heteroplasmy
  • Basic Protocol 2: Assessing PCR Efficiency and Optimizing DNA Template Concentration
  • Basic Protocol 3: Establishment of the Heteroplasmic Mixtures and the Calculation of the Level of Heteroplasmy
  • Support Protocol 1: Generation of Wild‐Type and Mutant Controls
  • Support Protocol 2: Preparation and Quantification of DNA Samples
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: ARMS Real‐Time qPCR for the Quantification of mtDNA Point Mutation Heteroplasmy

  Materials
  • Clinical sample genomic DNA (see protocol 5)
  • DNA controls:
    • 2 × 10–2 to 6.2 × 10–6 ng/µl (based on dilution curves) of 100% wild‐type and 100% mutant plasmid DNA (see protocol 4)
    • Control DNA sample with known percentage of mutation heteroplasmy (previously tested, if available)
  • ARMS primers for wild‐type and mutant alleles (see Table 19.6.2) diluted in water for a working concentration of 5 µM
  • iTaq SYBR Green Supermix with ROX (Bio‐Rad, cat. no. 170‐8852)
  • PCR hood
  • 96‐well thin‐walled PCR plates
  • Adhesive plate sealer
  • ABI Prism 7900HT sequence detector system (Applied Biosystems)
  • SDS data analysis software (version 2.2)

Basic Protocol 2: Assessing PCR Efficiency and Optimizing DNA Template Concentration

  Materials
  • Wild‐type or control plasmid DNA template (see protocol 4)

Basic Protocol 3: Establishment of the Heteroplasmic Mixtures and the Calculation of the Level of Heteroplasmy

  Materials
  • Wild‐type and mutant control DNA
  • 96‐well plates

Support Protocol 1: Generation of Wild‐Type and Mutant Controls

  Materials
  • Target DNA
  • ARMS‐qPCR primers (Table 19.6.2)
  • pCR2.1‐TOPO plasmid (Invitrogen, cat. no. K4520‐01)
  • Qiagen plasmid mini kit (Qiagen, cat. no. 12123)

Support Protocol 2: Preparation and Quantification of DNA Samples

  Materials
  • Various tissue samples
  • Puregene reagents kit (Gentra Systems, cat. no. D50K1) containing:
    • Red cell lysis solution
    • Cell lysis solution
    • DNA hydration solution
    • Protein precipitation solution
  • Rapid hair digestion buffer: 10 mM Tris⋅Cl (pH 8.0)/1.0% Brij 58/35 mM DTT/1 mM CaCl 2
  • 100% isopropanol
  • 70% ethanol
  • Calf thymus DNA standards (type XV; Sigma, cat. no. D4522)
  • Centrifuge
  • NanoDrop ND‐1000 UV‐Vis spectrophotometer
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Figures

Videos

Literature Cited

Literature Cited
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