Molecular Analysis of Gene Amplification in Tumors

Jonathon C. Wasson1, Garrett M. Brodeur1

1 Washington University School of Medicine, St. Louis, Missouri
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 10.5
DOI:  10.1002/0471142905.hg1005s02
Online Posting Date:  May, 2001
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Abstract

Detection of gene amplification in human cancer cells has both clinical and biological importance. Amplified genes can be classified in one of two categoriesas oncogenes or as genes conferring drug resistance. Both types of gene amplification may alter clinical management of the patient. The basic protocol describes preparation and quantitation of DNA from tumor tissue and the use of conventional Southern blot hybridization analysis to detect and quantify gene amplification. The first alternate protocol provides an approach to quickly screen tumor samples for gene amplification using slot blot hybridization analysis. The second alternate protocol describes the use of the polymerase chain reaction (PCR) for analyzing tumors that may be difficult to analyze because of degradation or limited amounts of DNA. A explains the proper methods for obtaining, processing, storing, and shipping tumor tissue for DNA analysis.

     
 
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Table of Contents

  • Basic Protocol 1: Detection of Gene Amplification by Southern Blot Hybridization Analysis
  • Alternate Protocol 1: Detection of Gene Amplification by Slot Blot Hybridization Analysis
  • Alternate Protocol 2: Detection of Gene Amplification by Differential PCR
  • Support Protocol 1: Obtaining and Processing Tumor Tissue
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Detection of Gene Amplification by Southern Blot Hybridization Analysis

  Materials
    For recipes, see Reagents and Solutions in this unit (or cross‐referenced unit); for common stock solutions, see appendix 2A; for suppliers, see suppliers appendix.
  • Tumor tissue ( protocol 2)
  • Stratagene DNA Extraction Kit (Stratagene) or equivalent:
DNA extraction solution: 50 mM Tris. Cl (pH 8.0)/20 mM EDTA (pH 8.0)/2% (w/v) SDS
  • 225 mg/ml pronase
  • NaCl extraction solution: saturated NaCl solution
  • 10 mg/ml RNase
  • 100% and 70% ethanol, ice cold
  • TE buffer, pH 8.0
  • recipe10, 100, 250, and 500 µg/ml calf thymus DNA standards (see recipe)
  • recipeCapillary assay solution (see recipe), prepared just before use
  • recipe1× TNE buffer (see recipe)
  • recipe1 M spermidine (see recipe)
  • Single‐copy control DNA
  • Amplified control DNA
  • 10 U/µl restriction enzyme and appropriate 10× buffer
  • 1 mg/ml bovine serum albumin (BSA)
  • 4 M NaCl
  • 6× gel loading buffer ( appendix 2A)
  • Agarose
  • 1× TBE buffer ( appendix 2A)
  • 10 mg/ml ethidium bromide
  • Denaturing solution: 0.5 NaOH/1 M NaCl, prepared fresh and equilibrated 30 min
  • recipeNeutralizing solution (see recipe), prepared fresh
  • 6× SSC ( appendix 2A)
  • Quik‐Hyb solution (Stratagene)
  • recipe10 mg/ml herring sperm DNA, sonicated and boiled (see recipe)
Double‐stranded DNA probe, labeled with [α‐32P]dCTP by random oligonucleotide priming to a specific activity ≥1 × 109 cpm/µg DNA and spin‐column‐purified ( appendix 3E)
  • Wash solution I: 2× SSC/0.2% (w/v) SDS, prepared fresh, 55°C
  • Wash solution II: 0.5× SSC/0.2% (w/v) SDS, prepared fresh, 55°C
  • Wash solution III: 0.2×SSC/0.2% (w/v) SDS, prepared fresh, 55°C
  • 7.5‐ml ground‐glass tissue homogenizer or equivalent
  • 30‐ and 50‐ml centrifuge tubes
  • Sorvall centrifuge and JS‐5.2 rotor (or equivalent)
  • Large‐bore pipets
  • Vacuum pump apparatus (creating ≥30 mm Hg) or Speedvac
  • 2‐ or 5‐ml O‐ring screw‐cap tubes
  • Rocker platform
  • Mini‐fluorometer (Hoefer)
  • 0.5‐ and 1.5‐ml microcentrifuge tubes
  • Nitrocellulose membrane
  • 80°C vacuum oven or UV cross‐linker (e.g., Stratalinker, Stratagene)
  • Heat‐sealable polyethylene bags
  • 65°C shaking water bath
  • X‐AR autoradiographic film
  • Additional reagents and equipment for agarose gel electrophoresis and Southern blotting (unit 2.7)

Alternate Protocol 1: Detection of Gene Amplification by Slot Blot Hybridization Analysis

  Additional MaterialsFor recipes, see Reagents and Solutions in this unit (or cross‐referenced unit); for common stock solutions, see appendix 2A; for suppliers, see suppliers appendix.
  • 3.0 M NaOH, filter sterilized
  • 2.0 M ammonium acetate, pH 7.0, filter sterilized
  • Repeating pipettor (Brinkmann)
  • Dot or slot blotting manifold (e.g., Bio‐Dot SF, Bio‐Rad; or Minifold 2, Schleicher & Schuell)
  • Whatman 3MM paper

Alternate Protocol 2: Detection of Gene Amplification by Differential PCR

  Additional Materials
    For recipes, see Reagents and Solutions in this unit (or cross‐referenced unit); for common stock solutions, see appendix 2D; for suppliers, see suppliers appendix.
  • 10× Taq DNA polymerase buffer: 500 mM KCl/100 mM Tris‐Cl, pH 8.3 (Store ≤18 months at −20°C)
  • 25 mM MgCl 2
  • 1.25 mM 4dNTP mix ( appendix 2A)
  • 20 µM target and control oligonucleotide primers, + and − strand sequences of each
  • 5 U/µl Taq DNA polymerase
  • Mineral oil (not needed if thermal cycler has a heated lid)
  • 2% (w/v) agarose in TBE buffer ( appendix 2A)
  • Low‐molecular‐weight DNA size markers (e.g., BioMarker Low, Bioventures)
  • 0.5‐ml microcentrifuge tube
  • Automated thermal cycler
  • Additional reagents and equipment for agarose gel electrophoresis (unit 2.7)

Support Protocol 1: Obtaining and Processing Tumor Tissue

  Additional Materials
    For recipes, see Reagents and Solutions in this unit (or cross‐referenced unit); for common stock solutions, see appendix 2A; for suppliers, see suppliers appendix.
  • Tumor tissue
  • Liquid N 2
  • Cryotubes
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Figures

Videos

Literature Cited

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Key Reference
   Kellems, R.E., ed. 1993. Gene Amplification in Mammalian Cells. Marcel Dekker, New York.
  A recent, comprehensive review of gene amplification in mammalian cells, including amplification of oncogenes and drug resistance genes.
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