Comparative Genomic Hybridization (CGH)—Detection of Unbalanced Genetic Aberrations Using Conventional and Micro‐Array Techniques

Evelin Schröck1, Zoë Weaver2, Donna Albertson3

1 Institute of Genetic Medicine, Charité, Berlin, 2 National Cancer Institute (NCI/NIH), Bethesda, Maryland, 3 University of California, San Francisco, California
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 8.12
DOI:  10.1002/0471142956.cy0812s18
Online Posting Date:  November, 2001
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Abstract

This unit presents comparative genomic hybridization (CGH), a genome‐wide screening technique for genetic aberrations in tumor samples. Specific emphasis is placed on recent applications to the analysis of murine model systems for human cancer. CGH is an invaluable tool for identifying the characteristic genetic rearrangements in these models. The authors discuss an exciting new method currently being developed, array CGH, which results in a tremendous increase in resolution. Oncogene amplifications and deletions of tumor‐suppressor genes are detected on a single‐gene level. Detailed protocols are supplied for CGH analysis of both human and mouse chromosomes. Keywords: comparative genomic hybridization; tumor genetics; unbalanced chromosomal aberrations; DNA copy number changes; gene amplification; oncogenes; tumor suppressor genes; mouse models of human cancer; array technology; array‐CGH This unit presents comparative genomic hybridization (CGH), a genome‐wide screening technique for genetic aberrations in tumor

     
 
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Table of Contents

  • CGH on Human Chromosomes and Mouse Chromosomes
  • Basic Protocol 1: Tumor DNA Preparation from Frozen Tissue
  • Alternate Protocol 1: Tumor DNA Preparation from Paraffin‐Embedded Tissue
  • Basic Protocol 2: Preparation of Control DNA from Peripheral Blood
  • Basic Protocol 3: Preparation of Normal Target Metaphase Chromosomes
  • Alternate Protocol 2: Preparation of Normal Target Chromosomes from Mouse Spleen
  • Basic Protocol 4: Nick Translation of Tumor and Control DNA for Both Human and Mouse Chromosomes
  • Alternate Protocol 3: Mouse Chromosome Identification Probes
  • Basic Protocol 5: Pretreatment of Target Chromosome Slides for Both Human and Mouse Chromosomes
  • Basic Protocol 6: Fluorescence In Situ Hybridization for Both Human and Mouse Chromosomes
  • Basic Protocol 7: Detection of Hybridized DNA Sequences for Both Human and Mouse Chromosomes
  • Basic Protocol 8: CGH Image Acquisition
  • Basic Protocol 9: Image Analysis
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Tumor DNA Preparation from Frozen Tissue

  Materials
  • Frozen tissue sample(s) of interest
  • Culture medium (e.g., RPMI 1640)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • DNA buffer I (see recipe)
  • 20 mg/ml proteinase K
  • 10% (w/v) sodium dodecyl sulfate (SDS)
  • Phenol
  • 24:1 (v/v) chloroform/isoamyl alcohol
  • 3 M sodium acetate, pH 5.2 (see recipe in unit 8.3)
  • 100% and 70% ethanol
  • Petri dishes
  • 15‐ml centrifuge tubes, sterile and phenol‐safe
  • 55°C water bath
  • 1.5‐ml microcentrifuge tubes (e.g., Eppendorf)
  • Speedvac evaporator (Savant)
  • Rotating shaker
  • Additional reagents and equipment for assessing DNA concentration (unit 4.5) and running a 1% agarose gel (unit 8.3)

Alternate Protocol 1: Tumor DNA Preparation from Paraffin‐Embedded Tissue

  • Formalin‐fixed, paraffin‐embedded tissue sample(s) of interest
  • Xylene
  • 100% methanol
  • 1 M sodium thiocyanate
  • DNA buffer II (see recipe)
  • 100 µg/ml RNase A in 2× SSC (optional; see recipe for 20 mg/ml RNase A stock in Reagents and Solutions; see appendix 2A for 20× SSC)
  • Isopropanol

Basic Protocol 2: Preparation of Control DNA from Peripheral Blood

  Materials
  • Normal whole blood
  • Lysis buffer (see recipe)
  • SE buffer (see recipe)
  • 20 mg/ml proteinase K
  • 20% (w/v) SDS
  • Phenol
  • 24:1 (v/v) chloroform/isoamyl alcohol
  • 3 M sodium acetate, pH 5.2 (see recipe in unit 8.3)
  • Isopropanol
  • 70% ethanol
  • 50‐ml blood collection tube containing EDTA, heparin, or sodium citrate anticoagulant
  • 37°C water bath
  • 1.5‐ml microcentrifuge tubes (e.g., Eppendorf)
  • Speedvac evaporator (Savant)
  • Additional materials and equipment for assessment of DNA concentration (unit 4.5) and 1% agarose gel electrophoresis (unit 8.3)

Basic Protocol 3: Preparation of Normal Target Metaphase Chromosomes

  Materials
  • RPMI 1640 medium (Life Technologies)
  • 100× antibiotic‐antimycotic: 10,000 U/ml penicillin G sodium, 10,000 µg/ml streptomycin sulfate, 25 µg/ml amphotericin B (Life Technologies)
  • Fetal bovine serum (FBS): qualified, heat‐inactivated, sterile‐filtered (Life Technologies)
  • Phytohemagglutinin (PHA; Murex Diagnostics Ltd.)
  • Normal whole blood (heparin anticoagulated)
  • 10 µg/ml KaryoMAX colcemid solution (Life Technologies)
  • 0.4% (w/v) KCl, 37°C
  • 3:1 (v/v) methanol/glacial acetic acid fixative, freshly prepared
  • 1:1 (v/v) ethanol/ether
  • 70%, 90%, and 100% ethanol
  • 75‐cm2 tissue culture flasks
  • 50‐ml and 15‐ml centrifuge tubes
  • 37°C and 60°C water bath
  • Microscope slides
NOTE: All incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Alternate Protocol 2: Preparation of Normal Target Chromosomes from Mouse Spleen

  Materials
  • RPMI 1640 medium
  • 100× antibiotic‐antimycotic: 10,000 U/ml penicillin G sodium, 10,000 µg/ml streptomycin sulfate, 25 µg/ml amphotericin B (Life Technologies)
  • Fetal bovine serum qualified, heat‐inactivated, sterile‐filtered (FBS; Life Technologies)
  • 0.4% (v/v) KCl, 37°C
  • Mouse spleen
  • 1 mg/ml concanavalin A (Sigma; see recipe)
  • 25 mg/ml lipopolysaccharide (LPS; see recipe)
  • 0.5% 2‐mercaptoethanol
  • 10 mg/ml 5‐bromo‐2′‐deoxyuridine (BrdU; see recipe)
  • 0.1 mg/ml 5‐fluoro‐2′‐deoxyuridine (FUdR; see recipe)
  • 10 µg/ml KaryoMAX colcemid solution (Life Technologies)
  • Fixative solution: 3:1 (v/v) methanol/glacial acetic acid, freshly prepared
  • Mouse spleen homogenizer
  • 15‐ml conical centrifuge tube
  • 125‐cm2 tissue culture flasks
  • 37°C, 5% CO 2 incubator
  • 37°C water bath
  • 3‐ml plastic transfer pipet

Basic Protocol 4: Nick Translation of Tumor and Control DNA for Both Human and Mouse Chromosomes

  Materials
  • 1 mg/ml DNase I from bovine pancreas (see recipe)
  • Genomic DNA
  • 10× NT buffer (see recipe)
  • 10× dNTP mix (see recipe)
  • 0.1 M 2‐mercaptoethanol (see recipe)
  • Biotin‐16‐dUTP (Roche Diagnostics)
  • Digoxigenin‐11‐dUTP (Roche Diagnostics)
  • Kornberg polymerase (Roche Diagnostics)
  • Lambda HindIII DNA marker
  • 0.5 M EDTA
  • 1.5‐ml microcentrifuge tube (e.g., Eppendorf)
  • Additional reagents and equipment for DNA concentration and 1% agarose gel electrophoresis (unit 8.3)

Alternate Protocol 3: Mouse Chromosome Identification Probes

  Materials
  • Metaphase chromosome preparations (see protocol 4)
  • 2× SSC (see appendix 2A for 20× recipe)
  • 20 mg/ml RNase A stock (see recipe)
  • 10% pepsin (see recipe)
  • 0.01 M HCl, pH 2.0, 37°C
  • PBS ( appendix 2A)
  • 1× PBS/MgCl 2 (see recipe)
  • Formalin (37% formaldehyde)
  • 24 × 60–mm coverslip
  • 37°C incubator
  • Coplin jars
  • 37°C water bath

Basic Protocol 5: Pretreatment of Target Chromosome Slides for Both Human and Mouse Chromosomes

  Materials
  • 500 ng tumor DNA labeled with biotin via nick translation (see protocol 6)
  • 500 ng control DNA labeled with digoxigenin via nick translation (see protocol 6)
  • 1 mg/ml human Cot‐1 DNA (Life Technologies)
  • 10 mg/ml salmon testes DNA (Sigma)
  • 3 M sodium acetate, pH 5.2 (unit 8.3)
  • 100%, 90%, and 70% ethanol, ice cold and room temperature
  • Hybridization mixture (see recipe)
  • Probe DNA
  • 70% formamide/2× SSC (see recipe)
  • Centromere enumeration probe (see )
  • Rubber cement
  • 1.5‐ml microcentrifuge tubes (Eppendorf)
  • Speedvac evaporator (Savant)
  • 24 × 60–mm coverslips
  • 18 × 18–mm coverslips

Basic Protocol 6: Fluorescence In Situ Hybridization for Both Human and Mouse Chromosomes

  Materials
  • Hybridized slides (see protocol 9)
  • 50% formamide/2× SSC solution (see recipe), prewarmed
  • 0.1× SSC (see appendix 2A for 20× recipe)
  • 4× SSC/Tween 20 (see recipe)
  • Blocking solution (see recipe)
  • Antibodies/fluorescent dye solution 1 (see recipe)
  • Antibodies/fluorescent dye solution 2 (see recipe)
  • Antibodies/fluorescent dye solution 3 (see recipe)
  • DAPI working solution (see recipe)
  • 70%, 90%, and 100% ethanol
  • Antifade: 1,4‐phenylenediamine (see recipe)
  • Coplin jars
  • Hybridization chamber
  • 37°C water bath
  • 24 × 60–mm coverslips
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Figures

Videos

Literature Cited

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Key References
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