Comparative Genomic Hybridization

Jane Bayani1, Jeremy A. Squire1

1 Princess Margaret Hospital and The Ontario Cancer Institute University of Toronto, Toronto, Ontario
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 22.6
DOI:  10.1002/0471143030.cb2206s25
Online Posting Date:  January, 2005
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Abstract

Comparative genomic hybridization (CGH) is a screening method based on fluorescence in situ hybridization (FISH). In contrast to conventional FISH, the metaphase target is derived from a normal peripheral blood lymphocyte culture. This target is hybridized to the test or tumor DNA, which is labeled/detected by one fluorochrome (i.e., green), and to an equal amount of labeled normal or reference DNA, which is labeled/detected by a different fluorochrome (red). It is the difference in these green/red ratios (determined by specialized software) along the length of each karyotyped chromosome that indicates the relative copy number changes in the test/tumor DNA. The basic FISH techniques reviewed in this section, the parameters for which also apply to obtaining satisfactory results for CGH, include cytogenetic preparation and slide‐making, DNA extraction (from fresh or paraffin‐embedded tissues) and labeling, slide pretreatment, hybridization, post‐hybridization washes, and detection.

Keywords: comparative genomic hybridization; CGH; FISH

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Comparative Genomic Hybridization
  • Support Protocol 1: Extraction of DNA from Fresh/Frozen Tissues or Cell Lines
  • Support Protocol 2: DNA Extraction from Paraffin Section
  • Support Protocol 3: Labeling DNA Extracted from Fresh Tissues for CGH
  • Support Protocol 4: Labeling DNA Extracted from Paraffin‐Embedded Tissues for CGH
  • Support Protocol 5: Preparation of Normal Metaphase Slides: Pretreatment and Denaturation
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Comparative Genomic Hybridization

  Materials
  • 10 ng/µl tumor DNA: indirectly labeled with biotin or directly labeled with Spectrum Green, FITC, or other green‐fluorescing dye (see unit 22.4 and Support Protocols in this unit) in hybridization buffer (see unit 22.4 for buffer)
  • 10 ng/µl normal reference DNA: indirectly labeled with digoxigenin or directly labeled with Spectrum Orange, rhodamine, or other red‐fluorescing dye (see unit 22.4 and Support Protocols in this unit) in hybridization buffer (see unit 22.4 for buffer)
  • Pretreated and denatured male metaphase slides (unit 22.4)
  • Rubber cement
  • 50% (v/v) formamide/2× SSC (see unit 18.6 for 20× SSC), 45°C
  • 2× SSC (unit 18.6), 45°C
  • Blocking solution: 1% BSA (w/v)/0.1% (v/v) Tween 20 in 4× SSC (store indefinitely at –20°C or up to several months at 4°C)
  • FITC‐labeled avidin and rhodamine‐labeled anti‐digoxigenin antibody (unit 22.4; for detection of indirectly labeled probes only)
  • 0.1% (v/v) Tween‐20/4× SSC (see unit 18.6 for 20× SSC), 45°C
  • DAPI in antifade (unit 22.4)
  • 75°C water bath
  • 37°C dry incubator or oven
  • Coverslips
  • Hybridization box: e.g., black videocassette box containing slightly dampened moist paper towel or gauze
  • Coplin jars
  • Fluorescent microscope with appropriate filter sets (units 4.2& 22.4)
  • Image acquisition software (unit 4.2)
  • CGH analysis software (e.g., Leica, Applied Imaging, Metasystems)
  • Additional reagents and equipment for FISH (unit 22.4)

Support Protocol 1: Extraction of DNA from Fresh/Frozen Tissues or Cell Lines

  Materials
  • Tissue sample or cell pellet
  • TE buffer, pH 7.5 ( appendix 2A)
  • 10% (w/v) SDS
  • 14 mg/ml proteinase K
  • Molecular‐biology‐grade phenol, TE‐saturated (unit 18.3)
  • 1:1 (v/v) phenol/chloroform (see recipe)
  • Chloroform
  • 3 M sodium acetate ( appendix 2A)
  • 100% and 70% ethanol, cold
  • DNase‐free H 2O
  • Scalpels and scalpel blades
  • 15‐ml polypropylene conical centrifuge tubes
  • Centrifuge
  • Platform rotator

Support Protocol 2: DNA Extraction from Paraffin Section

  Materials
  • Paraffin sections
  • Xylene
  • Methanol
  • 1 M sodium thiocyanate, 55°C (prepare fresh)
  • DNA extraction buffer (see recipe), 55°C (prepare fresh)
  • Proteinase K
  • Microtome
  • Polypropylene tubes
  • Oven maintained at 55°C

Support Protocol 3: Labeling DNA Extracted from Fresh Tissues for CGH

  Materials
  • Isolated (microdissected) cells (20 to 200) or DNA extracted from paraffin section
  • 1× DOP‐PCR buffer (see recipe) containing 0.5 µg/µl proteinase K, freshly added
  • 10× DOP‐PCR buffer (see recipe)
  • 2 mM dNTP mix (see recipe)
  • 10× DOP primer (see recipe)
  • Taq DNA polymerase (Perkin‐Elmer)
  • TBE buffer ( appendix 2A)
  • 500 µl PCR reaction tube
  • 50° and 95°C ovens
  • Thermal cycler
  • Additional reagents and equipment for agarose gel electrophoresis (unit 6.7) and labeling probes by nick translation (unit 22.4)
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Figures

  •   FigureFigure 22.6.1 Schematic representation of CGH analysis. CGH analysis involves the labeling of normal reference DNA and the tumor/test DNA such that they can be identified by either green or red fluorescence (tumor and normal reference, respectively). Equal amounts of these labeled DNAs are hybridized and detected by standard in situ methods to normal metaphase spreads. The metaphase cells are then imaged and karyotyped. CGH analysis determines the change in green/red ratio along the length of each chromosome, corresponding to the net genomic change of DNA at a specific chromosomal locus in the tumor in comparison to the normal reference. The metaphase is imaged using three filters: a DAPI filter to identify chromosomes for karyotyping purposes (the blue image is converted into a pseudo G‐banded image), a green filter (FITC/Spectrum Green) to detect the tumor DNA, and a red filter (rhodamine/Spectrum Orange) to detect the normal DNA hybridized to the normal chromosomes. These images can be overlayed as shown. Areas of genomic gain or amplification are seen as fluorescing green, while areas of loss are seen as fluorescing red. Regions of no genomic change are seen as yellow/orange. The three‐color merged image (DAPI/FITC/rhodamine) is also displayed. Analysis of 7 to 10 metaphase spreads results in a CGH karyotype as shown at the bottom of the figure. The CGH karyotype of this tumor is as follows: +1q, amp 2p24‐qter, +3q,−4q,+5q33‐qter, +6p22‐qter,+7p13‐qter, +8q,amp9p,+10p,−13,+14q12‐qter,+16q23‐qter,−17p,+Xp11‐14, +Xq221‐22.
  •   FigureFigure 22.6.2 Examples of poor hybridization resulting in a speckled or patchy appearance of fluorescence across the metaphase. From left to right: DAPI‐stained metaphase, FITC‐detected tumor DNA, and rhodamine‐detected normal reference DNA.

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Literature Cited

Literature Cited
   Bayani, J., Brenton, J.D., Macgregor, P.F., Beheshti, B., Albert, M., Nallainathan, D., Karaskova, J., Rosen, B., Murphy, J., Laframboise, S., Zanke, B., and Squire, J.A. 2002. Parallel analysis of sporadic primary ovarian carcinomas by spectral karyotyping, comparative genomic hybridization, and expression microarrays. Cancer Res. 15:62:3466‐3476.
   Beatty, B., Mai, S., and Squire, J.A. 2002. FISH: A Practical Approach. Oxford University Press, New York.
   Crabtree, J.S., Scacheri, P.C., Ward, J.M., McNally, S.R., Swain, G.P., Montagna, C., Hager, J.H., Hanahan, D., Edlund, H., Magnuson, M.A., Garrett‐Beal, L., Burns, A.L., Ried, T., Chandrasekharappa, S.C., Marx, S.J., Spiegel, A.M., and Collins, F.S. 2003. Of mice and MEN1: Insulinomas in a conditional mouse knockout. Mol. Cell. Biol. 23:6075‐6085.
   du Manoir, S., Schrock, E., Bentz, M., Speicher, M.R., Joos, S., Ried, T., Lichter, P., and Cremer, T. 1995a. Quantitative analysis of comparative genomic hybridization. Cytometry. 19:27‐41.
   du Manoir, S., Kallioniemi, O.P., Lichter, P., Piper, J., Benedetti, P.A., Carothers, A.D., Fantes, J.A., Garcia‐Sagredo, J.M., Gerdes, T., and Giollant, M. 1995b. Hardware and software requirements for quantitative analysis of comparative genomic hybridization. Cytometry 19:4‐9.
   Kallioniemi, A., Kallioniemi, O.P., Sudar, D., Rutovitz, D., Gray, J.W., Waldman, F., and Pinkel, D. 1992. Comparative genomic hybridization for molecular cytogenetic analysis of solid tumors. Science 258:818‐821.
   Kirchhoff, M., Gerdes, T., Rose, H., Maahr, J., Ottesen, A.M., and Lundsteen, C. 1998. Detection of chromosomal gains and losses in comparative genomic hybridization analysis based on standard reference intervals. Cytometry 31:163‐173.
   Knutsen, T., Pack, S., Petropavlovskaja, M., Padilla‐Nash, H., Knight, C., Mickley, L.A., Ried, T., Elwood, P.C., and Roberts, S.J. 2003. Cytogenetic, spectral karyotyping, fluorescence in situ hybridization, and comparative genomic hybridization characterization of two new secondary leukemia cell lines with 5q deletions, and MYC and MLL amplification. Genes Chromosomes Cancer 37:270‐281.
   Lundsteen, C., Maahr, J., Christensen, B., Bryndorf, T., Bentz, M., Lichter, P., and Gerdes, T. 1995. Image analysis in comparative genomic hybridization. Cytometry 19:42‐50.
   Piper, J., Rutovitz, D., Sudar, D., Kallioniemi, A., Kallioniemi, O.P., Waldman, F.M., Gray, J.W., and Pinkel, D. 1995. Computer image analysis of comparative genomic hybridization. Cytometry 19:10‐26.
   Speicher, M.R., du Manoir, S., Schrock, E., Holtgreve‐Grez, H., Schoell, B., Lengauer, C., Cremer, T., and Ried, T. 1993. Molecular cytogenetic analysis of formalin‐fixed, paraffin‐embedded solid tumors by comparative genomic hybridization after universal DNA‐amplification. Hum. Mol. Genet. 2:1907‐1914.
   Stoecklein, N.H., Erbersdobler, A., Schmidt‐Kittler, O., Diebold, J., Schardt, J.A., Izbicki, J.R., and Klein, C.A. 2002. SCOMP is superior to degenerated oligonucleotide primed‐polymerase chain reaction for global amplification of minute amounts of DNA from microdissected archival tissue samples. Am. J. Pathol. 161:43‐51.
   Zielenska, M., Bayani, J., Pandita, A., Toledo, S., Marrano, P., Andrade, J., Petrilli, A., Thorner, P., Sorensen, P., and Squire, J.A. 2001. Comparative genomic hybridization analysis identifies gains of 1p35 approximately p36 and chromosome 19 in osteosarcoma. Cancer Genet. Cytogenet. 130:14‐21.
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