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Comparative Genomic Hybridization

Sandy DeVries1,  Joe W. Gray1,  Daniel Pinkel1,  Frederic M. Waldman1,  Damir Sudar2

1University of California at San Francisco, San Francisco, California
2Lawrence Berkeley National Laboratory, Berkeley, California


Publication Name: 
Current Protocols in Human Genetics
Unit Number: 
Unit 4.6
DOI: 
10.1002/0471142905.hg0406s06
Online Posting Date: 
May, 2001
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Abstract

Comparative Genomic Hybridization (CGH) is a powerful molecular cytogenetic technique that permits assessment of DNA copy number on a genome-wide scale. Of note, this methodology uses tumor DNA as a probe for fluorescence in situ hybridization (FISH) to normal metaphase chromosomes and does not require dividing cells from the tumor specimen. This unit provides protocols for CGH, for preparation of metaphase chromosomes, tumor and normal DNAs for FISH and for the microscopy and image analysis of CGH experiments.

     
 
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Table of Contents

  • Unit Introduction
  • Basic Protocol 1: Comparative Genomic Hybridization Using Directly Labeled DNA
  • Alternate Protocol: Comparative Genomic Hybridization Using Indirectly Labeled DNA
  • Support Protocol 1: Preparation of Metaphase Chromosomes for CGH
  • Support Protocol 2: Preparation of Genomic DNA for CGH
  • Support Protocol 3: Preparation of Labeled DNA Probes for CGH
  • Basic Protocol 2: Microscopy, Imaging, and Image Analysis for CGH
  • Reagents and Solutions
  • Commentary
  • Bibliography
  • Figures
     
 
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Materials

Basic Protocol 1: Comparative Genomic Hybridization Using Directly Labeled DNA

 Materials
  • 20 µg/ml fluorescein-labeled test probe DNA (e.g., whole-genomic tumor DNA; see Support Protocol 3)
  • 20 µg/ml Texas red–labeled reference probe DNA (normal whole-genomic DNA; see Support Protocol 3)
  • 1 µg/µl human Cot-1 DNA (Life Technologies)
  • 3 M sodium acetate, pH 5.2 (appendix 2D)
  • 75% and 85% ethanol (room temperature) and 100% (ice-cold and room temperature) ethanol
  • Master hybridization solution (see recipe)
  • Slides containing normal human metaphase chromosomes (unit 4.1; also see Support Protocol 1)
  • Formamide (ultrapure; e.g., Life Technologies or deionized, unit 4.3)
  • Denaturation solution (see recipe)
  • Proteinase K solution (see recipe)
  • Hybridization wash buffer (see recipe), 45°C and room temperature
  • 2× SSC (appendix 2D), 45°C and RT
  • PN buffer (see recipe)
  • DAPI in antifade (see recipe)
  • Diamond pen
  • 37°C slide warmer
  • 45°, 70°, and 75°C water baths
  • 18-mm2 and 22-mm2 coverslips
  • Fine-tipped forceps
  • Rubber cement
  • Moist chamber (empty pipet-tip holder containing wet Kimwipe or setup as illustrated in Fig. 4.3.3).

Alternate Protocol: Comparative Genomic Hybridization Using Indirectly Labeled DNA

 Additional Materials (also see Basic Protocol 1)
  • 20 µg/ml biotin-labeled test probe DNA (e.g., whole-genomic tumor DNA; see Support Protocol 3)
  • 20 µg/ml digoxigenin-labeled reference probe DNA (normal whole-genomic DNA; see Support Protocol 3)
  • Blocking solution: 4× SSC (appendix 2D)/1% (w/v) BSA
  • Detection solution (see recipe)
  • 4× SSC (appendix 2D)
  • 4× SSC (appendix 2D) /0.1% (v/v) Triton X-100
  • 24 × 50–mm coverslips

Support Protocol 3: Preparation of Labeled DNA Probes for CGH

 Materials
  • 10× nucleotide mix (see recipe) or BioNick 10× dNTP mix containing biotin-14-dATP (Life Technologies)
  • Labeled dUTP for normal reference DNA: 1 µmol/ml Texas red–5-dUTP (direct label; Du Pont NEN) or digoxigenin-11-dUTP (indirect label; Boehringer Mannheim)
  • Labeled dUTP for test (tumor) DNA: 1 µmol/ml fluorescein-12-dUTP (direct label; Du Pont NEN)
  • Test (tumor) genomic DNA (see Support Protocol 2)
  • Normal (reference) genomic DNA (see Support Protocol 2)
  • 5 to 10 U/µl DNA polymerase I (Life Technologies)
  • BioNick 10× enzyme mix (Life Technologies)
  • 1% agarose minigel (unit 2.7)
  • 15° and 70°C water baths
  • Additional reagents and equipment for agarose gel electrophoresis using minigels (unit 2.7)

NOTE: The BioNick 10× dNTP mix and BioNick 10× enzyme mix are available from Life Technologies either separately or as components of the BioNick Labeling System.

Looking for materials?  Suppliers Guide
     
 
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Figures

  • Figure 4.6.1
    Comparative genomic hybridization (CGH). Tumor DNA (labeled with green fluorochrome) and normal reference DNA (labeled with red fluorochrome) are hybridized to normal human metaphase chromosomes in the presence of Cot-1 DNA (to suppress binding of labeled DNA to repetitive elements). Regions of DNA gain are seen as an increased green fluorescence intensity on the target chromosomes; regions of DNA loss are seen as increased red fluorescence intensity. Regions on the chromosome that are stained equally for both green and red indicate equal copy number for tumor and reference DNA. A ratio of 1.0 indicates no copy-number change. High green to red ratio (>1.0) indicates DNA gain, and low green to red ratio (<1.0) indicates DNA deletion (Kallioniemi et al., 1993; reprinted with permission of Academic Press).

    View Image
  • Figure 4.6.2
    CGH analysis of breast-cancer cell line MPE600. (A) Three-color digital image after hybridization of fluorescein-dUTP labeled MPE600 DNA (green) and Texas red-dUTP labeled reference DNA (red) to a normal lymphocyte metaphase counterstained with DAPI (blue). (B) Images of individual chromosomes from panel A with their green/red ratio profiles (also see Fig. 4.6.1) to the right. Chromosomes are oriented from p arm to q arm (left to right). Centromeres appear blue as a result of suppression of repetitive elements during hybridization. Deletions appear red and gains green. Plots of green/red profile show mean (dashed line) and standard deviations (dotted line) of green to red ratios from four separate chromosomes. Chromosomal aberrations detected include a small deletion near 1pter and gain of 1q; loss of 9p; proximal gain on 11q and a 11q distal deletion; two gains in 13q, one proximal, one distal; and loss of 16q.

    View Image

Literature Cited

 Literature Cited
    Aikens, R.S., Agard, D.A., and Sedat, J.W. 1989. Solid-state imagers for microscopy. Methods Cell Biol. 29:291-313.
    Dong, H.K., Mohapatra, G., Bollen, A., Waldman, F.M., and Feuerstein, B.G. 1995. Chromosomal abnormalities in glioblastoma multiforme tumors and glioma cell lines detected by comparative genomic hybridization. Int. J. Cancer 60:812-819.
    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. 1995a. Hardware and software requirements for quantitative analysis of comparative genomic hybridization. Cytometry 19.1:4-9.
    Du Manoir, S., Schroeck, E., Bentz, M., Speicher, M.R., Joos, S., Ried, T., Lichter, P., and Cremer, T. 1995b. Quantitative analysis of comparative genomic hybridization. Cytometry 19.1:27-41.
    Inoue, S. 1986. Video Microscopy. Plenum Press, New York.
    Isola, J., DeVries, S., Chu, L., Ghazvini, S., and Waldman, F. 1994. Analysis of DNA sequence copy number changes from archival paraffin embedded tumor samples by comparative genomic hybridization. Am. J. Pathol. 145:1301-1308.
    James, J. and Tanke, H.J. 1991. Biomedical Light Microscopy. Kluwer Academic Publishers, Boston.
    Ji, L. 1994. Fully automatic chromosome segmentation. Cytometry 17:196-208.
    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.
    Kallioniemi, O.P., Kallioniemi, A., Sudar, D., Rutovitz, D., Gray, J., Waldman, F., and Pinkel, D. 1993. Comparative genomic hybridization: A rapid new method for detecting and mapping DNA amplification in tumors. Sem. Cancer Biol. 4:41-46.
    Kallioniemi, O.P., Kallioniemi, A., Piper, J., Isola, J., Waldman, F.M., Gray, J.W., and Pinkel, D. 1994a. Optimizing comparative genomic hybridization for analysis of DNA sequence copy number changes in solid tumors. Genes Chrom. Cancer 10:231-243.
    Kallioniemi, A., Kallioniemi, O., Sudar, D., Rutovitz, D., Chen, L., Smith, H., Gray, J., Pinkel, D., and Waldman, F. 1994b. Identification of amplified regions in breast cancer by comparative genomic hybridization. Proc. Natl. Acad. Sci. 91:2156-2160.
    Kallioniemi, A., Kallioniemi, O., Citro, G., Sauter, G., DeVries, S., Kerschmann, R., Carroll, P., and Waldman, F. 1995. Identification of gains and losses of DNA sequences in primary bladder cancer by comparative genomic hybridization. Genes Chrom. Cancer 12:213-219.
    Morton, N.E. 1991. Parameters of the human genome. Proc. Natl. Acad. Sci. 88:7474-7476.
    Piper, J. and Granum, E. 1989. On fully automatic feature measurement for banded chromosome classification. Cytometry 10:242-255.
    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.
    Piper, J., Sudar, D., Peters, D., and Pinkel, D. 1994. Inter-cellular fluorescence background on microscope slides: Some problems and solutions for automatic analysis. In Image Acquisition and Scientific Imaging Systems Proc. SPIE (H.C. Titus and A. Waks, eds.). 2173:28-35.
    Reznikoff, C., Belair, C., Savelieva, E., Zhai, Y., Pfeifer, K., Yeager, T., Thompson, K.J., DeVries, S., Newton, M.A., Sekhon, G., and Waldman, F. 1994. Chromosome instability in HPV16 E6, but not E7 immortal human epithelial cells. Genes Devel. 8:2227-2240.
    Rutovitz, D. 1978. Expanding picture components to natural density boundaries by propagation methods. The notions of fall set and fall distance. Proceedings of the 4th International Japanese Conference on Pattern Recognition, pp. 657-664. Kyoto, Japan.
    Tanner, M.M., Tirkkonen, M., Kallioniemi, A., Collins, C., Stokke, T., Karhu, R., Kowbel, D., Shadraven, F., Hintz, M., Kuo, W., Waldman, F.M., Isola, J.J., Gray, J.W., and Kallioniemi, O.P. 1994. Increased copy number at 20q13 in breast cancer: Defining the critical region and exclusion of candidate genes. Cancer Res. 54:4257-4260.
    Verna, R.S. and Babu, A. 1989. Human Chromosomes: Manual of Basic Techniques, pp. 71-72. Pergamon Press, New York.
 Key References
    Du Manoir et al. 1995a. See above.

Complete description of image-analysis requirements for CGH.

    Kallioniemi et al. 1994a. See above.

Review article on optimizing CGH.

    Piper et al. 1995. See above.

Detailed description of CGH analysis procedure and profile interpretation.

     
 
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