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

Evelin Schröck1,  Zoë Weaver2,  Donna Albertson3

1Institute of Genetic Medicine, Charité, Berlin, Germany
2National Cancer Institute (NCI/NIH), Bethesda, Maryland
3University 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

  • Unit Introduction
  • 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

 Additional Materials (also see Basic Protocol 1)
  • 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% CO2 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% CO2 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)

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

 Materials
  • Metaphase chromosome preparations (see Basic Protocol 3)
  • 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/MgCl2 (see recipe)
  • Formalin (37% formaldehyde)
  • 24 × 60–mm coverslip
  • 37°C incubator
  • Coplin jars
  • 37°C water bath

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

 Materials
  • 500 ng tumor DNA labeled with biotin via nick translation (see Basic Protocol 4)
  • 500 ng control DNA labeled with digoxigenin via nick translation (see Basic Protocol 4)
  • 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 Background Information)
  • Rubber cement
  • 1.5-ml microcentrifuge tubes (Eppendorf)
  • Speedvac evaporator (Savant)
  • 24 × 60–mm coverslips
  • 18 × 18–mm coverslips

Basic Protocol 7: Detection of Hybridized DNA Sequences for Both Human and Mouse Chromosomes

 Materials
  • Hybridized slides (see Basic Protocol 6)
  • 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

  • Figure 8.12.1
    Principle of CGH. (A) Hybridization of tumor and control DNA onto normal human chromosomes. Chromosomal regions shown in cross-hatched lines are gained in the tumor genome, e.g., chromosomes 7 and 12, chromosome arms 3q and 20q, and bands 11q14-22. In contrast, losses (portrayed in a pattern of dots) are mapped to chromosomes 5 and Y, chromosome arms 3p, 8p, and 17p, and on band 13q14. (B) Hybridization of tumor and control DNA onto arrayed BAC clones demonstrating gains (cross-hatched lines) and losses (dotted pattern) with a resolution of ~100 kb. Chromosomal regions and DNA clones that are recurrently gained or lost are targets for molecular genetic approaches towards identification and cloning of potential oncogenes and tumor suppressor genes as well as for diagnostic and prognostic purposes.

    View Image
  • Figure 8.12.2
    Flow chart outlining the course of CGH protocols.

    View Image
  • Figure 8.12.3
    Typical metaphase spread suited for CGH experiments. (A) Chromosomes are surrounded by cytoplasm (gray layer around metaphase chromosomes) before pretreatment. (B) After pepsin treatment note the lower amount of cytoplasm.

    View Image
  • Figure 8.12.4
    Karyogram of a normal metaphase used as CGH target. Note that centromere enumeration probes (labeled with Cy5-near infrared, indicated by arrows) were added for several chromosomes that are difficult to differentiate from their neighbors (e.g., nos. 4 and 5; nos. 7, 8, 9, 10, and X; nos. 13, 14, and 15; nos. 19 and 20; nos. 21 and 22). One chromosome 4 was missing in this metaphase.

    View Image
  • Figure 8.12.5
    Scheme for chromosome identification based on DAPI banding, indicating the typical landmarks.

    View Image
  • Figure 8.12.6
    Array-CGH mapping of copy number alterations occurring on chromosome 20 in breast cancer. (A) Copy number profiles on chromosome 20 from two breast tumors determined using arrays of genomic clones spaced at 1 to 3 Mbp along chromosome 20. Both tumors show high-level amplification at 20q13.2 (after Pinkel et al., 1998). (B) Higher resolution analysis of copy number in the breast tumors using an array of contiguous genomic clones across a ~2 Mbp region at 20q13.2. Each clone in the array is represented by a horizontal bar that indicates the location and length of the clone as determined by STS content mapping. The high-resolution copy number profiles show narrow peaks, suggesting selection for amplification of genes in these regions. The peak in tumor S50 maps to the proximal region of the contig and is centered on ZNF217. In tumor S21, on the other hand, there is elevated copy number at ZNF217, but the copy number maximum maps more distally and includes CYP24, a gene involved in regulation of vitamin D signaling. Note that the array used for the low-resolution analysis of S21 did not contain the clones (RMC20B421 and RMC20B4087) that mapped to the copy number maximum in S21. The names of some clones are shown with the RMC20 prefix omitted (after Albertson et al., 2000).

    View Image
  • Figure 8.12.7
    Example of a CGH analysis of a mammary tumor obtained from a mouse conditionally mutated for Brca1. (A) DAPI-banding picture including chromosome identification. (B) FITC image showing the hybridization pattern of the tumor DNA. (C) TRITC image indicating the normal control-DNA hybridization. (D) FITC/TRITC overlay demonstrating, e.g., a gain on chromosome 6 and a gain close to the centromere on chromosome 9 (arrowhead). In contrast, increased intensity values are found on chromosome 1 in the FITC and the TRITC images (arrow), reflecting hybridization of repetitive DNA sequences in both images that do not result in a change of DNA copy numbers in the average ratio profile (compare to Figure 8.12.8; Xu et al., 1999).

    View Image
  • Figure 8.12.8
    Average ratio profile of the mouse mammary tumor shown in Figure 8.12.7. Numerous gains were identified on almost all chromosomes except for chromosome 17. Specifically, a gain was detected on chromosome 11 in band E, a region that is syntenic to the long arm of chromosome 17 in humans. This region is recurrently gained in human breast cancers and harbors the oncogene Her2-neu. Interphase cytogenetics using the DNA clone for Her2-neu reveals a consistent gain and amplification of this gene in mouse mammary tumors (Weaver et al., manuscript in preparation).

    View Image
  • Figure 8.12.9
    (A) Karyogram of an overlaid FITC/Texas Red image visualizing the DNA copy number changes in the tumor (SKBR3, breast cancer cell line) compared to the control DNA (normal DNA). Gains in the tumor DNA appear in green (e.g., gene amplifications on 8q, gain of chromosome arms 5p and 7p), whereas losses appear in red (e.g., loss of chromosome arms 4p and 9p). Regions with equal copy numbers show an orange mixed color (e.g., regions on chromosome arms 2q and 12q). The second chromosome 4 was missing in this metaphase. (B) Average ratio profile (black) indicating numerous gains and losses of specific chromosomal regions. The straight dotted dark lines to the right of the ratio value of 1.0 show the threshold for a gain (ratio value of 1.2), whereas the straight dashed lighter lines to the left visualize the border for a loss (ratio value of 0.8). Similarly, the gains and losses of chromosomal regions are also depicted by straight dotted dark and straight dashed lighter bars next to the chromosome ideogram based on the comparison of the average ratio values in B with the appearance of gains and losses in a single metaphase spread shown in A.

    View Image

Literature Cited

Literature Cited
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    Kallioniemi, O.P., Kallioniemi, A., Piper, J., Isola, J., Waldman, F.M., Gray, J.W., and Pinkel, D. 1994b. Optimizing comparative genomic hybridization for analysis of DNA sequence copy number changes in solid tumors. Genes Chrom. Cancer 10:231-243.
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    Korenberg, J.R., Chen, X.-N., Sun, Z., Shi, Z.-Y., Ma, S., Vataru, E., Yimlamai, D., Weissenbach, J.S., Shizuya, H., Simon, M.I., Gerety, S.S., Nguyen, H., Zemsteva, I.S., Hui, L., Silva, J., Wu, X., Birren, B.W., and Hudson, T.J. 1999. Human genome anatomy: BACs integrating the genetic and cytogenetic maps for bridging genome and biomedicine. Genome Res. 9:994-1001.
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    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.
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    Weaver, Z.A., McCormack, S.J., Liyanage, M., du Manoir, S., Coleman, A., Schröck, E., Dickson, R.B., and Ried, T. 1999. A recurring pattern of chromosomal aberrations in mammary gland tumors of MMTV-cmyc transgenic mice. Genes Chrom. Cancer 25:251-260.
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 Key References
 Development of CGH
    du Manoir et al., 1993. See above.
    du Manoir et al., 1995a. See above.
    du Manoir et al., 1995b. See above.
    Kallioniemi et al., 1992. See above.
    Piper et al., 1995. See above.
 CGH using DNA from formalin-fixed tissue
    Speicher et al., 1993. See above.
 Genotype-phenotype correlation
    Heselmeyer et al., 1996. See above.
    Ried et al., 1997. See above.
    Ried et al., 1999. See above.
 Single-cell CGH analysis
    Klein et al., 1999. See above.
 Array CGH development
    Heiskanen et al., 2000. See above.
    Pinkel et al., 1998. See above.
    Solinas-Toldo et al., 1997. See above.
 Internet Resources
    http://www.aicorp.com

Web site for Applied Imaging.

    http://www.leica-microsystems.com

Web site for Leica Microsystems Imaging Solutions Ltd., Cambridge, U.K.

    http://www.metasystems.de

Web site for Metasystems GmbH.

    http://www.vysis.com

Web site for Vysis, Inc.

The authors wish to thank Dr. Dan Pinkel (Cancer Center, UCSF, San Francisco, CA) and Dr. Thomas Ried (Genetics Department, NCI/NIH, Bethesda, MD) for continued support and collaboration.

     
 
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