Analysis of Genomic Aberrations Using Comparative Genomic Hybridization of Metaphase Chromosomes

  • Melanie A. CarlessEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1288)


Comparative genomic hybridization (CGH) allows the global screening of copy number aberrations within a sample. Specifically, large (>20 mb) deletions and amplifications are detected, based on utilization of test and reference (karyotypically normal) DNA. These samples are whole-genome amplified by DOP-PCR and then differentially labeled with fluorophores via nick translation. Test and reference samples are competitively hybridized to normal metaphase chromosomes. The relative amount of each DNA that binds to a chromosomal locus is indicative of the abundance of that DNA. Thus, if a chromosomal region is amplified, the test DNA will out-compete the reference DNA for binding and fluorescence will indicate amplification. Conversely, if a region is deleted, more reference DNA will bind and fluorescence will indicate a deletion. The following chapter outlines the protocols used for CGH analysis of metaphase chromosomes. These protocols include metaphase chromosome slide preparation, DNA extraction (from blood, cell lines, and microdissected formalin-fixed paraffin-embedded tissue), DOP-PCR, nick translation, in situ hybridization, and fluorescence microscopy and image analysis.

Key words

Comparative genomic hybridization (CGH) Microdissection Degenerate oligonucleotide primed-polymerase chain reaction (DOP-PCR) Nick translation Fluorescence microscopy Copy number aberrations (CNAs) 


  1. 1.
    Kallioniemi A, Kallioniemi OP, Sudar D, Rutovitz D, Gray JW, Waldman F, Pinkel D (1992) Comparative genomic hybridization for molecular cytogenetic analysis of solid tumors. Science 258(5083):818–821CrossRefPubMedGoogle Scholar
  2. 2.
    Kallioniemi OP, Kallioniemi A, Piper J, Isola J, Waldman FM, Gray JW, Pinkel D (1994) Optimizing comparative genomic hybridization for analysis of DNA sequence copy number changes in solid tumors. Genes Chromosomes Cancer 10(4):231–243CrossRefPubMedGoogle Scholar
  3. 3.
    Xu J, Chen Z (2003) Advances in molecular cytogenetics for the evaluation of mental retardation. Am J Med Genet C: Semin Med Genet 117(1):15–24CrossRefGoogle Scholar
  4. 4.
    Pollack JR, Perou CM, Alizadeh AA, Eisen MB, Pergamenschikov A, Williams CF, Jeffrey SS, Botstein D, Brown PO (1999) Genome-wide analysis of DNA copy-number changes using cDNA microarrays. Nat Genet 23(1):41–46CrossRefPubMedGoogle Scholar
  5. 5.
    Beheshti B, Park PC, Braude I, Squire JA (2002) Microarray CGH. Methods Mol Biol 204:191–207PubMedGoogle Scholar
  6. 6.
    Al-Mulla F (2011) Microarray-based CGH, and copy number analysis of FFPE samples. Methods Mol Biol 724:131–145CrossRefPubMedGoogle Scholar
  7. 7.
    Singh RR, Cheung KJ, Horsman DE (2011) Utility of array comparative genomic hybridization in cytogenetic analysis. Methods Mol Biol 730:219–234CrossRefPubMedGoogle Scholar
  8. 8.
    Baladandayuthapani V, Ji Y, Talluri R, Nieto-Barajas LE, Morris JS (2010) Bayesian random segmentation models to identify shared copy number aberrations for array CGH data. J Am Stat Assoc 105(492):1358–1375, PMC3079218CrossRefPubMedCentralPubMedGoogle Scholar
  9. 9.
    Hsu FH, Chen HI, Tsai MH, Lai LC, Huang CC, Tu SH, Chuang EY, Chen Y (2011) A model-based circular binary segmentation algorithm for the analysis of array CGH data. BMC Res Notes 4:394, PMC3224564CrossRefPubMedCentralPubMedGoogle Scholar
  10. 10.
    Houldsworth J, Chaganti RS (1994) Comparative genomic hybridization: an overview. Am J Pathol 145(6):1253–1260PubMedCentralPubMedGoogle Scholar
  11. 11.
    Gallo JH, Ordonez JV, Brown GE, Testa JR (1984) Synchronization of human leukemic cells: relevance for high-resolution chromosome banding. Hum Genet 66(2–3):220–224CrossRefPubMedGoogle Scholar
  12. 12.
    Barch M, Knutsen T, Spurbeck J. Lippincott-Raven Publishers. The AGT Cytogenetics Laboratory Manual. 3rd ed. 1997Google Scholar
  13. 13.
    Miller SA, Dykes DD, Polesky HF (1988) A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 16(3):1215CrossRefPubMedCentralPubMedGoogle Scholar
  14. 14.
    Kuukasjärvi T, Tanner M, Pennanen S, Karhu R, Visakorpi T, Isola J (1997) Optimizing DOP-PCR for universal amplification of small DNA samples in comparative genomic hybridization. Genes Chromosomes Cancer 18(2):94–101CrossRefPubMedGoogle Scholar
  15. 15.
    Shaffer LG, McGowan-Jordan J, Schmid M (eds) (2003) An international system for human cytogenetic nomenclature (2013). Recommendations of the international standing committee on human cytogenetic nomenclature. Karger, BaselGoogle Scholar
  16. 16.
    Schreck RR, Disteche C (2001) Karyotyping. Curr Protoc Hum Genet. Appendix 4: Appendix 4AGoogle Scholar
  17. 17.
    Schreck RR, Disteche CM, Adler D (2001) ISCN standard idiograms. Curr Protoc Hum Genet. Appendix 4: Appendix 4B.Google Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Texas Biomedical Research InstituteSan AntonioUSA

Personalised recommendations