Abstract
Classical banding methods provide basic information about the identities and structures of chromosomes on the basis of their unique banding patterns. Spectral karyotyping (SKY), and the related multiplex fluorescence in situ hybridization (M-FISH), are chromosome-specific multicolor FISH techniques that augment cytogenetic evaluations of malignant disease by providing additional information and improved characterization of aberrant chromosomes that contain DNA sequences not identifiable using conventional banding methods. SKY is based on cohybridization of combinatorially labeled chromosome-painting probes with unique fluorochrome signatures onto human or mouse metaphase chromosome preparations. Image acquisition and analysis use a specialized imaging system, combining Sagnac interferometer and CCD camera images to reconstruct spectral information at each pixel. Here we present a protocol for SKY analysis using commercially available SkyPaint probes, including procedures for metaphase chromosome preparation, slide pretreatment and probe hybridization and detection. SKY analysis requires approximately 6 d.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Rent or buy this article
Prices vary by article type
from$1.95
to$39.95
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Seabright, M. Rapid banding technique for human chromosomes. Lancet II, 971–972 (1971).
Caspersson, T. et al. Chemical differentiation along metaphase chromosomes. Exp. Cell Res. 49, 219–222 (1968).
Arrigghi, F.F. & Hsu, T.C. Localization of heterochromatin in human chromosomes. Cytogenetics 10, 81–86 (1971).
Landegent, J.E. Jansen in de Wal, N., Dirks, R. W., Baas, F. & van der Ploeg, M. Use of the whole cosmid cloned genomic sequences for chromosomal localization by non-radioactive in situ hybridization. Hum. Genet. 77, 366–370 (1987).
Pinkel, D. et al. Fluorescence in situ hybridization with human chromosome specific libraries: detection of trisomy 21 and translocation of chromosome 4. Proc. Natl. Acad. Sci. USA 85, 9138–9142 (1988).
Cremer, T., Lichter, P., Borden, J., Ward, D.C. & Mannuelidis, L. Detection of chromosome aberrations in metaphase and interphase tumor cells by in situ hybridization using chromosome-specific library probes. Hum. Genet. 80, 235–246 (1988).
Schröck, E. et al. Multicolor spectral karyotyping of human chromosomes. Science 273, 494–497 (1996).
Veldman, T., Vignon, C., Schrock, E., Rowley, J.D. & Ried, T. Hidden chromosome abnormalities in haematological malignancies detected by multicolour spectral karyotyping. Nat. Genet. 15, 406–410 (1997).
Macville, M. et al. Comprehensive and definitive molecular cytogenetic characterization of HeLa cells by spectral karyotyping. Cancer Res. 59, 141–150 (1999).
Hilgenfeld, E., Padilla-Nash, H., Schrock, E. & Ried, T. Analysis of B-cell neoplasias by spectral karyotyping (SKY). Curr. Top. Microbiol. Immunol. 246, 169–174 (1999).
Padilla-Nash, H.M. et al. Jumping translocations are common in solid tumor cell lines and result in recurrent fusions of whole chromosome arms. Genes Chromosom. Cancer 30, 349–363 (2001).
Knutsen, T. et al. The interactive online SKY/M-FISH & CGH database and the Entrez cancer chromosomes search database: linkage of chromosomal aberrations with the genome sequence. Genes Chromosom. Cancer 44, 52–64 (2005).
Speicher, M.R., Ballard, S.G. & Ward, D.C. Karyotyping human chromosomes by combinatorial multi-fluor FISH. Nat. Genet. 12, 368–375 (1996).
Geigl, J.B., Uhrig, S. & Speicher, M.R. Multiplex-fluorescence in situ hybridization for chromosome karyotyping. Nat. Prot. 1, 1172–1184 (2006).
Padilla-Nash, H.M. et al. Molecular cytogenetic analysis of the bladder carcinoma cell line BK-10 by spectral karyotyping. Genes Chromosom. Cancer 25, 53–59 (1999).
ISCN: An International System for Human Cytogenetic Nomenclature (Shaffer, L.G. & Tommerup, N. (eds); S Karger, Basel, Switzerland, 2005).
Barenboim-Stapleton, L. et al. Pediatric pancreatoblastoma: histopathologic and cytogenetic characterization of tumor and derived cell line. Cancer Genet. Cytogenet. 157, 109–117 (2005).
Schröck, E. et al. Spectral karyotyping refines cytogenetic diagnostics of constitutional chromosomal abnormalities. Hum. Genet. 101, 255–262 (1997).
Haddad, B.R. et al. Identification of de novo chromosomal markers and derivatives by spectral karyotyping. Hum. Genet. 103, 619–625 (1998).
Ning, Y., Laundon, C.H., Schrock, E., Buchanan, P. & Ried, T. Prenatal diagnosis of a mosaic extra structurally abnormal chromosome by spectral karyotyping. Prenat. Diagn. 19, 480–482 (1999).
Cotter, P.D. et al. Prenatal diagnosis of minute supernumerary marker chromosomes. Gynecol. Obstet. Invest. 60, 27–38 (2005).
Schrock, E. et al. Spectral karyotyping of human, mouse, rat and ape chromosomes–applications for genetic diagnostics and research. Cytogenet. Genome Res. 114, 199–221 (2006).
Muller, S. & Wienberg, J. “Bar-coding” primate chromosomes: molecular cytogenetic screening for the ancestral hominoid karyotype. Hum. Genet. 109, 85–94 (2001).
Rens, W., Fu, B., O'Brien, P.C.M. & Ferguson-Smith, M. Cross-species chromosome painting. Nat. Prot. 1, 783–790 (2006).
Buwe, A. et al. Multicolor spectral karyotyping of rat chromosomes. Cytogenet. Genome Res. 103, 163–168 (2003).
Barkan, D., Montagna, C., Ried, T. & Green, J.E. Mammary gland cancer. in Mouse Models of Human Cancer (ed. Holland, E.C.) 103–131 (Wiley-Liss, Hoboken, New Jersey, 2004).
Liyanage, M. et al. Multicolour spectral karyotyping of mouse chromosomes. Nat. Genet. 14, 312–315 (1996).
Weaver, Z. et al. Mammary tumors in mice conditionally mutant for Brca1 exhibit gross genomic instability and centrosome amplification yet display a recurring distribution of genomic imbalances that is similar to human breast cancer. Oncogene 21, 5097–5107 (2002).
Montagna, C. et al. The Septin 9 (MSF) gene is amplified and overexpressed in mouse mammary gland adenocarcinomas and human breast cancer cell lines. Cancer Res. 63, 2179–2187 (2003).
Coleman, A.E. et al. Previously hidden chromosome aberrations in T(12;15)-positive BALB/c plasmacytomas uncovered by multicolor spectral karyotyping. Cancer Res. 57, 4585–4592 (1997).
Barch, M., Knutsen, T. & Spurbeck, J. (eds.). The AGT Cytogenetics Laboratory Manual (Raven Press, New York, 1997).
Telenius, H. et al. Cytogenetic analysis by chromosome painting using DOP-PCR amplified flow sorted chromosomes. Genes Chromosom. Cancer 4, 257–263 (1992).
Amplification of flow sort DNA using DOP-PRC (SKY). Laboratory of Thomas Ried Protocols http://www.riedlab.nci.nih.gov/protocols.asp (2005).
DOP-PCR secondary (SKY). Laboratory of Thomas Ried Protocols http://www.riedlab.nci.nih.gov/protocols.asp (2005).
DOP-PCR labeling (SKY). Laboratory of Thomas Ried Protocols http://www.riedlab.nci.nih.gov/protocols.asp (2005).
McNamara, G., Difilippantonio, M.J. & Ried, T. Microscopy and image analysis. in Current Protocols in Human Genetics (ed. Miranker, L.) 4.4.1–4.4.34 (John Wiley & Sons, New York, 2005).
Acknowledgements
We thank E. Schröck and M. Liyange for their input into the initial development of SKY for human and mouse, respectively, T. Knutsen for editing of the original SKY protocols and B. Chen and J. Cheng for their technical assistance.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Rights and permissions
About this article
Cite this article
Padilla-Nash, H., Barenboim-Stapleton, L., Difilippantonio, M. et al. Spectral karyotyping analysis of human and mouse chromosomes. Nat Protoc 1, 3129–3142 (2006). https://doi.org/10.1038/nprot.2006.358
Published:
Issue Date:
DOI: https://doi.org/10.1038/nprot.2006.358
This article is cited by
-
Tools used to assay genomic instability in cancers and cancer meiomitosis
Journal of Cell Communication and Signaling (2022)
-
SMURF2 prevents detrimental changes to chromatin, protecting human dermal fibroblasts from chromosomal instability and tumorigenesis
Oncogene (2020)
-
A truncating mutation in the autophagy gene UVRAG drives inflammation and tumorigenesis in mice
Nature Communications (2019)
-
Long-term treatment with the PARP inhibitor niraparib does not increase the mutation load in cell line models and tumour xenografts
British Journal of Cancer (2018)
-
HPV positive neuroendocrine cervical cancer cells are dependent on Myc but not E6/E7 viral oncogenes
Scientific Reports (2017)
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.