Spectral karyotyping analysis of human and mouse chromosomes

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.

[1]  K. Sirotkin,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 , 2005, Genes, chromosomes & cancer.

[2]  Iscn.  International System for Human Cytogenetic Nomenclature , 1978 .

[3]  S. Uhrig,et al.  Multiplex-fluorescence in situ hybridization for chromosome karyotyping , 2006, Nature Protocols.

[4]  M. Ferguson-Smith,et al.  Cross-species chromosome painting , 2006, Nature Protocols.

[5]  M. Ploeg,et al.  Use of whole cosmid cloned genomic sequences for chromosomal localization by non-radioactive in situ hybridization , 1987, Human Genetics.

[6]  T. Caspersson,et al.  Chemical differentiation along metaphase chromosomes. , 1968, Experimental cell research.

[7]  C. Deng,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 , 2002, Oncogene.

[8]  F. Arrighi,et al.  Localization of heterochromatin in human chromosomes. , 1971, Cytogenetics.

[9]  N. Dracopoli,et al.  Current protocols in human genetics , 1994 .

[10]  L. Manuelidis,et al.  Detection of chromosome aberrations in metaphase and interphase tumor cells by in situ hybridization using chromosome-specific library probes , 1988, Human Genetics.

[11]  Thomas Ried,et al.  The Septin 9 (MSF) gene is amplified and overexpressed in mouse mammary gland adenocarcinomas and human breast cancer cell lines. , 2003, Cancer research.

[12]  D. Ledbetter,et al.  Multicolor Spectral Karyotyping of Human Chromosomes , 1996, Science.

[13]  D. Pinkel,et al.  Fluorescence in situ hybridization with human chromosome-specific libraries: detection of trisomy 21 and translocations of chromosome 4. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[14]  E. Schröck,et al.  Analysis of B-cell neoplasias by spectral karyotyping (SKY). , 1999, Current topics in microbiology and immunology.

[15]  A. Helmrich,et al.  Spectral karyotyping of human, mouse, rat and ape chromosomes – applications for genetic diagnostics and research , 2006, Cytogenetic and Genome Research.

[16]  T. Ried,et al.  Mammary Gland Cancer , 2004 .

[17]  J. Wienberg,et al.  "Bar-coding" primate chromosomes: molecular cytogenetic screening for the ancestral hominoid karyotype , 2001, Human Genetics.

[18]  Alan Tunnacliffe,et al.  Cytogenetic analysis by chromosome painting using dop‐pcr amplified flow‐sorted chromosomes , 1992, Genes, chromosomes & cancer.

[19]  M. Norton,et al.  Prenatal Diagnosis of Minute Supernumerary Marker Chromosomes , 2005, Gynecologic and Obstetric Investigation.

[20]  E. Schröck,et al.  Comprehensive and definitive molecular cytogenetic characterization of HeLa cells by spectral karyotyping. , 1999, Cancer research.

[21]  Eric C. Holland,et al.  Mouse models of human cancer , 2004 .

[22]  E. Schröck,et al.  Previously hidden chromosome aberrations in T(12;15)-positive BALB/c plasmacytomas uncovered by multicolor spectral karyotyping. , 1997, Cancer research.

[23]  E. Schröck,et al.  Identification of de novo chromosomal markers and derivatives by spectral karyotyping , 1998, Human Genetics.

[24]  T. Ried,et al.  Pediatric pancreatoblastoma: histopathologic and cytogenetic characterization of tumor and derived cell line. , 2005, Cancer genetics and cytogenetics.

[25]  E. Schröck,et al.  Prenatal diagnosis of a mosaic extra structurally‐abnormal chromosome by spectral karyotyping , 1999, Prenatal diagnosis.

[26]  C. Steinlein,et al.  Multicolor spectral karyotyping of rat chromosomes , 2003 .

[27]  Thomas Ried,et al.  Multicolour spectral karyotyping of mouse chromosomes , 1996, Nature Genetics.

[28]  E. Schröck,et al.  Jumping translocations are common in solid tumor cell lines and result in recurrent fusions of whole chromosome arms , 2001, Genes, chromosomes & cancer.

[29]  B. Horton.  Microscopy and image analysis , 1997, Nature.

[30]  W. Nash,et al.  Molecular cytogenetic analysis of the bladder carcinoma cell line BK‐10 by spectral karyotyping , 1999, Genes, chromosomes & cancer.

[31]  D. Ward,et al.  Karyotyping human chromosomes by combinatorial multi-fluor FISH , 1996, Nature Genetics.

[32]  Thomas Ried,et al.  Hidden chromosome abnormalities in haematological malignancies detected by multicolour spectral karyotyping , 1997, Nature Genetics.

[33]  L. Shaffer,et al.  Spectral karyotyping refines cytogenetic diagnostics of constitutional chromosomal abnormalities , 1997, Human Genetics.

[34]  M. Seabright.  A rapid banding technique for human chromosomes. , 1971, Lancet.