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  • Review Article
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The new cytogenetics: blurring the boundaries with molecular biology

Nature Reviews Genetics volume 6, pages 782–792 (2005)Cite this article

Key Points

  • An important aim of recent cytogenetic developments has been to increase resolution. This has been achieved by advances that relate to the two elements of cytogenetic analysis, the target and the probe.

  • Cytogenetic methods are now available at resolutions that allow the identification of even single-nucleotide changes in the genome. Therefore, the traditional distinction between cytogenetics and molecular genetics is fading.

  • The development of procedures for the hybridization of multiple probes, each labelled in a different colour, to targets such as metaphase spreads and interphase nuclei, has had a tremendous effect on the application of cytogenetics in the clinic and for basic research.

  • Multicolour approaches are now available that allow even complex chromosomal rearrangements, as frequently seen in solid tumours, to be analysed. This can be carried out in both metaphase spreads and interphase nuclei.

  • The recent introduction of array technologies for genome analysis has had a huge effect on cytogenetic applications. A wide variety of array platforms have been developed including genome-wide scanning for polymorphisms and sub-microscopic copy number changes.

  • The identification of single base pair changes for genotyping purposes and the identification of epigenetic modifications is also possible using these techniques.

  • An important feature of cytogenetic analyses is the ability to yield information at the resolution of a single cell. Although this has been possible for some time using fluorescence in situ hybridization technology, recent developments in unbiased DNA amplification now allow single cells to be analysed using genome scanning applications such as comparative genomic hybridization.

  • The many cytogenetic technologies that are now available allow far more than a simple description of the chromosomal or DNA status of a cell or tissue. Three-dimensional interphase cytogenetics and the mapping on microarrays of DNA that has been enriched by chromatin immunoprecipitation are starting to provide new insights into the functional organization of the genome.

  • Multicolour interphase fluorescence in situ hybridization has helped us understand better how the genome is organized in three-dimensions. Recent advances in four-dimensional fluorescence in situ hybridization will contribute to a better understanding of the dynamic interplay between the genome and its regulatory factors in living cells.

Abstract

Exciting advances in fluorescence in situ hybridization and array-based techniques are changing the nature of cytogenetics, in both basic research and molecular diagnostics. Cytogenetic analysis now extends beyond the simple description of the chromosomal status of a genome and allows the study of fundamental biological questions, such as the nature of inherited syndromes, the genomic changes that are involved in tumorigenesis and the three-dimensional organization of the human genome. The high resolution that is achieved by these techniques, particularly by microarray technologies such as array comparative genomic hybridization, is blurring the traditional distinction between cytogenetics and molecular biology.

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Figure 1: Principles of fluorescence in situ hybridization.
Figure 2: Fluorescence in situ hybridization probes for different applications.
Figure 3: Comparison of cytogenetic techniques for identifying chromosomal abnormalities.
Figure 4: Combining cytogenetic approaches to understand a complex chromosomal rearrangement.
Figure 5: Studying genome organization using three-dimensional fluorescence in situ hybridization.

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Acknowledgements

We are grateful to T. Cremer, A. Bolzer, J. Kraus and H. Fiegler for providing images. Research in the laboratory of M.R.S. is supported by the Deutsche Forschungsgemeinschaft (DFG), the Deutsche Krebshilfe, the Wilhelm Sander-Stiftung, and the BMBF (Bundesministerium für Bildung und Forschung). N.P.C. is supported by the Wellcome Trust.

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Authors and Affiliations

  1. Institut für Humangenetik, Technische Universität München, Trogerstrasse 32, München, D-81675

    Michael R. Speicher

  2. GSF-Nationales Forschungszentrum für Umwelt und Gesundheit, Ingolstädter Landstrasse 1, Neuherberg, D-85764, Germany

    Michael R. Speicher

  3. The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SA, United Kingdom

    Nigel P. Carter

Authors
  1. Michael R. Speicher
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Correspondence to Michael R. Speicher.

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DATABASES

Entrez

HER2

OMIM

B-cell leukaemia

CHARGE

Down syndrome

Duchenne muscular dystrophy

SwissProt

H2AX

H3 histone

FURTHER INFORMATION

Charité CGH database

Chroma filter sets

Ensembl Cytoview

Mitelman Database of Chromosome Aberrations in Cancer

NCBI Map-Viewer

NCBI SKY/M-FISH & CGH database

NCBI SNP-data base

Probe resources from the University of Bari Cytogenetics Unit

Progenetix CGH database

Tokyo Medical and Dental University CGH database

UCSC genome browser

Glossary

BANDING

A method that uses chemical treatments to produce differentially stained regions on chromosomes.

BIOTIN

A vitamin and mobile carrier of activated CO2 that has a high affinity for avidin and is used for non-radioactive labelling.

COT-1 DNA

DNA that is mainly composed of repetitive sequences. It is produced when short fragments of denatured genomic DNA are re-annealed.

METAPHASE SPREAD

Preparations of chromosomes in dividing cells that have been artificially arrested at metaphase, when chromosomes are highly condensed and shortened, so that they are visible under a light microscope.

DEGENERATE OLIGONUCLEOTIDE-PRIMED PCR

A method for the unbiased amplification of any DNA source using partially degenerate primers.

CHROMOSOME MICRODISSECTION

A technique in which an entire chromosome or region of a chromosome (for example, a chromosome arm or a chromosome band) is isolated using a micro-manipulated glass needle or highly focused laser beam. The sample is then transferred to a tube for subsequent amplification and probe labelling.

FLUOROCHROMES

Non-radioactive labels that can emit fluorescence after excitation by light. Also known as fluorophores.

NICK TRANSLATION

A widely used method for DNA-probe labelling. Nick translation uses a combination of DNase I to nick double-stranded probe DNA, and the polymerase and endonuclease activity of DNA polymerase I to proceed along the target strand from the nicks, incorporating labelled nucleotides.

RANDOM-PRIMED LABELLING

A method for labelling single-stranded probe DNA that uses a mixture of random short oligonucleotides to prime the incorporation of labelled nucleotides using polymerase.

HAPTEN

A small molecule that has binding affinity for a protein receptor.

MULTIPLEX-FISH

Painting of the entire chromosome complement such that each chromosome is labelled with a different combination of fluorophores. Images are collected with a fluorescence microscope that has filter sets for each fluorochrome, and a combinatorial labelling algorithm allows separation and identification of all chromosomes, which are visualized in characteristic pseudocolours.

SPECTRAL KARYOTYPING

Similar to M-FISH, except that an interferometer is used for fluorochrome discrimination and imaging.

COMBINED BINARY RATIO LABELLING

A multicolour karyotyping system that uses a combination of combinatorial and ratio labelling for probe discrimination.

CROSS-SPECIES COLOUR SEGMENTATION

A FISH-based multicolour banding technology that uses flow-sorted gibbon chromosome paints, which generate a cross-species banding pattern when hybridized to human metaphase spreads.

PSEUDOCOLOUR BANDING PATTERN

A fluorescence multicolour banding pattern along chromosomes that is generated by hybridization of multiple differentially labelled region-specific probes.

COOLED CHARGE-COUPLED DEVICE

A highly sensitive area imager that is widely used for capturing FISH images. Cooling, which reduces random noise during long exposures, is often not required as modern fluorochromes with microscope optics usually require only short exposure times.

MINIMAL RESIDUAL DISEASE

The low numbers of tumour cells that remain after therapy, which are often below the detection limits of classical morphological methods.

TRASTUZUMAB

A monoclonal antibody that targets cancer cells that overexpress HER2, which is found on the surface of some cancer cells.

TELOMERE CRISIS

The erosion of the telomeres so that chromosome ends are no longer protected, resulting in unstable chromosomes.

MOLECULAR COMBING

High molecular-weight DNA in solution is stretched at the meniscus as a glass slide is removed from the solution at a constant rate, generating fields of evenly stretched DNA fibres that have a parallel orientation.

LOSS OF HETEROZYGOSITY

A loss of one of the alleles at a given locus as a result of a genomic change, such as mitotic deletion, gene conversion or chromosome missegregration.

UNIPARENTAL DISOMY

A condition in which an individual or embryo carries two chromosomes that are inherited from the same parent.

CONSTITUTIONAL REARRANGEMENTS

Chromosomal rearrangements that are present in an individual at birth.

TILING CLONE ARRAY

A high-resolution array consisting of multiple overlapping clones.

FLOW SORTING

After staining with base-pair-specific fluorochromes, cells or chromosomes are sorted according to their DNA content and base pair ratio using a flow cytometer.

DOUBLE MINUTE

An acentric, extra-chromosomally amplified chromatin, which usually contains a particular chromosomal segment or gene. Double minutes occur frequently in cancer cells.

ACROCENTRIC CHROMOSOME

A chromosome with a near-terminal centromere so that one arm is very short. The short arms of acrocentric chromosomes consist mainly of repetitive DNA sequences.

CpG ISLANDS

Sequences of 200 bp or more that have high GC content and a high frequency of CpG dinucleotides. CpG islands are found upstream of many mammalian genes.

REPLICATION BANDING

Chromosome banding using differences in staining between early and late replicating regions of the genome after timed incorporation of bromodeoxyuridine (BrdU), a nucleoside that substitutes for thymidine in DNA.

CHROMOSOME TERRITORIES

Compartments within a cell nucleus that are occupied by a chromosome.

DECONVOLUTION ALGORITHMS

Computational techniques for removing out-of-focus haze from stacks of optical sections, so restoring sharpness and clarity to an image.

KARYOTYPING

A process in which metaphase chromosomes are ordered and numbered according to morphology, size, arm-length ratio and banding pattern.

DIGITAL KARYOTYPING

A technique that provides quantitative analysis of DNA copy number by isolation and enumeration of short sequence tags from specific genomic loci.

PADLOCK PROBE

A probe with two target-complementary segments, which on hybridization are brought close to each other so that they can be covalently linked, resulting in a circularized probe.

ROLLING-CIRCLE AMPLIFICATION

A method for the general amplification of DNA by DNA polymerase, which replicates circularized oligonucleotide probes with either linear or geometrical kinetics under isothermal conditions.

HYPERBRANCHED STRAND-DISPLACEMENT AMPLIFICATION

Isothermal amplification of genomic DNA that is driven by strand-displacing polymerases, such as phage φ29, for random-primed amplification of human genomic DNA.

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Speicher, M., Carter, N. The new cytogenetics: blurring the boundaries with molecular biology. Nat Rev Genet 6, 782–792 (2005). https://doi.org/10.1038/nrg1692

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