A method for nucleic acid hybridization to isolated chromosomes in suspension

Dudin, Gertrud; Cremer, Thomas; Schardin, Margit; Hausmann, Michael; Bier, Frank F.; Cremer, Christoph

doi:10.1007/bf00283626

Cited by 39 publications

(13 citation statements)

References 9 publications

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“…As pointed out by several authors [6], [23], [24], [25], [39], [40], [41], [42], [43], all previous attempts to directly translate FISH protocols into chromosomes in suspension have failed, due to many problems, such as chromosome clumping, paucity in suspension, poor hybridization pattern reproducibility, and loss of chromosome morphology. Here we present a straightforward wash-less method for fluorescence in situ hybridization of plant chromosomes in suspension (FISHIS), using synthetic, fluorescence-labeled DNA probes ( Figure 1 ) that quantitatively assess specific hybridization patterns, thus allowing for precise flow sorting of individual chromosomes to high purity ( Figure 2 ).…”

Section: Discussionmentioning

confidence: 99%

FISHIS: Fluorescence In Situ Hybridization in Suspension and Chromosome Flow Sorting Made Easy

et al. 2013

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The large size and complex polyploid nature of many genomes has often hampered genomics development, as is the case for several plants of high agronomic value. Isolating single chromosomes or chromosome arms via flow sorting offers a clue to resolve such complexity by focusing sequencing to a discrete and self-consistent part of the whole genome. The occurrence of sufficient differences in the size and or base-pair composition of the individual chromosomes, which is uncommon in plants, is critical for the success of flow sorting. We overcome this limitation by developing a robust method for labeling isolated chromosomes, named Fluorescent In situ Hybridization In suspension (FISHIS). FISHIS employs fluorescently labeled synthetic repetitive DNA probes, which are hybridized, in a wash-less procedure, to chromosomes in suspension following DNA alkaline denaturation. All typical A, B and D genomes of wheat, as well as individual chromosomes from pasta (T. durum L.) and bread (T. aestivum L.) wheat, were flow-sorted, after FISHIS, at high purity. For the first time in eukaryotes, each individual chromosome of a diploid organism, Dasypyrum villosum (L.) Candargy, was flow-sorted regardless of its size or base-pair related content. FISHIS-based chromosome sorting is a powerful and innovative flow cytogenetic tool which can develop new genomic resources from each plant species, where microsatellite DNA probes are available and high quality chromosome suspensions could be produced. The joining of FISHIS labeling and flow sorting with the Next Generation Sequencing methodology will enforce genomics for more species, and by this mightier chromosome approach it will be possible to increase our knowledge about structure, evolution and function of plant genome to be used for crop improvement. It is also anticipated that this technique could contribute to analyze and sort animal chromosomes with peculiar cytogenetic abnormalities, such as copy number variations or cytogenetic aberrations.

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Section: Discussionmentioning

confidence: 99%

FISHIS: Fluorescence In Situ Hybridization in Suspension and Chromosome Flow Sorting Made Easy

et al. 2013

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“…The morphology of chromosomes following fluorescence hybridization in Suspension was well preserved (see also Dudin et al, 1987a). Integrated density profiles obtained for randomly selected FITC and/or PI stained chromosomes in Suspension from several Chinese hamster x human hybrid cell lines were very similar to profiles obtained from conventional metaphase spreads (see also Hausmann et al, 1989).…”

Section: Resultsmentioning

confidence: 61%

“…The chromosomes were hybridized in Suspension with biotinylated human total genomic DNA as described (Dudin et al, 1987a). Human material was detected due to the binding of FITC labelled antibodies (goat anti rabbit -rabbit anti biotin) or a streptavidin -FITC complex to the biotinylated hybridized DNA Hausmann et al, 1989).…”

Section: Methodsmentioning

confidence: 99%

Chromosome Aberration Detection with Hybridized DNA Probes: Digital Image Analysis and Slit Scan Flow Cytometry

Cremer¹,

Hausmann²,

Díaz³

et al. 1989

Automation of Cytogenetics

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“…In principle, labeling of particular DNA sequences should provide a general approach to identify any chromosome. After Trask et al (1985) succeeded in labeling a specific DNA sequence using FISH on interphase nuclei in suspension and quantifying bound probe by flow cytometry, Dudin et al (1987) successfully applied the same method to chromosomes in suspension. They used human genomic DNA as probe for FISH to chromosomes isolated from Chinese hamster × human hybrid cell line.…”

Section: Sorting Chromosomes That Cannot Be Resolvedmentioning

confidence: 99%

Chromosomes in the flow to simplify genome analysis

Doležel

Vrána

Šafář

et al. 2012

Funct Integr Genomics

103

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Nuclear genomes of human, animals, and plants are organized into subunits called chromosomes. When isolated into aqueous suspension, mitotic chromosomes can be classified using flow cytometry according to light scatter and fluorescence parameters. Chromosomes of interest can be purified by flow sorting if they can be resolved from other chromosomes in a karyotype. The analysis and sorting are carried out at rates of 102–104 chromosomes per second, and for complex genomes such as wheat the flow sorting technology has been ground-breaking in reducing genome complexity for genome sequencing. The high sample rate provides an attractive approach for karyotype analysis (flow karyotyping) and the purification of chromosomes in large numbers. In characterizing the chromosome complement of an organism, the high number that can be studied using flow cytometry allows for a statistically accurate analysis. Chromosome sorting plays a particularly important role in the analysis of nuclear genome structure and the analysis of particular and aberrant chromosomes. Other attractive but not well-explored features include the analysis of chromosomal proteins, chromosome ultrastructure, and high-resolution mapping using FISH. Recent results demonstrate that chromosome flow sorting can be coupled seamlessly with DNA array and next-generation sequencing technologies for high-throughput analyses. The main advantages are targeting the analysis to a genome region of interest and a significant reduction in sample complexity. As flow sorters can also sort single copies of chromosomes, shotgun sequencing DNA amplified from them enables the production of haplotype-resolved genome sequences. This review explains the principles of flow cytometric chromosome analysis and sorting (flow cytogenetics), discusses the major uses of this technology in genome analysis, and outlines future directions.

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A method for nucleic acid hybridization to isolated chromosomes in suspension

Cited by 39 publications

References 9 publications

FISHIS: Fluorescence In Situ Hybridization in Suspension and Chromosome Flow Sorting Made Easy

FISHIS: Fluorescence In Situ Hybridization in Suspension and Chromosome Flow Sorting Made Easy

Chromosome Aberration Detection with Hybridized DNA Probes: Digital Image Analysis and Slit Scan Flow Cytometry

Chromosomes in the flow to simplify genome analysis

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