Chromosome sorting and DNA sequence localization

Lebo, Roger V.

doi:10.1002/cyto.990030302

Cited by 53 publications

(14 citation statements)

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“…Chromosome sorting, a powerful technique for studying human genetics (1,2), has been used for karyotype analysis (3), structural gene mapping (4), and construction of chromosome-specificrecombinant DNA libraries (5). The procedure reported earlier to map genes by chromosome sorting (4) only separated 12 chromosome fractions and required long sorting times to isolate 2 million chromosomes for restriction enzyme analysis.…”

mentioning

confidence: 99%

“…However, by using a dual laser sorter' and improved methods, we are now able to map a gene to any one of 21 human chromosomes in a single hybridization step. 'A gene mapped to the fraction containing' chromosomes 10, 11, and 12 may be localized subchromosomally by testing derivative chromosomes (2,13). Somatic cell hybridization has been used to map genes by testing protein expression and, more recently, by DNA hybridization to cloned gene probes (20).…”

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confidence: 99%

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High-Resolution Chromosome Sorting and DNA Spot-Blot Analysis Assign McArdle's Syndrome to Chromosome 11

Lebo

Gorin²,

Fletterick³

et al. 1984

Science

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223

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A rapid gene-mapping system uses a high-resolution, dual-laser sorter to identify genes from separate human chromosomes prepared with a new stain combination. This system was used to sort 21 unique chromosome types onto nitrocellulose filter papers. Several labeled gene probes hybridized to the sorted chromosomal DNA types predicted by their previous chromosome assignments. The skeletal muscle glycogen phosphorylase gene was then mapped to a portion of chromosome 11 by spot blotting normal and translocated chromosomes.

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confidence: 99%

mentioning

confidence: 99%

High-Resolution Chromosome Sorting and DNA Spot-Blot Analysis Assign McArdle's Syndrome to Chromosome 11

Lebo

Gorin²,

Fletterick³

et al. 1984

Science

Self Cite

223

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“…Initially, DNA was isolated from purified chromosomes and used for Southern blotting with labeled DNA probes (Lebo 1982). Later, chromosome dot-blots were prepared from only 10 4 chromosomes sorted onto a nitro-cellulose filter disk and the chromosomal DNA was hybridized with labeled DNA probe (Lebo et al 1984; Arumuganathan et al 1994).…”

Section: The Many Important Uses Of Flow-sorted Chromosomesmentioning

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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“…Of all cell lines, Mel-57 showed the most abnormal flow karotype. Here, the normal 13-16 peak appears to be replaced by a large peak with an accessory smaller peak that have filled also the valley between the 9-12 peak and the normal [13][14][15][16] peak. These changes are even more apparent when the chromosomes are stained with PI instead of with EB/MMC (Fig.…”

Section: Flow Karyotypesmentioning

confidence: 99%

“…Potentially, more detailed information can be obtained by flow cytometric measurement of the DNA content of isolated chromosomes. The evolution of this technique during the last 5 years has now made it possible to identify a variety of chromosomal polymorphisms and abnormalities and to sort individual chromosomes for gene mapping and cloning experiments (13,201. So far, only few reports on the application of flow cytometry for karyotyping of solid tumors have appeared (11,141.…”

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Flow karyotyping of human melanoma cell lines

Arkesteijn¹,

Dierendonck²,

Vossepoel³

et al. 1986

Cytometry

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Single-laser flow cytometry has been used to study the feasibility of flow karyotyping of human solid tumors. As a model, seven human melanoma cell lines have been used with varying numerical chromosome composition as verified by FCM DNA content measurements and chromosome countings. For all seven cell lines, flow karyotypes that showed a variety of consistent deviations from the normal diploid flow karyotype could be obtained although the resolution of the flow system and varying debris continuum limited the number of resolvable peaks. The predominant changes observed involved the regions normally representing chromosomes 3-8, 9-12, and 13-16. It is concluded that at present the preparation procedure is the main limiting factor for exploring the full potential of flow karyotyping for cytogenetic analysis of solid-tumor cell lines.Key terms: Chromosomes, flow cytometry Flow cytometry is widely used for studying the diagnostic and prognostic significance of DNA content differences in human tumors. The advantage of this technique is that rapid ploidy determinations can be made on large numbers of cells. However, the information provided by measuring total DNA content gives only a rather crude estimate for numerical chromosome abnormalities. Potentially, more detailed information can be obtained by flow cytometric measurement of the DNA content of isolated chromosomes. The evolution of this technique during the last 5 years has now made it possible to identify a variety of chromosomal polymorphisms and abnormalities and to sort individual chromosomes for gene mapping and cloning experiments (13,201. So far, only few reports on the application of flow cytometry for karyotyping of solid tumors have appeared (11,141.The purpose of the present study was to explore the possibility of using flow karyotyping for characterizing genetic differences between seven different human melanoma cell lines. We chose this neoplasm because melanoma shows a high degree of genetic instability, and cell lines from this tumor can be relatively easily maintained in tissue culture. The variation in ploidy level of the melanoma cell lines was determined by chromosome Table 1. Human foreskin fibroblasts (VH-25, 10th passage) were kindly provided by Dr. P. Abraham, Department of Medical Biochemistry, University Medical Center, Leiden. The cell lines were maintained in continuous cultures in 75-cm3 plastic culture flasks on Ham's F-12 medium supplemented with 10% foetal calf serum and antibiotics and passaged twice a week.Chromosome Isolation Confluent cultures were trypsinized and plated out in a 140-4 ratio into new culture flasks. After reaching 75% confluency (usually in about 48 h) colchicin (Colcemid, Gibco, Madison, WI) was added to the culture medium to a final concentration of 0.05 hg/ml. Mitotic cells were harvested 15-18 h later by gently tapping the side of the culture flask. Next the cells were centrifuged at 175g for 5 min at room temperature, and the pellet was washed once with hypotonic KC1 solution (0.075 M). After that, the c...

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Chromosome sorting and DNA sequence localization

Cited by 53 publications

References 57 publications

High-Resolution Chromosome Sorting and DNA Spot-Blot Analysis Assign McArdle's Syndrome to Chromosome 11

High-Resolution Chromosome Sorting and DNA Spot-Blot Analysis Assign McArdle's Syndrome to Chromosome 11

Chromosomes in the flow to simplify genome analysis

Flow karyotyping of human melanoma cell lines

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