Gene mapping by in situ hybridization

Trask, Barbara J.

doi:10.1016/0959-437x(91)80046-o

Cited by 26 publications

(6 citation statements)

References 21 publications

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“…Since radioactively labeled probes were used in those studies, the localization of hybridiza tion signals might have been affected by emulsion shifting and signal scattering. On the other hand, while FISH mapping is generally considered to be more precise than that offered by radioactive labeling (Trask, 1991), our data show that the posi tion of FISH signals could be greatly affected by overbaking or over-denaturization of the metaphase chromosomes. These un desirable results are probably due to destruction of the high order structure of the chromatin fibers.…”

mentioning

confidence: 78%

Fluorescence in situ hybridization mapping of the cystic fibrosis transmembrane conductance regulator (CFTR) gene to 7q31.3

Heng

Shi²,

Tsui³

1993

Cytogenet Genome Res

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mentioning

confidence: 78%

Fluorescence in situ hybridization mapping of the cystic fibrosis transmembrane conductance regulator (CFTR) gene to 7q31.3

Heng

Shi²,

Tsui³

1993

Cytogenet Genome Res

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“…FISH is a reliable technique for detecting chromosomal abnormalities (3,4) and allowed a rapid diagnosis of the most frequent abnormalities. It is the routine method of choice for rapid prenatal diagnosis.…”

Section: Discussionmentioning

confidence: 99%

“…Thus, there is a real need for a rapid method to detect common aneuploidies at prenatal diagnosis. FISH-based analysis of interphase nuclei with chromosome-specific probes allows fast analysis of chromosome copy number and structural chromosome abnormalities (2)(3)(4). However, the commercially available FISH probes for prenatal diagnosis are too expensive and then, not accessible in all cytogenetic laboratories.…”

mentioning

confidence: 99%

Simultaneous identification of chromosomes 18, X and Y in uncultured amniocytes by using multi-primed in situ labelling technique

2005

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The aim of this study is to validate the multi-PRINS (primed in situ labelling) technique for simultaneous detection of chromosomes 18, X and Y in uncultured amniocytes for prenatal diagnosis of aneuploidy. The sites of the newly synthesized DNA sequences were showed as fluorescent signals by using immunochemistry. A multi-PRINS technique was specifically performed for simultaneous detection in the same cells of three chromosome targets, e.g. chromosomes 18, X and Y. Fluorescent signals corresponding to chromosomes 18, X and Y were showed as yellow, red and green colour spots, respectively. A multi-FISH technique using chromosome 18, X and Y probes was performed for comparison. Sixty cases were analysed using both multi-PRINS and multi-FISH. Fifty to two hundred nuclei were scored for each case for each technique. In all cases, there was no significant difference in the detection of chromosomes 18, X and Y regarding the sensitivity, the specificity and the efficiency; multi-PRINS and multi-FISH yield a similar distribution of the number of spots per nucleus. Both techniques were able to identify aneuploid cases without any ambiguity. Both multi-PRINS and multi-FISH can accurately and reliably detect aneuploidies involving chromosomes 18, X and Y in uncultured amniocytes. Finally, multi-PRINS represents a faster and more cost-effective alternative to FISH for prenatal testing of aneuploidy in uncultured amniocytes.

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“…In conclusion, the present protocol for preparation of human chromosomes results in a structural preservation which should have sufficient quality to serve as a basis for gene mapping with SEM (TRASK, 1991).…”

Section: Results and Commentsmentioning

confidence: 99%

Preparation of Human Chromosomes for High Resolution Scanning Electron Microscopy.

Kättström

Nilsson

1992

Archives of Histology and Cytology

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Summary. The addition of ethidium bromide during the last 2.5-3h of lymphocyte culturing restricted chromosome contraction and preserved the banding structure in scanning electron microscopy. Treatment of the chromosomes with trypsin and use of impregnation with osmium tetroxide and thiocarbohydrazide resulted in a structural preservation of high resolution quality.A method for scanning electron microscopy (SEM) of metaphase chromosomes from human lymphocytes was designed by HARRISON et al. (1985). Their standard technique for the preparation of chromosomes included incubation of the lymphocytes with colcemid for the collection of metaphases. Usually, however, the chromosomes contract when held in metaphase by colchicine, thus making many bands confluent. Since high resolution banding techniques, which give elongated chromosomes with many distinguishable bands, have been developed for light microscopy (ZABEL et al., 1983;IKEUCHI, 1984), one aim of this work was to investigate whether a similar technique could be usf ul for SEM.The fine surface structure of chromosomes can be examined by SEM if the metal coating of the preparations is replaced by an impregnation with osmium tetroxide and tiocarbohydrazide (IP and FISCHMAN, 1979;HARRISON et al., 1985). However, the attainning of high resolution imaging also requires a short trypsin digestion (SEABRIGHT, 1971), the duration of which has to be found emperically. Therefore, another of our aims was to study the effect of different durations of the trypsinization. MATERIALS AND METHODSPeripheral vein blood was sampled from humans in tubes containing heparin and was processed immediately. The erythrocytes were agglutinated for 30 min at room temperature with phytohemagglutinin and then separated by centrifugation. The leucocytes were cultured in RPMI 1640 medium, supplemented with phytohemagglutinin, penicillin, streptomycin, and 20% fetal calf serum at +3TC in humidified air with 5% CO2.A differential replication staining procedure, modified after BENN and PERLE (1986), was applied after a culture period of 3 days. 5-bromodeoxyuridin (BrdU) was added at a concentration of 100uM/ml medium, and the cells were grown for 16-17 h. Deoxycytidine was also added at the same time to the same concentration to reduce the toxicity of BrdU.The cells were then rinsed and transferred into a prewarmed thymidine-enriched medium consisting of RPMI 1640, supplemented with penicillin, streptomycin, 20% fetal calf serum, and 2ug/ml thymidine, and the cells were harvested by centrifugation 6 1/2h later. Ethidium bromide (7ug/ml medium) was added during the last 2.5-3h of culturing to restrict the chromosome contraction.Fifteen min before harvest, colcemid was added at a concentration of 5ng/ml medium. The cells were then rinsed and placed into a solution of 75mM KCI, where they were left for 15min. After careful washings, the cells were fixed in a freshly made solution of methanol: glacial acetic acid (3:1). After centrifugation, the cells were suspended in a small volume of fixative, with ...

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Gene mapping by in situ hybridization

Cited by 26 publications

References 21 publications

Fluorescence in situ hybridization mapping of the cystic fibrosis transmembrane conductance regulator (CFTR) gene to 7q31.3

Fluorescence in situ hybridization mapping of the cystic fibrosis transmembrane conductance regulator (CFTR) gene to 7q31.3

Simultaneous identification of chromosomes 18, X and Y in uncultured amniocytes by using multi-primed in situ labelling technique

Preparation of Human Chromosomes for High Resolution Scanning Electron Microscopy.

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