Basic Preparative Techniques for Fluorescence In Situ Hybridization

Wiegant, J.; Raap, Anton K.

doi:10.1002/0471142956.cy0802s00

Cited by 2 publications

(3 citation statements)

References 29 publications

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“…The slides were air‐dried and maintained at 20°C until used for stretching. Different slides were tested: (i) slides coated with 2% AES in acetone (Wiegant and Raap, 1997); (ii) slides cleaned using chromic acid (Schwarzacher and Heslop‐Harrison, 2000); and (iii) untreated slides.…”

Section: Methodsmentioning

confidence: 99%

High‐resolution FISH on super‐stretched flow‐sorted plant chromosomes

et al. 2004

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SummaryA novel high-resolution¯uorescence in situ hybridisation (FISH) strategy, using super-stretched¯ow-sorted plant chromosomes as targets, is described. The technique that allows longitudinal extension of chromosomes of more than 100 times their original metaphase size is especially attractive for plant species with large chromosomes, whose pachytene chromosomes are generally too long and heterochromatin patterns too complex for FISH analysis. The protocol involves¯ow cytometric sorting of metaphase chromosomes, mild proteinase-K digestion of air-dried chromosomes on microscopic slides, followed by stretching with ethanol:acetic acid (3 : 1). Stretching ratios were assessed in a number of FISH experiments with super-stretched chromosomes from barley, wheat, rye and chickpea, hybridised with 45S and 5S ribosomal DNAs and the [GAA] n microsatellite, the [TTTAGGG] n telomeric repeat and a bacterial arti®cial chromosome (BAC) clone as probes. FISH signals on stretched chromosomes were brighter than those on the untreated control, resulting from better accessibility of the stretched chromatin and maximum observed sensitivity of 1 kbp. Spatial resolution of neighbouring loci was improved down to 70 kbp as compared to 5±10 Mbp after FISH on mitotic chromosomes, revealing details of adjacent DNA sequences hitherto not obtained with any other method. Stretched chromosomes are advantageous over extended DNA ®bres from interphase nuclei as targets for FISH studies because they still retain chromosomal integrity. Although the method is con®ned to species for which chromosome¯ow sorting has been developed, it provides a unique system for controlling stretching degree of mitotic chromosomes and high-resolution bar-code FISH.

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Section: Methodsmentioning

confidence: 99%

High‐resolution FISH on super‐stretched flow‐sorted plant chromosomes

et al. 2004

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“…Consequently, using the COBRA principle, a spectrally easily distinguishable fifth fluorochrome can be used to detect and map defined targets, such as integrated small viral DNA sequences, within the context of a 24-color FISH karyotype (7). In addition, FISH-to-linearized DNA molecules (fiber-FISH) was used to establish the copy number of the integrated adenoviral E1 sequences (8). The combination of the two molecular cytogenetic techniques is versatile and therefore optimally suited to monitor genome stability of producer cell lines in biotechnology.…”

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confidence: 99%

“…Fiber-FISH was done on cells attached to aminosilane-coated microscope slides that were lysed to produce linear DNA fibers (8). The plasmid probe pIG.E1A.E1B was treated with Eco R1 and Bgl II to separate plasmid from insert sequences.…”

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Molecular cytogenetic characterization by combined COBRA-FISH and fiber-FISH: application to PER.C6 ^® cells

et al. 2004

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FISH Fest With the advent of multicolor fluoresence in situ hybridization (FISH) for chromosome staining, looking down the microscope at a metaphase spread can reveal colors almost as festive as an aquarium full of tropical fish. Although chromosome FISH is very useful for identifying translocations and viral integration sites, resolution at the sub-kilobase level requires fiber FISH, a staining technique that uses stretched chromosomes. Now, Wiegant et al. (p. 188) have brought together fiber FISH and combined binary ratio labeling (COBRA) FISH in order to precisely define the stability of a cell line used for production of biopharmaceuticals. PER.C6 cells are adenovirus packaging cells that have been shown to produce high levels of adenoviral vectors free of replication-competent virus, but their safety depends upon the long-term stability of the adenovirus sequences they contain. By using COBRA FISH to detect the integration site of the viral sequences and fiber FISH to establish the copy number of these adenoviral sequences, Wiegant et al. followed the genome stability of this producer cell line for nearly one hundred passages. Their work shows not only the reassuring stability of this cell line, but also reveals the power of this combined FISH method, which provides more information than Southern or PCR analyses.

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Basic Preparative Techniques for Fluorescence In Situ Hybridization

Cited by 2 publications

References 29 publications

High‐resolution FISH on super‐stretched flow‐sorted plant chromosomes

High‐resolution FISH on super‐stretched flow‐sorted plant chromosomes

Molecular cytogenetic characterization by combined COBRA-FISH and fiber-FISH: application to PER.C6 ^® cells

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Basic Preparative Techniques for Fluorescence In Situ Hybridization

Cited by 2 publications

References 29 publications

High‐resolution FISH on super‐stretched flow‐sorted plant chromosomes

High‐resolution FISH on super‐stretched flow‐sorted plant chromosomes

Molecular cytogenetic characterization by combined COBRA-FISH and fiber-FISH: application to PER.C6 ® cells

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Molecular cytogenetic characterization by combined COBRA-FISH and fiber-FISH: application to PER.C6 ^® cells