High resolution detection of DNA–RNA hybrids in situ by indirect immunofluorescence

Rudkin, George T.; Stollar, B D

doi:10.1038/265472a0

Cited by 278 publications

(130 citation statements)

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“…These procedures already show their superiority over autoradiographic techniques with respect to convenience and topological resolution (2, 5,27,34,39,43,47). However, the sensitivity of the nonautoradiographic procedures has not yet reached the level attainable with procedures using radioisotopes.…”

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Denaturation, renaturation, and loss of DNA during in situ hybridization procedures

et al. 1986

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With the aim of optimizing in situ hybridization methods, alkaline, acid, and thermal denaturation procedures have been studied for their ability to separate the DNA strands of nuclear DNA and for the DNA losses they induce. Isolated methanol/acetic acid-fixed mouse liver nuclei have been used as a biological object. The results, obtained with acridine orange staining and microfluorometry, show that all denaturations studied lead to almost complete strand separation. Quantitative DNA staining and cytometry indicated that with heat and alkaline denaturation about 40% of the DNA is lost. Acid denaturation led to about 20% DNA loss. For the alkaline denaturation, the DNA retention could be improved to a 20% DNA loss by adding 70% ethanol to the denaturation medium. During hybridization, another 20% DNA loss occurs.When denatured nuclei are brought under annealing conditions, a rapid renaturation of a considerable fraction of the remaining DNA occurs. The extent of renaturation was dependent on the type of denaturation used. For the ethanolic alkaline denaturation, it was estimated to be 35% Quantitative nonautoradiographic in situ hybridization experiments with acetylaminofluorene-modified mouse satellite DNA showed that alkaline denaturation procedures are superior to the heat and acid denaturation.As proven by acridine orange fluorescence measurements, hybridization conditions can be designed that permit DNA.RNA hybridization under in situ DNA.DNA denaturing conditions. These conditions should be very useful, especially for in situ hybridization with single-stranded RNA probes.Key terms: Acetyl aminofluorene, cytometry, acridine orange, gallocyanin chrome alum, nonradioactive hybridizationIn recent years, DNA and RNA labeling techniques have been developed to permit nonautoradiographic visualization of specific nucleic acid sequences in microscopic preparations. These procedures already show their superiority over autoradiographic techniques with respect to convenience and topological resolution (2,5,27,34,39,43,47). However, the sensitivity of the nonautoradiographic procedures has not yet reached the level attainable with procedures using radioisotopes. For example, by using (cloned) nucleic acid probes of high specific radioactivity (1-6 x lo7 3H d p d p g ) and exposure times of 1 to 4 weeks, it is possible, by statistical analysis of 10 to 100 metaphase plates, to assign unique DNA sequences of 1.0 to 15.0 kb to specific chromosomal locations (18,23,36,41) a sensitivity that is not yet obtained using nonautoradiographic procedures.Consequently, attempts are being made to increase the sensitivity of the nonautoradiographic methods. Improvement of sensitivity can be envisaged at the level of the nucleic acid hybridization reaction, the immunological detection system, and the microscope (28). However, the result of an in situ hybridization procedure is also dependent on processes such as denaturation, renaturation, and loss of DNA. We have analyzed these processes for several denaturations to get more informati...

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Denaturation, renaturation, and loss of DNA during in situ hybridization procedures

et al. 1986

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“…20,21 En 1996 dos grupos independientes desarrollaron técnicas derivadas del FISH, el cariotipo espectral (spectral karyotype, SKY) y el multi-FISH (M-FISH), que permiten identificar cada cromosoma con un color diferente. 22,23 La alta complejidad de los cariotipos de algunos cánceres y, fundamentalmente, la dificultad para obtener buenas preparaciones cromosómi-cas en los tumores sólidos, llevaron en 1992 al desarrollo de otra técnica derivada del FISH, la hibridación genómica comparativa (comparative genomic hybridization, CGH), que permitió la detección de desbalances genómicos, sin la necesidad de tener células en cultivo de la muestra a investigar.…”

Section: Introductionunclassified

Rol de la citogenética en pediatría

Gallego¹

2011

Arch Argent Pediat

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“…At the indirect way of labelling, chemically modified nucleotides are incorporated into DNA probes (Rudkin & 1977;Langer et al 1981;Raap et al 1989). The basic principle is that biotin or digoxigenin are covalently bound to dUTP that incorporates into the probe sequence instead of thymine.…”

Section: Indirect Way Of Labellingmentioning

confidence: 99%

Fluorescence in situ hybridisation

Lakatosova

Holečková

2007

Biologia

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Fluorescence in situ hybridisation (FISH) is a rapid and reliable technique for chromosomal investigations that is used for a wide variety of cytogenetic purposes at present. This molecular-cytogenetic method has been developed continuously for many years. As a consequence, various modifications with different kinds of fluorescently labelled probes have been introduced to optimise the detection of DNA and RNA sequences. This review articlepaper presents the general principles of in situ hybridisation, probe labelling and examples of proper use of different kinds of probes. In addition, some newer FISH methods and their usefulness in human molecular cytogenetics are described.

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High resolution detection of DNA–RNA hybrids in situ by indirect immunofluorescence

Cited by 278 publications

References 10 publications

Denaturation, renaturation, and loss of DNA during in situ hybridization procedures

Denaturation, renaturation, and loss of DNA during in situ hybridization procedures

Rol de la citogenética en pediatría

Fluorescence in situ hybridisation

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