Rapid and high resolution detection of in situ hybridisation to polytene chromosomes using fluorochrome-labeled RNA

Bauman, J. G. J.; Wiegant, J.; Duijn, P. Van; Lubsen, Nicolette H.; Sondermeijer, P. J. A.; Hennig, Wolfgang; Kubli, Eric

doi:10.1007/bf00293359

Cited by 30 publications

(8 citation statements)

References 37 publications

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“…Panel d shows specific staining of the single band that contains 5S DNA (red channel). Copy of figure 1 (c, d) from Bauman et al [29]. …”

Section: Figmentioning

confidence: 99%

The origin of in situ hybridization – A personal history

Gall

2016

Methods

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In situ hybridization is the technique by which specific RNA or DNA molecules are detected in cytological preparations. Basically it involves formation of a hybrid molecule between an endogenous single-stranded RNA or DNA in the cell and a complementary single-stranded RNA or DNA probe. In its original form the probe was labeled with 3H and the hybrid was detected by autoradiography. The first successful experiments in 1968 involved detection of the highly amplified ribosomal DNA in oocytes of the frog Xenopus, followed soon after by the reiterated “satellite DNA” in mouse and Drosophila chromosomes. Fluorescent probes were developed about ten years later.

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“…Panel d shows specific staining of the single band that contains 5S DNA (red channel). Copy of figure 1 (c, d) from Bauman et al [29]. …”

Section: Figmentioning

confidence: 99%

The origin of in situ hybridization – A personal history

Gall

2016

Methods

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“…Considerable efforts have been devoted to the development of in situ hybridization methods using nonradioactive probes (1)(2)(3)(4)(5). The main objective was to simplify and shorten the handling while increasing the sensitivity of the detection and resolution in comparison to conventional radioactive procedures.…”

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confidence: 99%

Mapping of single-copy DNA sequences on human chromosomes by in situ hybridization with biotinylated probes: enhancement of detection sensitivity by intensified-fluorescence digital-imaging microscopy.

Viegas‐Péquignot

Dutrillaux

Magdelénat

et al. 1989

Proc. Natl. Acad. Sci. U.S.A.

127

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Two single-copy DNA segments of 6 kilobases (kb) and 2.3 kb were labeled with biotin-labeled dUTP (Bio-11-dUTP) and hybridized to human chromosomes. These probes were detected by immunofluorescence and directly mapped on chromosomes by using classical fluorescence microscopy and a microchannel-plate-intensified video camera. By a subsequent R-banding, the 6-kb and 2.3-kb fragments were precisely localized to the 18pll.3 band and to the 22q11.2 band, respectively, in agreement with previous results obtained with radioactive probes. The adaptation of fluorescence intensification and digital image processing (frame integration to enhance signal-to-noise ratio and linear contrast stretching) to microscopy makes it possible to detect very weak fluorescent spots on chromosomes. This system allows a high spatial resolution (<0.6 ,Lm), even at very low fluorescence levels. The efficiency and the specificity of the hybridization and detection methodology give a direct and precise localization of the short single-copy sequences on human chromosomes.Considerable efforts have been devoted to the development of in situ hybridization methods using nonradioactive probes (1-5). The main objective was to simplify and shorten the handling while increasing the sensitivity of the detection and resolution in comparison to conventional radioactive procedures. At present large genomic fragments and highly repeated sequences can be easily and reproducibly mapped on chromosomes and interphase nuclei (6)(7)(8)(9)(10)(11)(12). Nevertheless, only a few results on the localization of single-copy genes or DNA sequences shorter than 10 kilobases (kb) have yet been reported for human chromosomes or nuclei. A 7-kb insert from Epstein-Barr viral DNA was detected by immunofluorescence in 70% of the metaphase cells and two copies of a 5-kb fragment of the same viral DNA were detected in interphase nuclei (11). By using alkaline phosphatase detection and a statistical analysis similar to that employed for radioactive probe, Garson et al. (13) reported the identification of a 7-kb nerve growth factor gene target inserted in pBr322 in =50% ofthe cells analyzed and a 1-kb N-myc insert in plasmid pNB-1 in 15-33% of the metaphase cells. Bhatt et al. (14) confirmed the localization of NRAS and alantitrypsin genes on the human karyotype by gold reaction and silver intensification.The feasibility of detecting and precisely localizing nonradioactive single-copy sequences in chromosomes and nuclei relies upon the specificity of the hybridization procedure and the sensitivity of the detection. We report here the results of a highly specific in situ hybridization methodology and the adaptation of a probe-detection system based on amplifiedfluorescence digital-imaging microscopy. We have used two biotinylated probes, 6 kb and 2.3 kb, that hybridize with single DNA sequences on chromosomes 18 and 22, respectively (15)(16)(17). These biotinylated probes were detected by immunofluorescence after in situ hybridization and localized to bands on chromosome...

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“…Cheung et aL (13) generated a fluorescent signal by coupling latex microspheres containing both poly(U) and dansylated fluorochromes to a polyadenylylated mRNA probe. Fluorescent signals were also produced by Rudkin and Stollar (14) by using antibodies against DNA-RNA hybrids in conjunction with an immunofluorescent antibody sandwich and by Bauman et aL (15)(16)(17) who used RNAs that were labeled at the 3' end with fluorescein or rhodamine. Davidson and associates (18)(19)(20) chemically crosslinked biotin to RNA with cytochrome c or polyamine bridges and used these RNA-biotin complexes as hybridization probes.…”

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Immunological method for mapping genes on Drosophila polytene chromosomes.

Langer-Safer¹,

Levine²,

Ward³

1982

Proc. Natl. Acad. Sci. U.S.A.

679

289

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A method is described for localizing DNA sequences hybridized in situ to Drosophila polytene chromosomes. This procedure utilizes a biotin-labeled analog of TTP that can be incorporated enzymatically into DNA probes by nick-translation. After hybridization in situ, the biotin molecules in the probe serve as antigens which bind affinity-purified rabbit antibiotin antibodies. The site of hybridization is then detected either fluorimetrically, by using fluorescein-labeled goat anti-rabbit IgG, or cytochemically, by using an anti-rabbit IgG antibody conjugated to horseradish peroxidase. When combined with Giemsa staining, the immunoperoxidase detection method provides a permanent record that is suitable for detailed cytogenetic analysis. This immunological approach offers four advantages over conventional autoradiographic procedures for detecting in situ hybrids: (i) the time required to determine the site of hybridization is decreased markedly; (ii) biotin-labeled probes are chemically stable and give -reproducible results for many months; (iii) biotin-labeled probes appear to produce less background noise than do radiolabeled probes; and (iv) the resolving power is equal to and often greater than that achieved autoradiographically.In situ hybridization, initially developed by Gall and Pardue (1) and John et at (2), has proven to be a valuable method for determining the cellular or chromosomal location of hybridized nucleic acids (3)(4)(5)(6)(7)(8)(9)(10). Standard in situ hybridization protocols use radiolabeled RNA or DNA probes and autoradiographic methods of detection or quantification. By using probes of high specific activity under conditions such that hybridization "networks" are formed (8-12), it is now possible to localize unique sequences in mammalian chromosome spreads after autoradiographic exposures of 5-22 days (9, 10). However, the inherent drawbacks of radiolabeled probes-notably chemical lability due to radiolytic decomposition, concern for personnel safety, and disposal problems-make it desirable to have sensitive methods for detecting polynucleotide sequences that do not rely on the use of radioisotopes, especially for routine applications in clinical medicine. Several

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Rapid and high resolution detection of in situ hybridisation to polytene chromosomes using fluorochrome-labeled RNA

Cited by 30 publications

References 37 publications

The origin of in situ hybridization – A personal history

The origin of in situ hybridization – A personal history

Mapping of single-copy DNA sequences on human chromosomes by in situ hybridization with biotinylated probes: enhancement of detection sensitivity by intensified-fluorescence digital-imaging microscopy.

Immunological method for mapping genes on Drosophila polytene chromosomes.

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