Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1989 Jan;86(2):582–586. doi: 10.1073/pnas.86.2.582

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.

E Viegas-Pequignot 1, B Dutrillaux 1, H Magdelenat 1, M Coppey-Moisan 1
PMCID: PMC286516  PMID: 2643118

Abstract

Two single-copy DNA segments of 6 kilobases (kb) and 2.3 kb were labeled with biotin-labeled dUTP (Bio11-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 18p11.3 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 (less than 0.6 micron), 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.

Full text

PDF
582

Images in this article

584Image
on p.584
585Image
on p.585
585Image
on p.585
585Image
on p.585
585Image
on p.585

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bauman J. G., Wiegant J., Van Duijn P., Lubsen N. H., Sondermeijer P. J., Hennig W., Kubli E. Rapid and high resolution detection of in situ hybridisation to polytene chromosomes using fluorochrome-labeled RNA. Chromosoma. 1981;84(1):1–18. doi: 10.1007/BF00293359. [DOI] [PubMed] [Google Scholar]
  2. Bhatt B., Burns J., Flannery D., McGee J. O. Direct visualization of single copy genes on banded metaphase chromosomes by nonisotopic in situ hybridization. Nucleic Acids Res. 1988 May 11;16(9):3951–3961. doi: 10.1093/nar/16.9.3951. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Burns J., Chan V. T., Jonasson J. A., Fleming K. A., Taylor S., McGee J. O. Sensitive system for visualising biotinylated DNA probes hybridised in situ: rapid sex determination of intact cells. J Clin Pathol. 1985 Oct;38(10):1085–1092. doi: 10.1136/jcp.38.10.1085. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Delattre O., Bernard A., Malfoy B., Marlhens F., Viegas-Pequignot E., Brossard C., Haguenauer O., Creau-Goldberg N., N'guyen V. C., Dutrillaux B. Studies on the human chromosome 3 centromere with a newly cloned alphoid DNA probe. Hum Hered. 1988;38(3):156–167. doi: 10.1159/000153777. [DOI] [PubMed] [Google Scholar]
  5. Garson J. A., van den Berghe J. A., Kemshead J. T. Novel non-isotopic in situ hybridization technique detects small (1 Kb) unique sequences in routinely G-banded human chromosomes: fine mapping of N-myc and beta-NGF genes. Nucleic Acids Res. 1987 Jun 25;15(12):4761–4770. doi: 10.1093/nar/15.12.4761. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Hopman A. H., Wiegant J., Tesser G. I., Van Duijn P. A non-radioactive in situ hybridization method based on mercurated nucleic acid probes and sulfhydryl-hapten ligands. Nucleic Acids Res. 1986 Aug 26;14(16):6471–6488. doi: 10.1093/nar/14.16.6471. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Johnson G. D., Nogueira Araujo G. M. A simple method of reducing the fading of immunofluorescence during microscopy. J Immunol Methods. 1981;43(3):349–350. doi: 10.1016/0022-1759(81)90183-6. [DOI] [PubMed] [Google Scholar]
  8. Landegent J. E., Jasen in de Wal N., Baan R. A., Hoeijmakers J. H., Van der Ploeg M. 2-Acetylaminofluorene-modified probes for the indirect hybridocytochemical detection of specific nucleic acid sequences. Exp Cell Res. 1984 Jul;153(1):61–72. doi: 10.1016/0014-4827(84)90448-8. [DOI] [PubMed] [Google Scholar]
  9. Langer P. R., Waldrop A. A., Ward D. C. Enzymatic synthesis of biotin-labeled polynucleotides: novel nucleic acid affinity probes. Proc Natl Acad Sci U S A. 1981 Nov;78(11):6633–6637. doi: 10.1073/pnas.78.11.6633. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Lawrence J. B., Villnave C. A., Singer R. H. Sensitive, high-resolution chromatin and chromosome mapping in situ: presence and orientation of two closely integrated copies of EBV in a lymphoma line. Cell. 1988 Jan 15;52(1):51–61. doi: 10.1016/0092-8674(88)90530-2. [DOI] [PubMed] [Google Scholar]
  11. Manuelidis L., Langer-Safer P. R., Ward D. C. High-resolution mapping of satellite DNA using biotin-labeled DNA probes. J Cell Biol. 1982 Nov;95(2 Pt 1):619–625. doi: 10.1083/jcb.95.2.619. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Manuelidis L., Ward D. C. Chromosomal and nuclear distribution of the HindIII 1.9-kb human DNA repeat segment. Chromosoma. 1984;91(1):28–38. doi: 10.1007/BF00286482. [DOI] [PubMed] [Google Scholar]
  13. Mattei M. G., Philip N., Passage E., Moisan J. P., Mandel J. L., Mattei J. F. DNA probe localization at 18p113 band by in situ hybridization and identification of a small supernumerary chromosome. Hum Genet. 1985;69(3):268–271. doi: 10.1007/BF00293038. [DOI] [PubMed] [Google Scholar]
  14. Perry P., Wolff S. New Giemsa method for the differential staining of sister chromatids. Nature. 1974 Sep 13;251(5471):156–158. doi: 10.1038/251156a0. [DOI] [PubMed] [Google Scholar]
  15. Pinkel D., Straume T., Gray J. W. Cytogenetic analysis using quantitative, high-sensitivity, fluorescence hybridization. Proc Natl Acad Sci U S A. 1986 May;83(9):2934–2938. doi: 10.1073/pnas.83.9.2934. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Rudkin G. T., Stollar B. D. High resolution detection of DNA-RNA hybrids in situ by indirect immunofluorescence. Nature. 1977 Feb 3;265(5593):472–473. doi: 10.1038/265472a0. [DOI] [PubMed] [Google Scholar]
  17. Tchen P., Fuchs R. P., Sage E., Leng M. Chemically modified nucleic acids as immunodetectable probes in hybridization experiments. Proc Natl Acad Sci U S A. 1984 Jun;81(11):3466–3470. doi: 10.1073/pnas.81.11.3466. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Viegas-Péquignot E., Malfoy B., Leng M., Dutrillaux B., Tchen P. In situ hybridization of an acetylaminofluorene-modified probe recognized by Z-DNA antibodies in vitro. Cytogenet Cell Genet. 1986;42(1-2):105–109. doi: 10.1159/000132260. [DOI] [PubMed] [Google Scholar]
  19. Zhang F. R., Monpezat J. P., Delattre O., Aurias A., Thomas G. RFLP identified by the anonymous DNA segment FZ VI A2 at 22q11.2 [HGM no. D22S20]. Nucleic Acids Res. 1988 Mar 25;16(6):2739–2739. doi: 10.1093/nar/16.6.2739. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES