Skip to main content
NCBI home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1996 Sep 15;24(18):3656–3658. doi: 10.1093/nar/24.18.3656

Detection of HIV-1 DNA in cells and tissue by fluorescent in situ 5'-nuclease assay (FISNA).

B K Patterson 1, D Jiyamapa 1, E Mayrand 1, B Hoff 1, R Abramson 1, P M Garcia 1
PMCID: PMC146143  PMID: 8836201

Abstract

The critical aspects of successful in situ amplification include fixation, permeabilization, amplification and detection. We address these aspects and present a novel detection scheme that eliminates hybridization following amplification. We use the 5'-nuclease activity of Taq polymerase to cleave in situ a 5'-reporter dye from an oligonucleotide probe which hybridizes to the target amplicon during amplification. The 5'-reporter dye is disassociated from the 3'-quenching dye and remains localized by charge interactions. In addition, we describe probe design constraints for 5'-nuclease assays both in solution and in situ. Using this technique, we show the sensitive and specific detection of HIV-1 DNA in cells lines and tissue from HIV-1-infected individuals.

Full Text

The Full Text of this article is available as a PDF (63.9 KB).

Selected References

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

  1. Embretson J., Zupancic M., Ribas J. L., Burke A., Racz P., Tenner-Racz K., Haase A. T. Massive covert infection of helper T lymphocytes and macrophages by HIV during the incubation period of AIDS. Nature. 1993 Mar 25;362(6418):359–362. doi: 10.1038/362359a0. [DOI] [PubMed] [Google Scholar]
  2. Frumkin L. R., Patterson B. K., Leverenz J. B., Agy M. B., Wolinsky S. M., Morton W. R., Corey L. Infection of Macaca nemestrina brain with human immunodeficiency virus type 1. J Gen Virol. 1995 Oct;76(Pt 10):2467–2476. doi: 10.1099/0022-1317-76-10-2467. [DOI] [PubMed] [Google Scholar]
  3. Ho D. D., Neumann A. U., Perelson A. S., Chen W., Leonard J. M., Markowitz M. Rapid turnover of plasma virions and CD4 lymphocytes in HIV-1 infection. Nature. 1995 Jan 12;373(6510):123–126. doi: 10.1038/373123a0. [DOI] [PubMed] [Google Scholar]
  4. Korber B. T., Kunstman K. J., Patterson B. K., Furtado M., McEvilly M. M., Levy R., Wolinsky S. M. Genetic differences between blood- and brain-derived viral sequences from human immunodeficiency virus type 1-infected patients: evidence of conserved elements in the V3 region of the envelope protein of brain-derived sequences. J Virol. 1994 Nov;68(11):7467–7481. doi: 10.1128/jvi.68.11.7467-7481.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Levy J. A., Ramachandran B., Barker E., Guthrie J., Elbeik T. Plasma viral load, CD4+ cell counts, and HIV-1 production by cells. Science. 1996 Feb 2;271(5249):670–671. doi: 10.1126/science.271.5249.670. [DOI] [PubMed] [Google Scholar]
  6. Livak K. J., Flood S. J., Marmaro J., Giusti W., Deetz K. Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridization. PCR Methods Appl. 1995 Jun;4(6):357–362. doi: 10.1101/gr.4.6.357. [DOI] [PubMed] [Google Scholar]
  7. Lyamichev V., Brow M. A., Dahlberg J. E. Structure-specific endonucleolytic cleavage of nucleic acids by eubacterial DNA polymerases. Science. 1993 May 7;260(5109):778–783. doi: 10.1126/science.7683443. [DOI] [PubMed] [Google Scholar]
  8. Mergny J. L., Boutorine A. S., Garestier T., Belloc F., Rougée M., Bulychev N. V., Koshkin A. A., Bourson J., Lebedev A. V., Valeur B. Fluorescence energy transfer as a probe for nucleic acid structures and sequences. Nucleic Acids Res. 1994 Mar 25;22(6):920–928. doi: 10.1093/nar/22.6.920. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Patterson B. K., Goolsby C., Hodara V., Lohman K. L., Wolinsky S. M. Detection of CD4+ T cells harboring human immunodeficiency virus type 1 DNA by flow cytometry using simultaneous immunophenotyping and PCR-driven in situ hybridization: evidence of epitope masking of the CD4 cell surface molecule in vivo. J Virol. 1995 Jul;69(7):4316–4322. doi: 10.1128/jvi.69.7.4316-4322.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Patterson B. K., Till M., Otto P., Goolsby C., Furtado M. R., McBride L. J., Wolinsky S. M. Detection of HIV-1 DNA and messenger RNA in individual cells by PCR-driven in situ hybridization and flow cytometry. Science. 1993 May 14;260(5110):976–979. doi: 10.1126/science.8493534. [DOI] [PubMed] [Google Scholar]
  11. Spira A. I., Marx P. A., Patterson B. K., Mahoney J., Koup R. A., Wolinsky S. M., Ho D. D. Cellular targets of infection and route of viral dissemination after an intravaginal inoculation of simian immunodeficiency virus into rhesus macaques. J Exp Med. 1996 Jan 1;183(1):215–225. doi: 10.1084/jem.183.1.215. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Teo I. A., Shaunak S. PCR in situ: aspects which reduce amplification and generate false-positive results. Histochem J. 1995 Sep;27(9):660–669. [PubMed] [Google Scholar]
  13. Wei X., Ghosh S. K., Taylor M. E., Johnson V. A., Emini E. A., Deutsch P., Lifson J. D., Bonhoeffer S., Nowak M. A., Hahn B. H. Viral dynamics in human immunodeficiency virus type 1 infection. Nature. 1995 Jan 12;373(6510):117–122. doi: 10.1038/373117a0. [DOI] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES