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

Bruce K. Patierson, Daniel Jiyamapa, Eric Mayrand, Bud Hoff, Richard Abramson, Patricia M. Garcia

Research output: Contribution to journalArticlepeer-review

11 Scopus citations

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 Tag 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.

Original languageEnglish (US)
Pages (from-to)3656-3658
Number of pages3
JournalNucleic acids research
Volume24
Issue number18
StatePublished - 1996

ASJC Scopus subject areas

  • Genetics

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