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 language | English (US) |
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Pages (from-to) | 3656-3658 |
Number of pages | 3 |
Journal | Nucleic acids research |
Volume | 24 |
Issue number | 18 |
State | Published - 1996 |
ASJC Scopus subject areas
- Genetics