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Detection of HIV-1 DNA in cells and tissue by fluorescent in situ 5'-nuclease assay (FISNA)
B K Patterson1, D Jiyamapa, E Mayrand
1Department of Obstetrics and Gynecology, Northwestern University Medical School, Chicago, IL 60611, USA.
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.