Detection of HIV-1 DNA in cells and tissue by fluorescent in situ 5'-nuclease assay (FISNA).
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.
Patterson
B K
BK
Department of Obstetrics and Gynecology, Northwestern University Medical School, Chicago, IL 60611, USA.
Jiyamapa
D
D
Mayrand
E
E
Hoff
B
B
Abramson
R
R
Garcia
P M
PM
D015720:5'-Nucleotidase / Q000378:metabolism*; D002460:Cell Line; D004279:DNA, Viral / Q000032:analysis*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D015497:HIV-1 / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D008198:Lymph Nodes / Q000821:virology; D016133:Polymerase Chain Reaction / Q000379:methods; D019914:Taq Polymerase
D004279:DNA, Viral; D019914:Taq Polymerase; D004259:DNA-Directed DNA Polymerase; D015720:5'-Nucleotidase