Figure 1
From: Visualising single molecules of HIV-1 and miRNA nucleic acids
Overview of the O-FISH mechanism. Target nucleic acids are initially hybridised with a biotintylated complimentary oligonucleotide probe (step 1). The biotin conjugate is then targeted with an anti-biotin monoclonal antibody (mAb; step 2). The proximal ligation assay (PLA) consisting of a + and – mAb, is then employed to target the specific IgG domain of the biotin-bound mAb (step 3). Oligos conjugated to each of the PLA mAbs are then hybridised to form circularised DNA (step 4) and then rolling circle amplification is used to effectively multiply the target sequence (step 5). Fluorescently labelled oligonucleotide probes are then hybridized with the rolling circle amplified DNA allowing the observation of target with fluorescence microscopy.