From Structure-Function Analyses to Protein Engineering for Practical Applications of DNA Ligase

Figure 6

Schematic representation of the target-primed Rolling-Circle Amplification (RCA) of circularized padlock probes. (1) 5′–3′ exonucleolysis: the target DNA is restriction digested 3′ of the target sequence and irreversibly made single stranded by strand-specific 5′–3′ exonucleolysis. (2) Hybridization: padlock probes (red) with the tag sequence (green) are hybridized to their target sequences. (3) Ligation & 5′–3′ exonucleolysis: adjacent padlock probes that are perfectly complementary to the target (left) are connected by DNA ligase, thus locking the probe onto the target molecule. After ligation, the RCA is initiated by the Phi29 DNA polymerase, by turning the target molecule into a primer through the 3′–5′ exonucleolysis of any 3′ end protruding beyond the padlock probe hybridization site. The padlock probe then serves as the template for DNA synthesis. The RCA product (black) is detected through the hybridization of fluorescently labeled probes (green) to tag sequences specific for the padlock probe. Arrowheads indicate 3′ ends of the RCA reaction.