Schematic diagram for real-time RAA

Recombinase was bound to primers to form complexes, and the complexes searched for a specific binding site on the target DNA at a constant temperature. By utilizing single-stranded binding proteins, the double strand of template DNA is opened, and a single-stranded molecule is formed. In the next step, DNA polymerase was initiated at the 3′ end of the primer and synthesized toward the 5′ end of the target DNA. Hence, two new double-stranded DNA molecules were formed, and the cycle was repeated to achieve amplification (Chen et al., 2021; Wang et al., 2021) (Figure 1A). When nucleic acid exonuclease is not activated, the probe is stable and does not emit fluorescence signals; however, when the probe binds to the target DNA site, exonuclease was activated. Upon recognizing tetrahydrofuran (THF) on the probe, nucleic acid exonuclease cleaves both the reporter group and the burst group, and the reporter group is released, allowing the fluorescence signal to be emitted. As the probe was amplified further to the 3′ end, the blocker was cut off, and the probe continued to amplify to the 5′ end of the target DNA. Consequently, real-time fluorescence signals were gathered and plotted (Figure 1B).

RAA process schematic diagram. (A) Mechanism based on the RAA process. (B) Mechanism of the real-time RAA probe amplification process. Exonuclease was activated when the probe and template DNA strands were bound. Exonuclease recognized tetrahydrofuran (THF) and cut at the site, separating reporter from quenched groups and generating a fluorescence signal to be collected. After the blocker separation, the probe was further extended in the template to complete the amplification. We drew this schematic diagram by Figdraw (www.figdraw.com).