Recombinase Polymerase Amplification (RPA)

1. Primer Recombination: Recombinase enzymes bind to single-stranded DNA primers, forming nucleoprotein complexes. These complexes scan double-stranded DNA for homologous sequences.
2. Strand Invasion: Once a homologous site is found, the primer-recombinase complex invades the DNA duplex, creating a D-loop structure and separating the strands. This creates a binding site for DNA polymerase.
3. DNA Synthesis and Amplification: A strand-displacing DNA polymerase binds to the primer and initiates synthesis, extending the primer and displacing the template strand. This process repeats, leading to exponential amplification of the target sequence. The key distinctions in the RPA vs PCR comparison are the absence of high-temperature denaturation and the elimination of thermal cycling, which simplifies instrumentation requirements significantly.

1. Prepare the Reaction Mix: Rehydrate the lyophilized RPA pellet, which contains all necessary enzymes, and add your primers and probes.
2. Add the Target Sample: Introduce the purified nucleic acid sample into the reaction mix.
3. Incubate at a Constant Temperature: Place the reaction tubes in a simple heating block or water bath set to a constant temperature, typically between 37°C and 42°C. Incubation times are short, often ranging from 5 to 20 minutes.
4. Detect the Amplified Product: Analyze the results using real-time fluorescence detection or lateral flow strips for end-point analysis.

For optimal results, proper RPA optimization is critical. Test multiple primer designs to ensure specificity and efficiency. Additionally, establish clean workflows to avoid contamination, a common focus of RPA troubleshooting.