Recombinase polymerase amplification (RPA) is a molecular biology technique used to rapidly amplify specific DNA or RNA sequences under isothermal conditions, meaning it does not require the temperature cycling typically associated with polymerase chain reaction (PCR). RPA is highly sensitive, specific, and can be performed using simple equipment, making it suitable for various applications in diagnostics, environmental monitoring, and research.
The basic principle of RPA involves the use of recombinase enzymes to facilitate the annealing of primers to target DNA or RNA sequences, followed by strand displacement DNA synthesis by a DNA polymerase enzyme. This process leads to the exponential amplification of the target sequence.
Key features of RPA include:
Isothermal amplification: RPA operates at a constant temperature (typically around 37-42°C), eliminating the need for thermal cycling equipment required by PCR.
Recombinase activity: Recombinase enzymes are used to facilitate primer annealing and strand exchange, enabling the amplification process to proceed efficiently.
Primer design: RPA primers are typically short (around 30-35 nucleotides) and designed to specifically bind to target sequences, similar to PCR primers.
Single-stranded DNA binding protein: RPA often utilizes single-stranded DNA binding proteins to stabilize the DNA strands during amplification, increasing the efficiency of the reaction.
Detection: Amplification products can be detected using various methods, such as real-time fluorescence detection, lateral flow strips, or gel electrophoresis.
RPA is particularly advantageous in scenarios where rapid and sensitive nucleic acid detection is required, such as point-of-care diagnostics for infectious diseases, field-based environmental monitoring, and rapid response to disease outbreaks. It has been adapted for use with a variety of sample types, including blood, saliva, urine, and environmental samples.