Focus on multiplexing using Recombinase Polymerase Amplification (RPA).
RPA is an isothermal nucleic acid amplification technology which works with a single primer pair, similar to PCR. A number of alternative isothermal methods, including LAMP, require more than two primers per target. As only two primers are required for RPA, multiplexing is relatively simple.
Recombinase Polymerase Amplification (RPA) an isothermal technique suitable for a range of applications - Evaluation of RPA volume tolerance.
In this application note we demonstrate RPA performance in reactions established in quite different total reaction volumes and performed in standard PCR tubes (0.2ml) without oil overlays or any other specialised consumables.
Evaluation of isothermal Recombinase Polymerase Amplification incubation temperature tolerance
RPA works at an optimal temperature range of 39-42˚C, typically in 3-15 minutes. Low temperature amplification reduces hardware requirements. The tolerance to further reduced temperatures was explored.
PCRD is a rapid, safe and sensitive alternative to ethidium bromide staining of agarose gels. A generic protocol for detection of RPA products on the PCRD is presented; better sensitivity and specificity for particular targets may be obtained by altering the dilution of amplicon in step 2.