Recombinase Polymerase Amplification in Minimally Buffered Conditions

Application of recombinase polymerase amplification (RPA) for pH-based detection of DNA amplification has been investigated. Commercial RPA kits from TwistDx are modified to minimize their pH buffering capacity. Due to the RPA's unique biochemistry, removal of tris from the amplification kit is not enough to lower the buffering capacity of the RPA assay. Even in the absence of tris, RPA components in the commercial kit intrinsically buffer the pH. We show different strategies to minimize the buffering capacity of the RPA kit, while maintaining the amplification efficiency. Even in minimally buffered conditions, it is noticed that RPA's amplification yield is not high enough to overcome the assay's intrinsic buffering capacity. The effect of pyrophosphate precipitation in RPA on the reaction's pH have also been addressed. In conclusion, this work highlights strategies and considerations for the development of pH-based assays from nucleic acid amplification methods which involve ancillary enzymes that catalyze nucleotide hydrolysis.