This disclosure describes related novel methods for
Recombinase-
Polymerase Amplification (RPA) of a target
DNA that
exploit the properties of
recombinase and related proteins, to invade double-stranded
DNA with single stranded homologous
DNA permitting sequence specific priming of
DNA polymerase reactions. The disclosed methods have the
advantage of not requiring thermocycling or thermophilic enzymes, thus offering easy and affordable implementation and portability relative to other amplification methods. Further RPA reactions using light and otherwise, methods to determine the nature of amplified species without a need for
gel electrophoresis, methods to improve and optimize
signal to
noise ratios in RPA reactions, methods to optimize
oligonucleotide primer function, methods to control carry-over
contamination, and methods to employ sequence-specific third ‘specificity’ probes. Further described are novel properties and approaches for use of probes monitored by light in dynamic recombination environments.