Hyperthermophilic polymerase enabled proximity extension assay

Abstract

The present invention relates to a proximity probe based detection assay ('proximity assay') for an analyte in a sample, specifically a proximity probe extension assay (PEA), an in particular to an improvement in the method to reduce non-specific 'background' signals, wherein the improvement comprises the use in such assays of a hyperthermophilic polymerase, said method comprising: (a) contacting said sample with at least one set of at least first and second proximity probes, which probes each comprise an analyte-binding domain and a nucleic acid domain and can simultaneously bind to the analyte; (b) allowing the nucleic acid domains of the proximity probes to interact with each other upon binding of said proximity probes to said analyte, wherein said interaction comprises the formation of a duplex; (c) extending the 3' end of at least one nucleic acid domain of said duplex to generate an extension product, wherein the extension reaction comprises increasing the temperature of assay above room temperature and uses a polymerase enzyme which is characterised as having less than 20% of its maximal enzyme activity at 40° C. and having less than 10% of its maximal enzyme activity at 25° C., wherein the optimum temperature for maximal activity of the polymerase is more than 40° C. and wherein the polymerase is selected from(Pfu) DNA polymerase and(Pwo) DNA polymerase or a derivative or mutant thereof, preferably wherein said derivative is a sequence-modified derivative; and (d) amplifying and detecting the extension product.