Biofidelity Ltd

Ana Luisa Bras Dos Santos Ribeiro Da Silva Weatherley

Cameron Alexander Frayling

Barnaby Balmforth

Magdalena Stolarek-Januszkiewicz

A method of detecting a target polynucleotide sequence in a given nucleic acid analyte characterised by the steps of: a. annealing the analyte to a single-stranded probe oligonucleotide Ato create a first intermediate product which is at least partially double-stranded and in which the 3' end of Aforms a double-stranded complex with the analyte target sequence; b. pyrophosphorolysing the first intermediate product with a pyrophosphorolysing enzyme in the 3′-5′ direction from the 3′ end of Ato create partially digested strand Aand the analyte; c. (i) annealing Ato a single-stranded trigger oligonucleotide B and extending the Astrand in the 5′-3′ direction against B; or (ii) circularising Athrough ligation of its 3′ and 5′ ends; or (iii) ligating the 3′ end of Ato the 5′ end of a ligation probe oligonucleotide C; in each case to create an oligonucleotide A; d. priming Awith at least one single-stranded primer oligonucleotide and creating multiple copies of A, or a region of A; and e. detecting a signal derived from the multiple copies and inferring therefrom the presence or absence of the polynucleotide target sequence in the analyte.

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Cambridge, United Kingdom

Ana Luisa Bras Dos Santos Ribeiro Da Silva Weatherley