Biofidelity Ltd

Other

Magdalena Stolarek-Januszkiewicz

Barnaby Balmforth

Cameron Alexander Frayling

Ana Silva-Weatherley

Robert Osborne

Paulina Powalowska

Ana Luisa Bras Dos Santos Ribeiro Da Silva Weatherley

Barnaby William Balmforth

Paulina Klaudyna Powalowska

Ana Luisa Bras Dos Santos R Da Silva-Weatherley

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.

Polynucleotide sequence detection method

Disclosed is a hybridisation capture method based on the pyrophosphorolysis reaction. According to the present invention, there is provided a method for increasing the ratio of a first nucleic add sequence to second nucleic add sequence in a sample.

Nucleic acid enrichment and detection

Methods of detecting target polynucleotide sequences may include introducing one or more nucleic acid analytes to a first reaction mixture comprising a single-stranded probe oligonucleotide A, a pyrophosphorolysing enzyme, and a ligase. The analyte may anneal to the single-stranded probe oligonucleotide Ato create a first intermediate product which is at least partially double-stranded, where the 3' end of Aforms a double-stranded complex with the analyte and where Ais pyrophosphorylsed in the 3&#x2032;-5&#x2032; direction from the 3&#x2032; end to create at least a partially digested strand A. Amay undergo ligation to form oligonucleotide A. The methods may also include detecting a signal derived from the formed oligonucleotides, and inferring therefrom the presence or absence of the target polynucleotide sequence in the analyte.

Simplified polynucleotide sequence detection method

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&#x2032;-5&#x2032; direction from the 3&#x2032; 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&#x2032;-3&#x2032; direction against B; or (ii) circularising Athrough ligation of its 3&#x2032; and 5&#x2032; ends; or (iii) ligating the 3&#x2032; end of Ato the 5&#x2032; 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

Magdalena Stolarek-Januszkiewicz