Oxford Nanopore Technologies Limited

Daniel George Fordham

James White

Clive Gavin Brown

Daniel John Turner

Andrew John Heron

James Anthony Clarke

Stuart William Reid

Rebecca Victoria Bowen

Richard Alexander Gutierrez

James Edward Graham

Roger Charles Gill

Nicholas Antony Smith

Stuart Reid

Nicholas Smith

The invention relates to a method of determining the presence or absence of a target analyte in a sample. The method comprises immobilising any target analyte present in the sample on a surface; contacting the surface with: (i) a first detection agent that binds specifically to the target analyte; and (ii) a reporter polynucleotide, wherein the reporter polynucleotide is bound to, or binds to, the first detection agent; and contacting a transmembrane pore with any reporter polynucleotide that has been immobilised on the surface, wherein the reporter polynucleotide is immobilised on the surface by binding of the first agent to the target analyte, and using the transmembrane pore to detect the reporter polynucleotide, thereby determining the presence or absence of the target analyte in the sample.

Method of determining the presence or absence of a target analyte comprising using a reporter polynucleotide and a transmembrane pore

The invention provides a method of detecting a target polynucleotide in a sample comprising: (a) contacting the sample with a guide polynucleotide that binds to a sequence in the target polynucleotide and a polynucleotide-guided effector protein, wherein the guide polynucleotide and polynucleotide-guided effector protein form a complex with any target polynucleotide present in the sample; (b) contacting the sample with a membrane comprising a transmembrane pore; (c) applying a potential to the membrane; and (d) monitoring for the presence or absence of an effect resulting from the interaction of the complex with the transmembrane pore to determine the presence or absence of the complex, thereby detecting the target polynucleotide in the sample.

Method for nucleic acid detection by guiding through a nanopore

The invention relates to a new method of determining in a sample the presence or absence of one or more analyte members of a group of two or more analytes. The invention therefore relates to a multiplex assay for determining the presence or absence of each analyte in a group of multiple analytes. The assay uses aptamers and transmembrane pores.

Aptamer method

A method for determining the presence, absence or amount of two or more target polynucleotides in a sample comprising additional components, the method comprising: (i) contacting the sample with a panel of two or more probes under conditions suitable for hybridisation of the target polynucleotides to the probes, wherein: (a) each probe comprises a non-hybridisation region and a hybridisation region that specifically hybridises to one of the target polynucleotides to form a hybridised probe; and (b) the hybridisation region of a probe of the panel comprises one or more non-natural nucleotides; (ii) contacting the sample prepared in step (i) with a transmembrane pore through which a single stranded polynucleotide but not a double stranded polynucleotide can pass and applying a potential difference to the transmembrane pore such that the hybridised probes in the sample interact with the pore; (iii) measuring current blockades having a duration within a defined window, wherein: (a) the one or more non-natural nucleotides present in the hybridisation region of the probe increase or decrease the duration of the current blockade due to the probe hybridised to its target polynucleotide such that the proportion of current blockades that occur within the window due to the interaction of the hybridised probes with the pore is increased compared to when the corresponding one or more natural nucleotides are present in the hybridisation region; and (b) each hybridised probe gives rise to a current blockade indicative of that probe; and (iv) correlating the measured current blockades with the probes, thereby determining the presence, absence or amount of the two or more target polynucleotides in the sample.

Method

The invention relates to a new method of determining in a sample the presence, absence or concentration of one or more target analytes, such as micro-ribonucleic acids (microRNAs or miRNAs). The invention may therefore relate to a multiplex assay for determining the presence or absence of each target analyte in a group of multiple analytess. The invention uses one or more probes comprising a quadruplex and transmembrane pores.

Quadruplex method

Growing community of inventors

Oxford, United Kingdom

Daniel George Fordham