Base4 Innovation Limited

Lightcast Discovery Ltd

Biofidelity Ltd

United Kingdom Research and Innovation

Other

Medical Research Council

Barnaby Balmforth

Cameron Alexander Frayling

Bruno Flavio Nogueira De Sousa Soares

Michele Amasio

Thomas Henry Isaac

Boris Breiner

Alessandra Natale

Magdalena Stolarek-Januszkiewicz

Paul Dear

Ana Silva-Weatherley

Gareth Podd

Tom Isaac

Kerr Francis Johnson

Maciej Sosna

Richard Ingham

Mark Dethlefsen

Jasmin Conterio

Bruno Flavio Soares

Robert Osborne

Ana Luisa Bras Dos Santos Ribeiro Da Silva Weatherley

Paulina Powalowska

Ana Luisa Bras Dos Santos Ribeiro Da Silva-Weatherley

Ana Luisa Bras Dos Santos Ribeiro Da Silva

A method of sequencing a nucleic acid comprising (1) generating a stream of single nucleoside triphosphates by progressive enzymatic digestion of the nucleic acid; (2) producing at least one oligonucleotide used probe by reacting, in the presence of a polymerase, at least one of the single nucleoside triphosphates with a corresponding biological probe comprising (a) a first single-stranded oligonucleotide including an exonuclease blocking-site, a restriction endonuclease recognition-site located on the 5' side of the blocking-site and including a single nucleotide capture-site e, and at least one fluorophore region and (b) a second and optionally a third single-stranded oligonucleotide each separate from the first oligonucleotide; (3) cleaving the first oligonucleotide strand of the used probe at the recognition-site with a restriction endonuclease; (4) digesting the first oligonucleotide component with an enzyme to yield fluorophores in a detectable state and (5) detecting the fluorophores released in step (4).

Single nucleotide detection method and associated probes

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

A method of manipulating microdroplets having an average volume in the range 0.5 femtolitres to 10 nanolitres comprised of at least one biological component and a first aqueous medium having a water activity of aof less than 1 is provided. It is characterised by the step of maintaining the microdroplets in a water-immiscible carrier fluid which further includes secondary droplets having an average volume less than 25% of the average volume of the microdroplets up to and including a maximum of 4 femtolitres and wherein the volume ratio of carrier fluid to total volume of microdroplets per unit volume of the total is greater than 2:1. The method may be employed for example with microdroplets containing biological cells or with microdroplets containing single nucleoside phosphate such as are prepared in a droplet-based nucleic acid sequencer. The method is suitable for controlling for example cellular, chemical or enzymatic processes and/or microdroplet size in microdroplets or single nucleotide nucleic acid sequencing.

Microdroplet manipulation method

A method of sequencing a nucleic acid comprising (1) generating a stream of single nucleoside triphosphates by progressive enzymatic digestion of the nucleic acid; (2) producing at least one oligonucleotide used probe by reacting, in the presence of a polymerase, at least one of the single nucleoside triphosphates with a corresponding biological probe comprising (a) a first single-stranded oligonucleotide including an exonuclease blocking-site, a restriction endonuclease recognition-site located on the 5′ side of the blocking-site and including a single nucleotide capture-site e, and at least one fluorophore region and (b) a second and optionally a third single-stranded oligonucleotide each separate from the first oligonucleotide; (3) cleaving the first oligonucleotide strand of the used probe at the recognition-site with a restriction endonuclease; (4) digesting the first oligonucleotide component with an enzyme to yield fluorophores in a detectable state and (5) detecting the fluorophores released in step (4).

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.

A microfluidic sequencing device in which a stream of microdroplets at least some of which contain a single nucleotide base are made to undergo reaction with a capture system to capture and detect an ordered sequence of single nucleotide bases generated by progressive pyrophosphorolysis.

Microfluidic device for characterzing polynucleotides

A method for determining the sequence of nucleotide bases in a polynucleotide analyte is provided. It is characterised by the steps of (1) generating a stream of single nucleotide bases from the analyte by pyrophosphorolysis; (2) producing captured molecules by reacting each single nucleotide base with a capture system labelled with detectable elements in an undetectable state; (3) releasing the detectable elements from each captured molecule in a detectable state and (4) detecting the detectable elements so released and determining the sequence of nucleotide bases therefrom. The method can be used advantageously in sequencers involving the use of microdroplets.

Single nucleotide detection method

A method of analysing a single nucleoside triphosphate comprising: (1) producing at least one substantially double-stranded oligonucleotide used probe by reacting in the presence of a polymerase and a ligase the single nucleoside triphosphate with a corresponding probe system comprising (a) a first single-stranded oligonucleotide labelled with detectable elements in an undetectable state and (b) second and third single-stranded oligonucleotides capable of hybridising to complementary regions on the first oligonucleotide; (2) digesting the used probe with an enzyme having double-stranded exonucleolytic activity to yield the detectable elements in a detectable state and a single-stranded fourth oligonucleotide which is at least in part the sequence complement of the first oligonucleotide; (3) reacting the fourth oligonucleotide with another first oligonucleotide to produce a substantially double-stranded oligonucleotide product corresponding to the used probe; (4) repeating steps (2) and (3) in a cycle and (5) detecting the detectable elements released in each iteration.

A probe system comprising (a) a first single-stranded oligonucleotide labelled with detectable elements in an undetectable state, and (b) second and third single-stranded oligonucleotides capable of hybridising to complementary regions on the first oligonucleotide.

A method for determining the sequence of nucleotide bases in a polynucleotide analyte is provided. It is characterised by the steps of (1) generating a stream of single nucleotide bases from the analyte; (2) producing captured molecules by reacting each single nucleotide base with a capture system; (3) amplifying at least part of the captured molecule to produce a plurality of amplicons characteristic of the single nucleotide base; (4) labelling the amplicons with a corresponding probe having a characteristic detectable element and (5) detecting a property characteristic of the detectable element.

A method for characterising a DNA analyte comprised of one or more polynucleotide types characteristic of a site of nucleotide polymorphism each of which includes a target region having the formula -X-Y-Z- wherein X and Z are respectively first and second characteristic flanking oligonucleotide regions and Y is one of the variants constituting the site is provided. The method is characterised by the steps of; (a) reacting a single-stranded oligonucleotide including the target region derived from at least one of the polynucleotide types with a set of unused probes comprised of (i) a single-stranded first aptamer terminating at its 3' end in a sequence complementary to that of -X or -X-Y and (ii) one or more second single-stranded aptamers terminating at their 5&#x2032; end in a sequence complementary to that of -Z-Y or -Z (as the case may be) and labelled with detectable elements which are in an undetectable state in the presence of a ligase to create a substantially double-stranded used probe comprised of the oligonucleotide, first aptamer and one of the second aptamers; (b) wholly or in part digesting the used probe with an exonuclease or polymerase exhibiting exonuclease activity in a 3&#x2032; to 5&#x2032; direction into its constituent single nucleotides at least one of which includes a detectable element now in a detectable state and (c) thereafter detecting the detection property associated with the now detectable element thereby identifying the nature of the Y variant and therefore the allele it gives rise to. A second mirror-image method is also disclosed. Also provided are vesicles in which the method can be carried out. The method is suitable for a range of diagnostic screening applications including the detection of mutant alleles associated with genetic disorders and cancer.

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Cambridge, United Kingdom

Barnaby Balmforth