Abstract: 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 (

Abstract: 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 acid sequence to second nucleic acid sequence in a sample.

Abstract: 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.

Abstract: 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 aw1 of 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.

Abstract: 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 aw1 of 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.

Abstract: 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 acid sequence to second nucleic acid sequence in a sample.

Abstract: 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 A0, a pyrophosphorolysing enzyme, and a ligase. The analyte may anneal to the single-stranded probe oligonucleotide A0 to create a first intermediate product which is at least partially double-stranded, where the 3? end of A0 forms a double-stranded complex with the analyte and where A0 is pyrophosphorylsed in the 3?-5? direction from the 3? end to create at least a partially digested strand A1. A1 may undergo ligation to form oligonucleotide A2. 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.

Abstract: 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 aw1 of 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.

Abstract: 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 A0, a pyrophosphorolysing enzyme, and a ligase. The analyte may anneal to the single-stranded probe oligonucleotide A0 to create a first intermediate product which is at least partially double-stranded, where the 3? end of A0 forms a double-stranded complex with the analyte and where A0 is pyrophosphorylsed in the 3?-5? direction from the 3? end to create at least a partially digested strand A1. A1 may undergo ligation to form oligonucleotide A2. 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.

Abstract: 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 A0 to create a first intermediate product which is at least partially double-stranded and in which the 3? end of A0 forms 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 A0 to create partially digested strand A1 and the analyte; c. (i) annealing A1 to a single-stranded trigger oligonucleotide B and extending the A1 strand in the 5?-3? direction against B; or (ii) circularising A1 through ligation of its 3? and 5? ends; or (iii) ligating the 3? end of A1 to the 5? end of a ligation probe oligonucleotide C; in each case to create an oligonucleotide A2; d. priming A2 with at least one single-stranded primer oligonucleotide and creating multiple copies of A2, or a region of A2; and e.

Abstract: 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 A0 to create a first intermediate product which is at least partially double-stranded and in which the 3? end of A0 forms 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 A0 to create partially digested strand A1 and the analyte; c. (i) annealing A1 to a single-stranded trigger oligonucleotide B and extending the A1 strand in the 5?-3? direction against B; or (ii) circularising A1 through ligation of its 3? and 5? ends; or (iii) ligating the 3? end of A1 to the 5? end of a ligation probe oligonucleotide C; in each case to create an oligonucleotide A2; d. priming A2 with at least one single-stranded primer oligonucleotide and creating multiple copies of A2, or a region of A2; and e.

Abstract: 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 A0 to create a first intermediate product which is at least partially double-stranded and in which the 3? end of A0 forms 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 A0 to create partially digested strand A1 and the analyte; c. (i) annealing A1 to a single-stranded trigger oligonucleotide B and extending the A1 strand in the 5?-3? direction against B; or (ii) circularising A1 through ligation of its 3? and 5? ends; or (iii) ligating the 3? end of A1 to the 5? end of a ligation probe oligonucleotide C; in each case to create an oligonucleotide A2; d. priming A2 with at least one single-stranded primer oligonucleotide and creating multiple copies of A2, or a region of A2; and e.

Abstract: A method of detecting whether the nucleobase of a given nucleoside triphosphate molecule in an analyte includes a given chemical or structural modification comprising (1) reacting the analyte in the presence of a polymerase with a biological probe comprised of (a) a pair of single-stranded first oligonucleotides each comprising an exonuclease blocking-site; a restriction endonuclease recognition-site; a single nucleotide capture-site and respectively first and second fluorophore(s) in a substantially undetectable state, and (b) at least one second single-stranded oligonucleotide to create a used-probe duplex; (2) reacting the duplex with a restriction endonuclease system comprised of at least one restriction endonuclease; (3) creating another used-probe duplex; (4) digesting the first oligonucleotide elements with an enzyme having 5?-3? exonucleolytic activity to produce fluorophore(s) in a detectable state; and (5) detecting the fluorophore(s) released.

Abstract: A method of sequencing a nucleic acid comprising the steps of (1) generating a stream of single nucleoside triphosphates; (2) producing at least one substantially double-stranded primary oligonucleotide used probe by reacting with a corresponding primary probe comprising (a) a first single-stranded oligonucleotide including a restriction endonuclease nicking-site, a single nucleotide capture site, and oligonucleotide flanking regions.

Abstract: 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 (

Abstract: 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.

Abstract: 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.

Abstract: 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)

Abstract: 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.

Abstract: 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.