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 causing the cellular proliferation of one or more cell types contained within a microdroplet having an average volume in the range 4 femtolitres to 10 nanolitres and comprised of an aqueous buffer is provided. It is characterised by the step of incubating the cell(s) inside the droplets in suitable environmental conditions and thereafter detecting the number of cells inside each droplet, characterised in that the microdroplets are suspended in an immiscible carrier fluid further comprising 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 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 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 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 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 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.