Abstract: A method for manipulating a microdroplet of a reaction medium in an immiscible carrier medium with a target molecule bound to a solid support for the purposes of effecting a chemical transformation is provided. It is characterised by the steps of (a) bringing the microdroplet into contact with the solid support under conditions where the microdroplet and solid support are caused to combine, (b) allowing the reaction medium to react with the target molecule and (c) thereafter exerting a force to induce the reaction medium to become detached from the solid support and reform a microdroplet in the carrier fluid. In one embodiment the solid support is a particle, bead or the like.

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 (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 device comprising: a first zone comprising an attachment site; a first pathway; a second pathway and a means for creating a second medium comprised of aqueous microdroplets in a carrier; a microdroplet manipulation zone comprising: a first composite wall comprised of a first transparent substrate; a first transparent conductor layer on the substrate; a photoactive layer activated by electromagnetic radiation; and a first dielectric layer on the conductor layer; a second composite wall comprised of a second substrate; a second conductor layer on the substrate; and optionally a second dielectric layer on the conductor layer; an A/C source; a source of first electromagnetic radiation; means for manipulating the points of impingement of the electromagnetic radiation on the photoactive layer; an detection zone disposed downstream of the microdroplet manipulation zone or integral therewith; and a fluorescence or Raman-scattering detection system.

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 device for manipulating microdroplets using optically-mediated electrowetting comprising: a first composite wall comprising: a first transparent substrate; a first transparent conductor layer on the substrate having a thickness of 70 to 250 nm; a photoactive layer activated by electromagnetic radiation in the wavelength range 400-1000 nm on the conductor layer having a thickness of 300-1000 nm; and a first dielectric layer on the conductor layer having a thickness of 120-160 nm; a second composite wall comprised of: a second substrate; a second conductor layer on the substrate having a thickness of 70 to 250 nm; and an A/C source to provide a voltage across the first and second composite walls connecting the first and second conductor layers; at least one source of electromagnetic radiation having an energy higher than the bandgap of the photoexcitable layer; and means for manipulating the points of impingement of the electromagnetic radiation on the photoactive layer.

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: An apparatus for sequencing a nucleic acid by printing droplets at least some of which contain single nucleotides derived from the nucleic acid is provided.

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.

Abstract: Disclosed is a method of identifying the contents of individual droplets in a droplet stream each droplet containing fluorophores in an initial non-fluorescing state characterized by the steps of introducing the droplets one-by-one into at least one open-ended tube to create a stack of droplets therein; activating the fluorophores within the droplets to cause them to fluoresce; releasing each droplet in the droplet stack in turn from the tube and detecting along the major axis of the tube fluorescence associated with each droplet as it emerges.

Abstract: Various method of isolating a modified polynucleotide having the formula A-P-B from a crude mixture of modified polynucleotides also including those having the formulae A-P-A and B-P-B, wherein P is a polynucleotide region and A and B are different modifying moieties or nucleotide sequences are provided. The methods include the steps of (a) reacting the crude mixture concurrently or consecutively with beads of one type capable of binding to moiety A but not B, and beads of a second type capable of binding to moiety B but not A; (b) fractionating the intermediate product based on the properties of each type of bead and of pairs of different types of bead conjoined by A-P-B polynucleotides, such that only or predominantly conjoined pairs of the two different types of bead are retained and (c) optionally, releasing one or both beads from such conjoined pairs such that the polynucleotide may be recovered.

Abstract: 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.

Abstract: An apparatus for sequencing a nucleic acid by printing droplets at least some of which contain single nucleotides derived from the nucleic acid is provided.

Abstract: A method of determining whether a given single nucleotide is methylated or not methylated characterized by the steps of (a) contacting the single nucleotide with one or more hybridization probe types each of which in its unused form; (b) for the relevant probe type causing (i) the single nucleotide to bind to the region resistant to exonucleolytic degradation and the single-stranded region and (ii) the second oligonucleotide to bind to the single nucleotide and the single-stranded nucleotide region; (c) treating the used probe with a methylation-dependent restriction or a methylation-sensitive restriction endonuclease to cleave adjacent the region resistant to exonucleolytic degradation; and thereafter (d) treating the product of step (c) with an exonuclease or a polymerase exhibiting exonuclease activity to liberate either only first or both first and second detectable elements in a detectable state to determine if the single nucleotide is methylated or not.

Abstract: Disclosed is a method of identifying the contents of individual droplets in a droplet stream each droplet containing fluorophores in an initial non-fluorescing state characterised by the steps of introducing the droplets one-by-one into at least one open-ended tube to create a stack of droplets therein; activating the fluorophores within the droplets to cause them to fluoresce; releasing each droplet in the droplet stack in turn from the tube and detecting along the major axis of the tube fluorescence associated with each droplet as it emerges.

Abstract: A method of storing a stream of droplets at least some of which comprise one or more single nucleotides and/or oligonucleotides, and a droplet fluid is provided. It is characterized by the step of introducing each droplet sequentially onto a surface of a substrate at a corresponding unique location and further characterized in that the stream of droplets is prepared by a process which includes the steps of generating an ordered stream of nucleotides from the analyte by progressive pyrophosphorolysis or exo nucleolysis and capturing each nucleotide in a corresponding droplet. The method can advantageously be used in association with microdroplet droplet sequencers and an analysis unit in which the sequence of nucleotides in a precursor polynucleotide analyte is determined using fluorescence spectroscopy. A device for carrying out the method is also described.

Abstract: Disclosed is a method for determining the sequence of nucleotide bases in a polynucleotide analyte characterised by the steps of: a. generating a stream of droplets at least some of which contain a single nucleotide and wherein the order of single nucleotides in the droplet stream corresponds to the sequence of nucleotides in the analyte; b. introducing into each droplet a plurality of biological probe types each type (i) comprising a different detectable element in an undetectable state and (ii) being adapted to capture a different complimentary single nucleotide from which the analyte is constituted; c. causing the single nucleotide contained in the droplet to bind to its complimentary probe to create a used probe; and d. causing the detectable element to be released from the used probe in a detectable state.

Abstract: A method of sequencing a nucleic acid such as DNA or RNA is provided.