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.

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

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 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 polynucleotide analyte is provided and comprises; •a first zone in which a stream of single nucleotides is generated by progressive digestion of a molecule of the analyte attached to a particle located therein and exposed to a flowing aqueous medium; •a second zone in which a corresponding stream of aqueous droplets is generated from the aqueous medium and the nucleotide stream and wherein at least some of the droplets contain a single nucleotide and •a third zone in which each droplet is stored and/or interrogated to reveal a property characteristic of the single nucleotide it may contain; characterised in that the first zone comprises a microfluidic channel through which the aqueous medium flows and the location comprises a hollow seating in a wall thereof to which suction can be applied and into which the particle can be close-fitted.

Abstract: A method of sequencing a nucleic acid involves (1) generating single nucleoside triphosphates by progressive pyrophosphorolysis of the nucleic acid; (2) producing a substantially double-stranded oligonucleotide used probe by reacting one of the single nucleoside triphosphates with a corresponding probe system comprising (a) a first single-stranded oligonucleotide labelled with characteristic detectable elements in an undetectable state and (b) second and third single-stranded oligonucleotides capable of hybridising to complementary regions on the first oligonucleotide; (3) 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 complement of the first oligonucleotide; (4) reacting the fourth oligonucleotide with another first oligonucleotide to produce a substantially double-stranded oligonucleotide product corresponding to the used probe; (5) repeati

Abstract: A method for determining the sequence of nucleotide bases in a polynucleotide analyte is provided. It is characterized 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: Disclosed is a method for sequencing a polynucleotide analyte comprising: •a. generating a stream of droplets containing a single nucleotide 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 comprising a different label in an undetectable state and being adapted to capture a different single nucleotide; •c. causing the single nucleotide contained in the droplet to bind to its complementary probe and •d. causing the label to be released from the probe that has bound the nucleotide in a detectable state. The probe is a dumbbell shaped probe comprising fluorescent donor and quencher labels and a single nucleotide gap. After gap repair by a polymerase and a ligase, a restriction enzyme recognition site is cleaved by a restriction enzyme, followed by exonuclease digestion to release the labels.

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

Abstract: The present invention provides an apparatus for analyzing the sequence of nucleotides in a nucleic acid sample, said apparatus comprising a substrate and a plurality of nanopores provided therein suitable for the passage of nucleic acid molecules therethrough; at least one sample holding chamber disposed upstream of the inlet of said nanopores, at least one detection window juxtaposed within or downstream of the outlet of each nanopore adapted to detect a property characteristic of one or more detectable elements associated with the nucleic acid as each nucleic acid molecule passes therethrough and a detector adapted to generate a data stream characteristic of the various detection events occurring in the detection window characterized in that the apparatus further comprises a means located within the sample holding chamber adapted to increase the local concentration of the nucleic acid sample adjacent the inlet of the nanopores relative to the bulk concentration thereof.

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 characterised by the step of introducing each droplet sequentially onto a surface of a substrate at a corresponding unique location and further characterised 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: 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: 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: Disclosed is a method for determining the sequence of nucleotide bases in a polynucleotide analyte. It is characterised by analyte characterised by the steps of: a. generating a stream of droplets at least some of which comprise both (1) a single nucleotide base and (2) colloidal metal particles capable of undergoing plasmon resonance and b. irradiating each droplet with electromagnetic radiation to cause (1) the metal particles also contained therein to undergo plasmon resonance and (2) the nucleotide base contained therein to Raman scatter light at one or more wavelengths characteristic of its type. Suitably, the order of the single nucleotides bases in the droplet stream corresponds to the sequence of nucleotide bases in the analyte.

Abstract: Disclosed is a biological probe characterised in that it comprises a single-stranded nucleotide region the ends of which are attached to two different oligonucleotide regions wherein at least one of the oligonucleotide regions comprises detectable elements having a characteristic detection property and wherein the detectable elements are so arranged on the oligonucleotide region that the detectable property is less detectable than when the same number detectable elements are bound to a corresponding number of single nucleotides. The biological probe is especially useful for capturing single nucleotides or single-stranded nucleotides to create a used probe which can be degraded by means of a restriction enzyme and an exonuclease to generate single nucleotides carrying a detectable element in a form which can be detected.

Abstract: Disclosed is a method for sequencing a polynucleotide analyte comprising:•a. generating a stream of droplets containing a single nucleotide 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 comprising a different label in an undetectable state and being adapted to capture a different single nucleotide;•c. causing the single nucleotide contained in the droplet to bind to its complementary probe and•d. causing the label to be released from the probe that has bound the nucleotide in a detectable state. The probe is a dumbbell shaped probe comprising fluorescent donor and quencher labels and a single nucleotide gap. After gap repair by a polymerase and a ligase, a restriction enzyme recognition site is cleaved by a restriction enzyme, followed by exonuclease digestion to release the labels.

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: The present invention provides an apparatus for analysing the sequence of nucleotides in a nucleic acid sample, said apparatus comprising a substrate and a plurality of nanopores provided therein suitable for the passage of nucleic acid molecules therethrough; at least one sample holding chamber disposed upstream of the inlet of said nanopores, at least one detection window juxtaposed within or downstream of the outlet of each nanopore adapted to detect a property characteristic of one or more detectable elements associated with the nucleic acid as each nucleic acid molecule passes therethrough and a detector adapted to generate a data stream characteristic of the various detection events occurring in the detection window characterised in that the apparatus further comprises a means located within the sample holding chamber adapted to increase the local concentration of the nucleic acid sample adjacent the inlet of the nanopores relative to the bulk concentration thereof.

Abstract: A method for mapping the number and location of restriction enzymes sites for a given restriction enzyme in a target nucleic acid comprises the steps of (1) translocating a target nucleic acid having detectable elements characteristic of the presence of the restriction enzyme sites therein through an analysing device comprising a nanopore and a detection window and (2) causing the detectable elements to be detected as they pass though the detection window. Typically the detectable elements are formed by attaching to the restriction enzyme sites a restriction enzyme to which one or more marker moieties have been added. The data or signal obtained from the detection is suitably in the form of a distribution profile of the detectable elements, and therefore the restriction enzyme sites along the length of the target nucleic acid and can be used to create a reference set of like distribution profiles against which new distributions can be compared.