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 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 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 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: 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: 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 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; (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 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.