Base4 Innovation Limited

United Kingdom Research and Innovation

Lightcast Discovery Ltd

Biofidelity Ltd

Medical Research Council

Cameron Alexander Frayling

Barnaby Balmforth

Thomas Henry Isaac

Bruno Flavio Nogueira De Sousa Soares

Michele Amasio

Boris Breiner

Alessandra Natale

Paul Dear

Magdalena Stolarek-Januszkiewicz

Ana Silva-Weatherley

Tom Isaac

Maciej Sosna

Mark Dethlefsen

Andreas Michael Waeber

Bruno Flavio Soares

Ana Luisa Bras Dos Santos Ribeiro Da Silva Weatherley

Emma Head

Paul Loeffen

Claire Marie Murzeau

Nele Marie Godette Dieckmann

Evangelia-Nefeli Athanasopoulou

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 (4).

Single nucleotide detection method and associated probes

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 Ato create a first intermediate product which is at least partially double-stranded and in which the 3' end of Aforms 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 Ato create partially digested strand Aand the analyte; c. (i) annealing Ato a single-stranded trigger oligonucleotide B and extending the Astrand in the 5′-3′ direction against B; or (ii) circularising Athrough ligation of its 3′ and 5′ ends; or (iii) ligating the 3′ end of Ato the 5′ end of a ligation probe oligonucleotide C; in each case to create an oligonucleotide A; d. priming Awith at least one single-stranded primer oligonucleotide and creating multiple copies of A, or a region of A; and e. detecting a signal derived from the multiple copies and inferring therefrom the presence or absence of the polynucleotide target sequence in the analyte.

Polynucleotide sequence detection method

Devices, systems, and associated methods are provided for manipulating and/or determining one or more characteristics of cells contained within a biological sample. In particular a device and methods of use thereof are provided, the device comprising a sorting component configured to separate cell-containing microdroplets from empty ones into a population of cell-containing first microdroplets; a microdroplet manipulation component configured to manipulate the first microdroplets using real or virtual electrowetting electrodes, and an optical detection system configured to detect an optical signal from the microdroplets via the one or more detection windows.

Device and method for microdroplet detection of cells

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 (4).

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 Ato create a first intermediate product which is at least partially double-stranded and in which the 3' end of Aforms a double-stranded complex with the analyte target sequence; b. pyrophosphorolysing the first intermediate product with a pyrophosphorolysing enzyme in the 3&#x2032;-5&#x2032; direction from the 3&#x2032; end of Ato create partially digested strand Aand the analyte; c. (i) annealing Ato a single-stranded trigger oligonucleotide B and extending the Astrand in the 5&#x2032;-3&#x2032; direction against B; or (ii) circularising Athrough ligation of its 3&#x2032; and 5&#x2032; ends; or (iii) ligating the 3&#x2032; end of Ato the 5&#x2032; end of a ligation probe oligonucleotide C; in each case to create an oligonucleotide A; d. priming Awith at least one single-stranded primer oligonucleotide and creating multiple copies of A, or a region of A; and e. detecting a signal derived from the multiple copies and inferring therefrom the presence or absence of the polynucleotide target sequence in the analyte.

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.

Microfluidic device for characterzing polynucleotides

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.

Single nucleotide detection method

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. It is characterised by comprising; &#x2022;a planar substrate having a face with droplet-receiving locations arranged in at least one track parallel to a first axis defining the substrate; &#x22c5;a printer head comprising a plurality of droplet-dispensing nozzle groups juxtaposed above the droplet-receiving locations so that each nozzle group can in turn dispense a droplet into the droplet-receiving locations; &#x2022;a means for stepping the printer head along the first axis relative to the droplet-receiving locations; &#x22c5;a nucleotide-generating site upstream of the printer head by means of which an ordered stream of single nucleotides is created from the nucleic acid and delivered to a first droplet-dispensing nozzle group; &#x2022;at least one source of incident electromagnetic radiation adapted to illuminate the droplet-receiving locations and &#x22c5;at least one photodetector to detect adapted to detect fluorescence radiation emitted by each droplet-receiving location after illumination.

Droplet sequencing device

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) detecting the detectable elements released in each iteration.

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.

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.

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. Also disclosed is a method suitable for sequencing a biopolymer characterized by the steps of (1) progressively digesting the biopolymer into an ordered stream of its constituent monomers; (2) converting the stream of monomers into a corresponding stream of monomer-containing aqueous droplets each droplet additionally containing a probe capable of (a) capturing the monomer and (b) thereafter being digested to release an unqueched fluorophore characteristic of the captured monomer; (3) introducing the stream of droplets created in step (2) into an inlet end of at least one open-ended tube to create a stack of droplets therein and (4) releasing each droplet in turn from an outlet end of the tube(s) and detecting fluorophores in each droplet as each droplet emerges. The method may be used in a corresponding apparatus for sequencing a biopolymer such as a nucleic acid or protein.

Method of identifying droplets in a stack and an associated sequencer

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.

Droplet storage method

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. Typically the biological probe employed 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 essentially undetectable in the probe's unused state. In a most preferred embodiment the probe is labelled with multiple fluorophores and further comprises a restriction enzyme recognition site generated by the binding of the target single nucleotide to the single-stranded nucleotide region. Suitably, step c is carried out in the presence of a polymerase and ligase and step d in the presence of a restriction enzyme and an exonuclease. Typically the flow rate of the droplets is 100 to 2000 droplets per second.

Growing community of inventors

Cambridgeshire, United Kingdom

Cameron Alexander Frayling