Provided herein is a method of chemically modifying a polypeptide or polynucleotide monomer. The monomer may be a monomer of a protein pore such as a monomeric or oligomeric protein pore. Also disclosed are related monomers and oligomers and methods of using the disclosed pores in characterising analytes.
A method is provided comprising, for each respective pair of one or more target nucleic acids and one or more reference nucleic acids, generating an alignment between a segment of the respective target nucleic acid a segment of the respective reference nucleic acid, wherein the respective reference nucleic acid comprises a respective barcode sequence and a respective first context sequence. For each pair, a sequence similarity is determined between a scoring region of the reference nucleic acid and a corresponding segment of the target nucleic acid. For each pair, it is determine whether the target nucleic acid comprises the barcode sequence of the reference nucleic acid based on the sequence similarity between the target nucleic acid and the scoring region of the reference nucleic acid.
A nanopore sensing device comprises a planar structure provided with plural fluidic passages extending between the first and second chambers. The planar structure supports nanopores in membranes across respective passages and sensor electrodes are arranged to sense a fluidic electrical potential in respective passages between the nanopores and the second chamber. The passages comprise planar fluidic resistor portions between the sensor electrode and the second chamber, the planar fluidic resistor portions extending in a planar direction of the planar structure and being configured to form a fluidic resistor.
There is disclosed a nanopore support structure comprising a wall layer comprising walls defining a plurality of wells, and overhangs extending from the walls across each of the wells, the overhang defining an aperture configured to support a membrane suitable for insertion of a nanopore. There is further disclosed a nanopore sensing device comprising a nanopore support structure, and methods of manufacturing the nanopore support structure and the nanopore sensing device.
The invention provides a method of selectively characterising polynucleotides of a desired property, such as length, using a nanopore, based on the translocation of the polynucleotide through or across the nanopore. Kits and systems for use in such methods are also provided. The methods of the invention are particularly suitable for sequencing polynucleotides such as DNA.
Provided herein is a method of moving a double-stranded polynucleotide with respect to a nanopore using a motor protein. The method allows a portion of the polynucleotide to be interrogated by the pore multiple times. Also provided are polynucleotide adapters and kits comprising such adapters. The methods find use in characterising polynucleotides, for example in sequencing.
Provided herein is a method of characterising a target polynucleotide as it moves with respect to a nanopore using a motor protein. Also provided are polynucleotide adapters and kits comprising such adapters. The methods, kits and adapters find use in characterising polynucleotides, for example in sequencing.
The invention resides in a microfluidic device for analysing a test liquid. The device has a preparation portion between an inlet and an upstream portion of the device, wherein said upstream portion is configured with a sensing chamber for housing a well array and for receiving via the inlet a test liquid to be sensed, and said preparation portion is configured having: a reagent port configured to receive a reagent for interacting with a test liquid to be sensed within the upstream portion; and/or an actuator, operably configured to induce a test liquid from the inlet towards the upstream portion. Between the inlet and respective outlet lies a sole channel. The channel can extend parallel to the substrate, preferably along its entire length. The preparation portion can be configured in a first part, and the upstream portion can be configured in a second part, wherein said first part and second part are connectable such that a sample received and processed in the first part can be passed to the second part for analysis.
Provided herein are methods of characterising a target polypeptide as it moves with respect to a nanopore. Also provided are related kits, systems and apparatuses for carrying out such methods.
Provided herein is a method of concentrating a tethering complex in a region of an amphiphilic layer, such as a lipid membrane. Also provided herein are methods of assembling a tethering complex; methods of concentrating an analyte in the region of a detector; amphiphilic layers; and arrays and devices for use in the disclosed methods.
Provided herein is a method of sequencing a target double stranded nucleic acid. The method comprises contacting the double stranded nucleic acid with a reagent as described herein to form a construct and sequencing the construct using a single-molecule sequencing technique as described herein. Associated products and kits are further provided.
The invention relates to a method of forming a sensing device (100) for supporting a plurality of nanopores upon an array of wells (10). The method involves providing a substrate, said substrate (102) having a surface (104) having an array of electrodes (6) located thereon for connecting to or for configuring upon an electronic circuit. Separately, a well array structure (108) is provided, which has an array of walls (110) defining through-holes (112) for defining wells (10). The substrate (102) and the well array structure (108) are aligning said array of electrodes (6) to define, at least in part, a portion of the bases of respective wells (10) at the bottom of the through holes (112).
A method of preparing a nucleic acid construct for single molecule characterisation, comprising contacting a target polynucleotide with: a polynucleotide-guided effector protein, a guide polynucleotide; a transposase; and a transposable element comprising a modified polynucleotide, wherein the polynucleotide-guided effector protein directs said transposase to a region of interest within the target polynucleotide and the transposase inserts the transposable element into the polynucleotide, thereby producing a nucleic acid construct for single molecule characterisation.
The invention relates to new methods for synthesising polynucleotide molecules according to a predefined nucleotide sequence. The invention also relates to methods for the assembly of synthetic polynucleotides following synthesis, as well as systems and kits for performing the synthesis and/or assembly methods.
Provided herein is a method of loading a motor protein onto a polynucleotide adapter. Also provided are polynucleotide adapters and kits comprising such adapters. The adapters find use in characterising analytes such as polynucleotides in methods in which the polynucleotide moves in respect of a nanopore.
A biochemical sensing system senses interactions between molecular entities and nanopores using a sensor device comprising an array of sensor elements that support the nanopores. A switch arrangement selectively connects detection channels for amplifying sensed electrical signals to respective sensor elements. On the basis of an analysis of the amplified electrical signal output from the detection channels, detection of completion of interactions at sensor elements occurs. In response thereto, the switch arrangement is controlled to connect the detection channel connected to a sensor element at which completion of an interaction has been detected to a further sensor element.
A method for detecting interactions between elements within one or more DNA molecules within a cell, wherein the elements are not adjacent in the primary DNA sequence, the method comprising: a) providing a cell in which elements within one or more DNA molecules that are in close proximity are cross-linked; b) simultaneously lysing the cell and mechanically fragmenting the DNA molecules within the cell; c) proximity ligating the one or more fragmented DNA molecules; d) reversing the crosslinks in the ligated DNA molecules; e) sequencing the ligated DNA molecules; and f) analysing the sequencing data to detect interactions between elements within the one or more DNA molecules within the cell.
A modified portal protein of a bacteriophage DNA packaging motor, wherein the modified portal protein is capable of direct insertion into a membrane and wherein the portal protein is modified compared to the wild type portal protein such that one or more amino acid residues on the outer surface of the portal protein is substituted by one or more other amino acid residue, and/or wherein a one or more amino acid residue is inserted on the outer surface of the portal protein so as to alter the outer surface hydrophobicity of the modified portal protein compared to the wild type portal protein.
Methods and apparatus for measuring current are provided. In one arrangement, a first charge amplifier integrates a current to be measured. A processing circuit filters an output from the first charge amplifier using a first low pass filter module and a second low pass filter module. A second charge amplifier integrates a current derived from the filtered output from the first charge amplifier. The apparatus is configured to reset the first charge amplifier at the start of each of a plurality of sensing frames. The processing circuit obtains at least a first sample of the output from the first charge amplifier in each sensing frame. The sampling of the f ir st sample alternates from one sensing frame to the next sensing frame between sampling via the first low pass filter module and sampling via the second low pass filter module.
A nanopore sensing device has a structure (100) arranged to separate an analyte reservoir (106) and an outlet chamber (108). The structure (100) includes an array of nanopore structures (104), each nanopore structure (104) comprising a passage (114) for fluid connection through the structure between the analyte reservoir (106) and outlet chamber (108). Control terminals (156) are arranged for applying a control signal to alter the electrical potential difference across that nanopore structure (104). An electronic circuit (152) is configured to detect a signal from an electrical transduction element (126) at each nanopore structure (104).
Provided herein are methods of encoding data on a polymer. Also provided are methods of reading data encoded on a polymer. Also provided are systems for encoding data on a polymer; systems for reading data encoded on a polymer; and data encoding / data reading platforms.
A signal derived from a polymer comprising a series of polymer units during translocation of the polymer with respect to a nanopore is analysed using a machine learning technique. A series of weight distributions is output, each comprising weights in respect of transitions between labels over a set of labels representing the possible types of polymer unit in the polymer. An estimate of the series of polymer units from the weight distributions. Use of weights in respect of the transitions increases the accuracy of the technique.
The invention relates to a sensing system comprising an electrowetting device, which electrowetting device comprises an array of actuation electrodes, and a control system configures to perform droplet operations on a system of droplets present in the sensing system. The invention also relates to a method of operating the sensing system of the invention. The invention also provides novel droplet constructs which can be made and manipulated in the sensing system of the invention.
A61K 47/69 - Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
A system for characterising a target polynucleotide, the system comprising a membrane and a pore complex; wherein the pore complex comprises: (i) a nanopore located in the membrane, and (ii) an auxiliary protein or peptide attached to the nanopore; wherein the nanopore and the auxiliary protein or peptide together form a continuous channel across the membrane, the channel comprising a first constriction region and a second constriction region; wherein the first constriction region is formed by a portion of the nanopore, and wherein the second constriction region is formed by at least a portion of the auxiliary protein or peptide.
The invention resides in a method of determining a sequence of a target polymer, or part thereof, comprising polymer units comprising canonical and non-canonical polymer units. The method comprises taking a series of measurements of a signal relating to the target polymer wherein a measurement of the signal is dependent upon a plurality of polymer units, and wherein the polymer units of the target polymer modulate the signal, and wherein a non- canonical polymer unit modulates the signal differently from a corresponding canonical polymer unit. The series of measurements are analysed using a machine learning technique that attributes a measurement of a non-canonical polymer unit to being a measurement of a respective corresponding canonical polymer unit. The sequence of the target polymer, or part thereof, is determined from the analysed series of measurements. A non-canonical polymer unit identified from the analysis can be additionally or alternatively determined. Two or more types of non-canonical polymer units corresponding to the two or more types of canonical polymer unit can be used. The polynucleotide can be DNA.
The invention relates to new methods for synthesising polynucleotide molecules according to a predefined nucleotide sequence. The invention also relates to methods for the assembly of synthetic polynucleotides following synthesis, as well as systems and kits for performing the synthesis and/or assembly methods.
The invention relates to new methods for synthesising polynucleotide molecules according to a predefined nucleotide sequence. The invention also relates to methods for the assembly of synthetic polynucleotides following synthesis, as well as systems and kits for performing the synthesis and/or assembly methods.
The invention relates to new methods for synthesising polynucleotide molecules according to a predefined nucleotide sequence. The invention also relates to methods for the assembly of synthetic polynucleotides following synthesis, as well as systems and kits for performing the synthesis and/or assembly methods.
Methods of characterizing an analyte using a detector such as a nanopore and an enzyme are provided. One aspect features methods for characterizing a double-stranded polynucleotide using a detector, e.g., without using a hairpin connecting a template and a complement of the double-stranded polynucleotide. Another aspect features methods for characterizing an analyte using a tag-modified nanopore with increased sensitivity and/or higher throughput. Compositions and systems including, e.g., adaptors for attachment to double-stranded polynucleotides and tag-modified nanopores, which can be used in the methods are also provided.
A component (8) adapted to engage with a receiver (6) has an array of contact pads (16) to removably connect with a corresponding array of connectors (18) on the receiver (6). Each contact pad (16) of the array is electrically connected to the electrode (26) of a corresponding recess or well (28) that is part of a sensor, wherein a membrane is formable across each recess. A conductive grid (102) is configured between the contact pads (16) of the array, to inhibit an electrostatic discharge (BSD) conducting across the recesses or wells and/or direct an BSD away from the recesses or wells.
The invention provides methods of selecting polynucleotides based on their size in globular form. The polynucleotides may be treated with a condensation medium, e.g. comprising polyethylene glycol, e.g. PEG8000, and salt, in order to adopt a globular form. The method may employing filtering in order to retain the larger nucleic acids and make the larger nucleic acids available for sequencing applications, such as nanopore sequencing. The invention also relates to kits for selecting polynucleotides.
A method of attaching an adapter to a polynucleotide comprising: providing a double stranded polynucleotide comprising a single stranded break point within its polynucleotide sequence; contacting said double stranded polynucleotide with an exonuclease to form a single stranded region initiated at said break point; attaching an adapter to said single stranded region.
The invention relates to new methods for synthesising polynucleotide molecules according to a predefined nucleotide sequence. The invention also relates to methods for the assembly of synthetic polynucleotides following synthesis, as well as systems and kits for performing the synthesis and/or assembly methods.
A method for selectively modifying a target polynucleotide in a sample of polynucleotides, the method comprising contacting a sample of polynucleotides with a guide polynucleotide that binds to a sequence in the target polynucleotide and a polynucleotide-guided effector protein such that the polynucleotide-guided effector protein cuts the target polynucleotide to produce a cut end comprising an overhang; and attaching an adapter to the cut end in the target polynucleotide.
A method of amplifying a target polynucleotide, comprising: providing a template polynucleotide comprising a 5' hairpin, a target polynucleotide and a 3' hairpin, wherein the 5' hairpin comprises one or more non-canonical nucleotides; and contacting the template polynucleotide with a polymerase and canonical nucleotides, wherein the polymerase extends, using the canonical nucleotides, the target polynucleotide from its 3' end to form a first extended polynucleotide comprising the 5' hairpin at its 5' end and the complement of the 5' hairpin at its 3' end, wherein the complement of the 5' hairpin forms a 3' hairpin; and the polymerase extends the first extended polynucleotide from its 3' end to form a second extended polynucleotide comprising the 5' hairpin at its 5' end and the complement of the 5' hairpin at its 3' end, wherein the complement of the 5' hairpin forms a 3' hairpin.
Disclosed herein are compositions and methods that involve inserting connector protein channels of bacteriophage DNA packaging motors into copolymeric membranes via liposome-polymer fusion, which can be used as nanopore sensors for biomedical applications such as high throughput protein sequencing or cancer diagnosis. For example, disclosed are compositions comprising a copolymeric membrane into which a connector protein channel of a bacteriophage packaging motor has been inserted.
in vitroin vitro methods for synthesising a polymer, particularly a polynucleotide molecule, having a pre-defined sequence of units such as nucleotides. For synthesising a polynucleotide molecule the methods involve a process of extending a polynucleotide synthesis molecule with a transfer nucleotide. The methods additionally involve repeating the extension process multiple times to iteratively extend the polynucleotide molecule with multiple transfer nucleotides to generate a new polynucleotide molecule having a pre-defined nucleotide sequence. The invention also relates to in vitro methods of joining multiple synthetic polynucleotides following synthesis to form larger synthetic polynucleotides, as well as devices and systems for performing the extension, synthesis and assembly methods of the invention.
A method for selecting polynucleotides, the method comprising: allowing a nucleic acid handling enzyme to move along multiple polynucleotides in a sample for a defined time period, wherein the enzyme is loaded onto each of the multiple polynucleotides and wherein one or more molecule of the enzyme moves along each of the multiple polynucleotides; and selecting polynucleotides based on whether or not the enzyme reaches the end of and/or unbinds from the polynucleotides in the defined time period.
Droplet interfaces are formed between droplets in an electro-wetting device comprising an array of actuation electrodes. Actuation signals are applied to selected actuation electrodes to place the droplets into an energised state in which the shape of the droplets is modified compared to a shape of the droplets in a lower energy state and to bring the two droplets into proximity. The actuation signals are then changed to lower the energy of the droplets into the lower energy state so that the droplets relax into the gap and the two droplets contact each other thereby forming a droplet interface. The use of sensing electrodes in the device permit electrical current measurements across the droplet interface. The sensing electrodes can be used for either (i) applying a reference signal during droplet actuation or (ii) recording electrical current measurements.
A microfluidic device for analysing a test liquid comprises: a sensor (235), such as a membrane provided with nanopores, provided in a sensing chamber (237); a sensing chamber inlet channel (261) and a sensing chamber outlet channel (262), each connecting to the sensing chamber for respectively passing liquid into and out of the sensing chamber, and a reservoir (233) forming a sample input port to the microfluidic device, the reservoir being in fluid communication with the sensing chamber inlet channel (261); a liquid collection channel (232); a barrier (231) between an end of the sensing chamber outlet channel (262) and the liquid collection channel (232); a first seal (251), covering the sample input port; a second seal (252), covering the end of the sensing chamber outlet channel (262), thereby preventing liquid from flowing from the sensing chamber (237), over the barrier (231), into the liquid collection channel (232); wherein the microfluidic device is filled with a liquid from the first seal (251) at the sample input port to the second seal (252), such that the sensor (235) is covered by liquid and unexposed to a gas or gas/liquid interface; and wherein the first and second seals (251, 252) are removable to cause the liquid to flow between the reservoir and the end of the sensing chamber outlet and over the barrier.
A kit comprising first and second component parts adapted for connection to each other, wherein: the first component part comprises: a first array of electrical connectors; two substantially parallel lateral walls, one provided on either side of the first array of electrical connectors at a predetermined position with respect to the first array of electrical connectors; two rails provided between the first array of electrical connectors and the lateral walls, one rail being on either side of the first array of electrical connectors, wherein each rails extends at least along a length of the first array of electrical connectors, and has a front tip positioned at a predetermined position with respect to the first array of electrical connectors; a front contact point; an overhang for receiving the second component; the second component part comprises: a second array of electrical connectors, for connection to the first array of electrical connectors; a front end being configured to fit to a width between the parallel lateral walls, lateral sides, each comprising a rail relief to allow the sides to fit around the two rails, and wherein a shoulder is formed by the front end of each rail relief; and wherein the first and component are configured such that they may be connected by sliding the front end of the second component along the two rails of the first component and under the overhang, so that the shoulders of the rail reliefs of the second component pass the front tips of the rails of the first component, and the front end of the second component bears against the front contact point of the first component, urging the shoulders of the rail reliefs against the front tips of the rails, thereby locating the second array of electrical connectors in the correct position for connecting to the first array of electrical connectors.
The present invention relates to novel protein pores and their uses in analyte detection and characterisation. The invention particularly relates to an isolated pore complex formed by a CsgG-like pore and a modified CsgF peptide, or a homologue or mutant thereof, thereby incorporating an additional channel constriction or reader head in the nanopore. The invention further relates to a transmembrane pore complex and methods for production of the pore complex and for use in molecular sensing and nucleic acid sequencing applications.
Provided is a method of characterising a polynucleotide using a transmembrane pore, wherein the pore is a double pore comprising a first Csg G pore, or a homologue thereof, and a second CsgG pore, or a homologue thereof.
The invention relates to a method of determining the presence or absence of a target analyte in a sample. The method comprises immobilising any target analyte present in the sample on a surface; contacting the surface with: (i) a first detection agent that binds specifically to the target analyte; and (ii) a reporter polynucleotide, wherein the reporter polynucleotide is bound to, or binds to, the first detection agent; and contacting a transmembrane pore with any reporter polynucleotide that has been immobilised on the surface, wherein the reporter polynucleotide is immobilised on the surface by binding of the first agent to the target analyte, and using the transmembrane pore to detect the reporter polynucleotide, thereby determining the presence or absence of the target analyte in the sample.
A series of measurements taken from a polymer during translocation through a nanopore is analysed using a machine learning technique using a recurrent neural network (RNN). The RNN may derive posterior probability matrices each representing, in respect of different respective historical sequences of polymer units corresponding to measurements prior to the respective measurement, posterior probabilities of plural different changes to the respective historical sequence of polymer units giving rise to a new sequence of polymer units. Alternatively, the RNN may output decisions on the identity of successive polymer units of the series of polymer units, wherein the decisions are fed back into the recurrent neural network. The analysis may comprise performing convolutions of groups of consecutive measurements using a trained feature detector such as a convolutional neural network to derive a series of feature vectors, on which the RNN operates.
Provided herein relate to modified or mutant forms of secretin and compositions comprising the same. In particular, the modified or mutant forms of secretin permits efficient capture and/or translocation of an analyte through the modified or mutant secretin nanopores. Methods for using unmodified secretin or the modified or mutant forms of secretin and compositions, for example, for characterizing an analyte, e.g., a target polynucleotide, are also provided.
The invention relates to new methods for synthesising polynucleotide molecules according to a predefined nucleotide sequence. The invention also relates to methods for the assembly of synthetic polynucleotides following synthesis, as well as systems and kits for performing the synthesis and/or assembly methods.
Methods of characterizing an analyte using a nanopore. One aspect features methods for characterizing a double-stranded polynucleotide using a nanopore, e.g., without using a hairpin connecting a template and a complement of the double-stranded polynucleotide. Another aspect features methods for characterizing an analyte using a tag-modified nanopore with increased sensitivity and/or higher throughput. Compositions and systems including, e.g., adaptors for attachment to double-stranded polynucleotides and tag-modified nanopores, which can be used in the methods are also provided.
Apparatus and methods for controlling the insertion of a membrane channel into a membrane are disclosed. In one arrangement a first bath holds a first liquid in contact with a first surface of a membrane. A second bath holds a second liquid in contact with a second surface of the membrane. The membrane separates the first and second liquids. A first electrode contacts the first liquid. A second electrode contacts the second liquid. A driving unit applies a potential difference across the membrane via the first and second electrodes to promote insertion of a membrane channel into the membrane from the first liquid or the second liquid. A membrane voltage reduction unit is connected in series with the membrane. The driving unit applies a driving voltage across the membrane voltage reduction unit and the membrane, the driving voltage providing the potential difference across the membrane. The membrane voltage reduction unit is configured such that a reduction in resistance through the membrane caused by insertion of a membrane channel intrinsically increases a potential difference across the membrane voltage reduction unit thereby lowering the potential difference across the membrane. The lowering of the potential difference across the membrane is sufficient to prevent or reduce promotion of insertion of a further membrane channel.
A method for determining the presence, absence or amount of two or more target polynucleotides in a sample comprising additional components, the method comprising: (i) contacting the sample with a panel of two or more probes under conditions suitable for hybridisation of the target polynucleotides to the probes, wherein: (a) each probe comprises a non-hybridisation region and a hybridisation region that specifically hybridises to one of the target polynucleotides to form a hybridised probe; and (b) the hybridisation region of a probe of the panel comprises one or more non-natural nucleotides; (ii) contacting the sample prepared in step (i) with a transmembrane pore through which a single stranded polynucleotide but not a double stranded polynucleotide can pass and applying a potential difference to the transmembrane pore such that the hybridised probes in the sample interact with the pore; (iii) measuring current blockades having a duration within a defined window, wherein: (a) the one or more non-natural nucleotides present in the hybridisation region of the probe increase or decrease the duration of the current blockade due to the probe hybridised to its target polynucleotide such that the proportion of current blockades that occur within the window due to the interaction of the hybridised probes with the pore is increased compared to when the corresponding one or more natural nucleotides are present in the hybridisation region; and (b) each hybridised probe gives rise to a current blockade indicative of that probe; and (iv) correlating the measured current blockades with the probes, thereby determining the presence, absence or amount of the two or more target polynucleotides in the sample.
The invention provides a method of detecting a target polynucleotide in a sample comprising: (a) contacting the sample with a guide polynucleotide that binds to a sequence in the target polynucleotide and a polynucleotide-guided effector protein, wherein the guide polynucleotide and polynucleotide-guided effector protein form a complex with any target polynucleotide present in the sample; (b) contacting the sample with a membrane comprising a transmembrane pore; (c) applying a potential to the membrane; and (d) monitoring for the presence or absence of an effect resulting from the interaction of the complex with the transmembrane pore to determine the presence or absence of the complex, thereby detecting the target polynucleotide in the sample.
A microfluidic device comprises: a sensor provided in a sensing chamber; a liquid inlet and liquid outlet connecting to the sensor chamber for respectively passing liquid into and out of the sensing chamber and; a sample input port in fluid communication with the liquid inlet; a liquid collection channel downstream of the sensing chamber outlet; a flow path interruption between the liquid outlet and the liquid collection channel, preventing liquid from flowing into the liquid collection channel from upstream; a buffer liquid filling from the sample input port to the sensing chamber, and filling the sensing chamber and filing from the liquid outlet to the flow path interruption; an activation system operable to complete the flow path between the liquid outlet and the liquid collection channel such that the sensor remains unexposed to gas or a gas/liquid interface.
The invention relates to a method for modifying a template double stranded polynucleotide, especially for characterisation using nanopore sequencing. The method produces from the template a plurality of modified double stranded polynucleotides. These modified polynucleotides can then be characterised.
The invention relates to a new method of characterising a target polynucleotide using a pore. The method involves controlling the formation of secondary structure by the target polynucleotide after the polynucleotide has moved through the pore.
The invention relates to a new method of characterising two or more target polynucleotides using a pore. The method involves sequentially attaching to a first polynucleotide one or more subsequent polynucleotides to form a concatenated polynucleotide.
Arrangements are disclosed for measuring small electrical currents with high sensitivity, for example in the context of sensing molecular entities, for example via interactions between the molecular entities and a membrane protein inserted in an amphiphilic membrane. In one arrangement there is provided a current sensing circuit (52) configured to integrate the current output by a sensor element (56) during each of a plurality of sensing frames (62). In each sensing frame (62) first and second analogue samples of the integral are taken during first and second time windows (71,72). A readout circuit (54) processes the first and second analogue samples to output a digital output signal representing the current output by the sensor element (56). The processing comprises analogue to digital conversion processing and output processing. The output processing is performed exclusively during periods outside of the first and second time windows.
The invention relates to hetero-oligomeric pores derived from Msp. The invention also relates to polynucleotide characterisation using the hetero-oligomeric pores.
C07K 14/35 - Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Mycobacteriaceae (F)
C12Q 1/68 - Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
The invention relates to a novel method of producing hetero-oligomeric pores. The invention also relates to hetero-oligomeric pores produced using the method and polynucleotide characterisation using the hetero-oligomeric pores.
C12N 15/00 - Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
The invention relates to a method for modifying a template double stranded polynucleotide, especially for characterisation using nanopore sequencing. The method produces from the template a plurality of modified double stranded polynucleotides. These modified polynucleotides can then be characterised.
Analysis Of A Polymer A biochemical analysis system analyses polymers by taking measurements of a polymer from a sensor element comprising a nanopore during translocation of the polymer through the nanopore. When a polymer has partially translocated, the series of measurements is analysed using reference data derived from a reference sequence to provide a measure of similarity. Responsive to the measure of similarity, the sensor element may be selectively operated to eject the polymer and thereby make the nanopore available to receive a further polymer. Where the biochemical analysis system comprises an array of sensor elements and is takes measurements from sensor elements selected in a multiplexed manner, responsive to the measure of similarity, the biochemical analysis system ceases taking measurements from the currently selected sensor element and to starts taking measurements from a newly selected sensor element.
C12Q 1/68 - Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
G01N 33/487 - Physical analysis of biological material of liquid biological material
G06F 19/22 - for sequence comparison involving nucleotides or amino acids, e.g. homology search, motif or Single-Nucleotide Polymorphism [SNP] discovery or sequence alignment
65.
METHODS FOR DELIVERING AN ANALYTE TO TRANSMEMBRANE PORES
A detachable electrical device can be formed from a kit comprising a pair of component parts adapted for connection to each other, wherein the connected components of the device may be subsequently disconnected, comprising: an array of electrical connectors, each electrical connector comprising an electrically conductive liquid; and an array of electrodes; wherein the arrays can be brought into contact with each other so as to provide a plurality of electrical connections between the electrically conductive liquid of the array of electrical connectors and the electrodes of the array of electrodes, and wherein the electrical connections may be subsequently broken by detaching the electrically conductive liquid from the electrodes of the array.
The invention relates to a new method of characterising a target RNA polynucleotide by taking one or more measurements as the target RNA polynucleotide moves with respect to a transmembrane pore. The movement is controlled by a DNA helicase. The invention also relates to a modified RNA construct wherein the RNApolynucleotidehas been modified to increase DNA helicase binding thereto.
The invention relates to modified Dda helicases which can be used to control the movement of polynucleotides and are particularly useful for sequencing polynucleotides.
C07K 14/35 - Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Mycobacteriaceae (F)
C12Q 1/68 - Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
C12Q 1/68 - Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
C07K 14/35 - Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Mycobacteriaceae (F)
The invention relates to mutant forms of the outer membrane-located lipoprotein CsgG, in particular, modifications at one or more of positions Tyr51; Asn55; and Phe56. The invention also relates to analyte detection and characterisation using said mutant CsgG.
An estimate of a target sequence of polymer units is generated from a series of measurements taken by a measurement system comprising nanopores during translocation of the polymer through a nanopore. A global model of the measurement system is stored, comprising transition weightings for possible transitions between k-mers on which successive measurements are dependent and emission weightings for possible values of measurements being observed when the measurement is dependent on possible identities of k-mer. The global model is adjusted, making reference to measurements taken using the measurement system such that the fit of the measurements to the adjusted model is improved. The estimate of a target sequence of polymer units is generated using the adjusted model. The adjustment of the model improves the quality of the estimation.
G06F 19/22 - for sequence comparison involving nucleotides or amino acids, e.g. homology search, motif or Single-Nucleotide Polymorphism [SNP] discovery or sequence alignment
G01N 33/487 - Physical analysis of biological material of liquid biological material
C07K 14/35 - Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Mycobacteriaceae (F)
C12Q 1/68 - Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
73.
METHOD OF IMPROVING THE MOVEMENT OF A TARGET POLYNUCLEOTIDE WITH RESPECT TO A TRANSMEMBRANE PORE
The invention relates to improving the movement of a target polynucleotide with respect to a transmembrane pore when the movement is controlled by a polynucleotide binding protein. The invention also relates to improved transmembrane pores and polynucleotide binding proteins.
The invention relates to a method for method of characterising, such as sequencing, a target double stranded polynucleotide. The polynucleotide is coupled to a membrane using at least two adaptors with different strengths of coupling to the membrane.
The invention relates to a new method of determining the presence, absence or one or more characteristics of multiple analytes. The invention concerns coupling a first analyte to a membrane containing a detector and investigating the first analyte using the detector. The invention also concerns coupling a second analyte to the membrane and investigating the second analyte. The first analyte is uncoupled from the membrane prior to investigating the second analyte. The invention also relates to polynucleotide sequencing.
A target sequence of polymer units is estimated from plural series of measurements taken from sequences of polymer units that comprise the target sequence or a complementary sequence. Each measurement is dependent on a k-mer (k polymer units). Models treat the measurements as observations of k-mer states, comprising transition weightings in respect of transitions between successive k-mer states and emission weightings for different measurements being observed. An estimated alignment mapping between the plural series of measurements is derived based on an application of the models to each series. An estimate of the target sequence of polymer units is generated by applying the models, treating the types of k-mer state of each model and the measurements as dimensions of a plural dimensional k-mer state and plural dimensional observations. Constraint of paths through the plural dimensional k-mer states using the derived alignment mapping greatly reduces the required processing.
G06F 19/22 - for sequence comparison involving nucleotides or amino acids, e.g. homology search, motif or Single-Nucleotide Polymorphism [SNP] discovery or sequence alignment
G01N 33/487 - Physical analysis of biological material of liquid biological material
The invention relates to an improved method for characterising a template polynucleotide. The method involves using a polymerase to prepare a modified polynucleotide which makes it easier to characterise than the template polynucleotide.
The invention relates to new methods of controlling the movement of polynucleotides through transmembrane pores. The invention also relates to new methods of characterising target polynucleotides using helicases.
The invention relates to new methods of attaching one or more polynucleotide binding proteins to a target polynucleotide. The invention also relates to new methods of characterising target polynucleotides.
The invention relates to a new method of characterising a target polynucleotide. The method uses a pore and a Dda helicase. The helicase controls the movement of the target polynucleotide through the pore. The invention also relates to modified Dda helicases which can be used to control the movement of polynucleotides and are particularly useful for sequencing polynucleotides.
The invention relates to a new method of characterising a target ribonucleic acid (RNA) involving forming a complementary polynucleotide. The method uses a transmembrane pore.
The invention relates to a method for modifying a template double stranded polynucleotide, especially for characterisation using nanopore sequencing. The method produces from the template a plurality of modified double stranded polynucleotides. These modified polynucleotides can then be characterised.
The invention relates to new methods of moving helicases past spacers on polynucleotides and controlling the loading of helicases on polynucleotides. The invention also relates to new methods of characterising target polynucleotides using helicases.
A target polynucleotide is expanded. In respect of each nucleotide in the target polynucleotide, the target polynucleotide comprises clock nucleotides and at least one signal nucleotide in a predetermined order. The clock nucleotides have a predetermined sequence common to each nucleotide in the target polynucleotide. The at least one signal nucleotide is characteristic of the identity of the respective nucleotide in the target polynucleotide. During translocation of the expanded polynucleotide through a nanopore, electrical measurements dependent on the polynucleotide within the pore are made, to derive an analysis signal. Clock signals derived from the clock nucleotides are identified. Relative to the positions of the identified clock signals, nucleotide signals derived from the least one signal nucleotide are derived to analyse the target polynucleotide. The predetermined sequence of the clock nucleotides comprises a restriction site for a restriction enzyme and at least one further nucleotide that extends the predetermined sequence.
The invention relates to a new method of determining in a sample the presence, absence or concentration of one or more target analytes, such as micro-ribonucleic acids (microRNAs or miRNAs). The invention may therefore relate to a multiplex assay for determining the presence or absence of each target analyte in a group of multiple analytess. The invention uses one or more probes comprising a quadruplex and transmembrane pores.
An array of membranes comprising amphipathic moleculesis formed using an apparatus comprising a support defining an array of compartments. Volumes comprising polar medium are provided within respective compartments and a layer comprising apolar medium is provided extending across the openings with the volumes. Polar medium is flowed across the support to displace apolar medium and form a layer in contact with the volumes, forming membranes comprising amphipathic molecules at the interfaces. In one construction of the apparatus, the support that comprises partitions which comprise inner portions and outer portions. The inner portions define inner recesses without gaps therebetween that are capable of constraining the volumes comprising polar medium contained in neighbouring inner recesses from contacting each other. The outer portions extend outwardly from the inner portions and have gaps allowing the flow of an apolar medium across the substrate.
The invention provides a method of forming a membrane between a first volume of polar medium and a second volume of polar medium, which method comprises: (a) providing a first volume comprising polar medium and a second volume comprising polar medium which are separated from one another by an apolar medium, wherein at least one of said first and second volumes comprises a layer comprising amphipathic molecules, at the interface between the polar medium and the apolar medium, wherein each of the amphipathic molecules comprises a first outer hydrophilic group, a hydrophobic core group, and a second outer hydrophilic group, wherein each of the first and second outer hydrophilic groups is linked to the hydrophobic core group; and (b) causing the first and second volumes to come into contact with one another to form a membrane comprising said amphipathic molecules between the first and second volumes. The invention also provides a system comprising a membrane between a first volume of a polar medium; and a second volume of a polar medium, which membrane comprises the amphipathic molecules, and wherein the first volume of polar medium is within an apolar medium.
B01D 69/00 - Semi-permeable membranes for separation processes or apparatus characterised by their form, structure or properties; Manufacturing processes specially adapted therefor
C12Q 1/68 - Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
G01N 33/50 - Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
The invention relates to a method for modifying a template polynucleotide for characterisation, especially for nanopore sequencing. The method produces a modified polynucleotide which is complementary to the template polynucleotide at some positions and which contains universal or abasic nucleotides at the other, and in some instances predicable, positions. The resulting modified polynucleotide can then be characterised.
The invention relates to a method of characterising a target polynucleotide using a single-stranded binding protein (SSB). The SSB is either an SSB comprising a carboxy-terminal (C-terminal) region which does not have a net negative charge or a modified SSB comprising one or more modifications in its C-terminal region which decreases the net negative charge of the C-terminal region.
The invention relates to modified helicases with reduced unbinding from polynucleotides. The helicases can be used to control the movement of polynucleotides and are particularly useful for sequencing polynucleotides.
The invention relates to methods using constructs comprising a helicase and an additional polynucleotide binding moiety. The helicase is attached to the polynucleotide binding moiety and the construct has the ability to control the movement of a polynucleotide. The constructs can be used to control the movement of polynucleotides and are particularly useful for sequencing polynucleotides.
C07K 14/435 - Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from humans
C12Q 1/00 - Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
The invention relates to a new method of determining in a sample the presence or absence of one or more analyte members of a group of two or more analytes. The invention therefore relates to a multiplex assay for determining the presence or absence of each analyte in a group of multiple analytes. The assay uses aptamers and transmembrane pores.
A time-ordered series of measurements of a polymer made during translocation of the polymer through a nanopore are analysed. The measurements are dependent on the identity of k-mers in the nanopore, a k-mer being k polymer units of the polymer, where k is a positive integer. The method involves deriving, from the series of measurements, a feature vector of time-ordered features representing characteristics of the measurements; and determining similarity between the derived feature vector and at least one other feature vector.
C12Q 1/68 - Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
B82Y 15/00 - Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
G01N 33/487 - Physical analysis of biological material of liquid biological material
G06F 19/22 - for sequence comparison involving nucleotides or amino acids, e.g. homology search, motif or Single-Nucleotide Polymorphism [SNP] discovery or sequence alignment
95.
APPARATUS COMPRISING AN ARRAY OF SENSOR WELLS AND AN ARRAY OF FLOW CONTROL WELLS FOR IMPROVING THE WETTABILITY AND DISTRIBUTION OF FLUIDS APPLIED TO THE SURFACE OF THE BODY OF THE APPARATUS AND METHOD OF FORMING AN ARRAY OF LAYERS OF AMPHIPHILIC MOLECULES
An apparatus for supporting an array of layers of amphiphilic molecules, the apparatus comprising: a body (11), formed in a surface of the body, an array of sensor wells (10) capable of supporting a layer of amphiphilic molecules (30) across the sensor wells, the sensor wells each containing an electrode (12) for connection to an electrical circuit, and formed in the surface of the body between the sensor wells, flow control wells capable of smoothing the flow of a fluid across the surface.
The invention relates to a new method of characterising a target polynucleotide. The method uses a pore and a RecD helicase. The helicase controls the movement of the target polynucleotide through the pore.
The invention relates to a new method of characterising a target polynucleotide. The method uses a pore and an XPD helicase. The helicase controls the movement of the target polynucleotide through the pore.
The invention relates to a new method of characterising a target polynucleotide. The method uses a pore and a Hel308 helicase or amolecular motor which is capable of binding to the target polynucleotide at an internal nucleotide. The helicase or molecular motor controls the movement of the target polynucleotide through the pore.
A sequence of polymer units in a polymer (3), eg. DNA, is estimated from at least one series of measurements related to the polymer, eg. ion current as a function of translocation through a nanopore (1), wherein the value of each measurement is dependent on a k-mer being a group of k polymer units (4). A probabilistic model, especially a hidden Markov model (HMM), is provided, comprising, for a set of possible k-mers: transition weightings representing the chances of transitions from origin k-mers to destination k-mers; and emission weightings in respect of each k-mer that represent the chances of observing given values of measurements for that k-mer. The series of measurements is analysed using an analytical technique, eg. Viterbi decoding, that refers to the model and estimates at least one estimated sequence of polymer units in the polymer based on the likelihood predicted by the model of the series of measurements being produced by sequences of polymer units. In a further embodiment, different voltages are applied across the nanopore during translocation in order to improve the resolution of polymer units.
C12Q 1/68 - Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
G01N 33/487 - Physical analysis of biological material of liquid biological material
G06F 19/22 - for sequence comparison involving nucleotides or amino acids, e.g. homology search, motif or Single-Nucleotide Polymorphism [SNP] discovery or sequence alignment
B82Y 15/00 - Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
A pump for use in low-profile applications comprises a barrel (30) for holding fluid; and a piston (20) that converts a rotational driving force into a longitudinal driving motion within the barrel. The pump provides space saving advantages by reducing the need for external equipment and mechanisms around the pump for providing actuation or moving the actuating mechanism.
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