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Небесная энциклопедия

Космические корабли и станции, автоматические КА и методы их проектирования, бортовые комплексы управления, системы и средства жизнеобеспечения, особенности технологии производства ракетно-космических систем

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Мониторинг СМИ

Мониторинг СМИ и социальных сетей. Сканирование интернета, новостных сайтов, специализированных контентных площадок на базе мессенджеров. Гибкие настройки фильтров и первоначальных источников.

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Поддерживает ввод нескольких поисковых фраз (по одной на строку). При поиске обеспечивает поддержку морфологии русского и английского языка
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Применить Всего найдено 1632. Отображено 100.
02-05-2013 дата публикации

Real-time redox sequencing

Номер: US20130109577A1
Автор: Jonas Korlach, Lei Sun, Stephen Turner
Принадлежит: Pacific Biosciences of California Inc

Real time redox sequencing methods, devices, and systems are described. Arrays of redox devices comprising one or two electrodes are used to provide sequence information about a template nucleic acid in a polymerase-template complex bound proximate to the electrode(s). A sequencing reaction mixture comprising nucleotide analogs comprising redox labels is introduced to the array of redox devices under conditions of polymerase mediated nucleic acid synthesis. The time sequence of incorporation of nucleotide analogs is determined by electrochemically identifying the redox labels of the nucleotide analogs that are incorporated into the growing strand.

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Номер записи: 1
05-09-2013 дата публикации

Capture of target dna and rna by probes comprising intercalator molecules

Номер: US20130230856A1
Автор: Jan Gorm Lisby, Nina JØHNK, Uffe Vest Schneider
Принадлежит: Quantibact AS

The present invention relates to a technology for specific capture of single stranded target Polynucleotide by a complementary probe comprising one or more intercalator molecules. The method further involves removal of one or more types of bases in the single stranded target Polynucleotide prior to interaction with the complementary probe. This results in generation of one or more abasic sites which can interact with and/or into where the intercalator molecule can be inserted.

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Номер записи: 2
06-01-2022 дата публикации

TAGMENTATION-ASSOCIATED MULTIPLEX PCR ENRICHMENT SEQUENCING

Номер: US20220002793A1
Автор: AMBUR Ole Herman, ELLONEN Pekka, KRAUS CHRISTIANSEN Irene, LAGSTRÖM Sonja, LEPISTÖ Maja, MEISAL Roger, ROUNGE Trine
Принадлежит:

The present invention is related to methods for parallel sequencings of nucleic acid target sequences of interest, and in particular to massively parallel sequencing of nucleic acid sequences such as viral sequences that may have been integrated into a genome. For example, the methods, systems and kits provided herein may be used to enrich and sequence viral DNA sequences such as HPV and HIV sequences. 1. A method of amplifying a target nucleic acid sequence for use in a parallel sequencing method comprising:tagmenting a target nucleic sample to provide a plurality of tagmented sequences comprising a transposon adapter sequence at the ends of the tagmented sequences;contacting a first sample of the tagmented sequences with 1) a tag primer comprising a tag sequence portion that anneals to the transposon adapter sequence and a tag primer adapter portion, 2) a plurality of forward primers, each forward primer comprising a target sequence portion that anneals to a preselected portion of the sense strand of the target nucleic acid sequence and a forward primer sequencing portion, and 3) a sequencing primer comprising a portion that anneals to the forward sequencing portion of the forward primer and a sequencing primer adapter portion;performing a forward amplification reaction on the first sample of the tagmented sequences to provide a first library of amplicons spanning the target nucleic acid sequence;contacting a second sample of the tagmented sequences with 1) a tag primer comprising a tag sequence portion that anneals to the transposon sequence and a tag primer adapter portion, 2) a plurality of reverse primers, each reverse primer comprising a target sequence portion that anneals to a preselected portion of the antisense strand of the target nucleic acid sequence and a reverse primer sequencing portion, and 3) a sequencing primer comprising a portion that anneals to the reverse sequencing portion of the forward primer and a sequencing primer adapter portion; ...

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Номер записи: 3
04-01-2018 дата публикации

METHODS OF CONSTRUCTING A CIRCULAR TEMPLATE AND DETECTING DNA MOLECULES

Номер: US20180002731A1
Автор: Tien Meng-Tsung, WU Mengchu
Принадлежит: Personal Genomics, Inc.

A method of constructing a circular template includes preparing a partially double stranded linear DNA molecule, and incubating the partially double stranded linear DNA molecule with a ligase capable of intra-molecular ligation of single stranded DNA molecules to generate a partially double stranded circular DNA molecule. A method of detecting DNA molecules includes the following steps. Target DNA molecules are isolated with probes to form partially double stranded linear DNA molecules. The partially double stranded linear DNA molecules are incubated with ligases capable of intra-molecular ligation of single stranded DNA molecules to generate partially double stranded circular DNA molecules. A circular sequencing of the partially double stranded circular DNA molecules is conducted by using the probes as primers. 1. A method of constructing a circular template comprising:preparing a partially double stranded linear DNA molecule, wherein the partially double stranded linear DNA molecule comprises single stranded protruding portions at both ends on a same strand; andincubating the partially double stranded linear DNA molecule with a ligase capable of intra-molecular ligation of single stranded DNA molecules to generate a partially double stranded circular DNA molecule.2. The method as claimed in claim 1 , wherein the step of preparing the partially double stranded linear DNA molecule comprises:providing a single stranded linear DNA molecule; andhybridizing a probe to the single stranded linear DNA molecule.3. The method as claimed in claim 2 , wherein the single stranded linear DNA molecule comprises 40 nucleotides to 500 nucleotides in length.4. The method as claimed in claim 2 , wherein the probe comprises 20 nucleotides to 120 nucleotides in length.5. The method as claimed in claim 2 , wherein the probe comprises a biotinylated probe.6. The method as claimed in claim 5 , after hybridizing the probe to the single stranded linear DNA molecule claim 5 , further ...

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Номер записи: 4
07-01-2021 дата публикации

QUANTITATIVE MICROBIAL COMMUNITY PROFILING USING MOLECULAR INVERSION PROBES WITH UNIQUE MOLECULAR IDENTIFIERS

Номер: US20210002718A1
Автор: HARTMAN Wyatt, Zwirko Zachary
Принадлежит: Trace Genomics, Inc.

The present disclosure relates to the profiling of microorganisms in an environmental sample. Specifically, the present disclosure relates to methods of using molecular inversion probes comprising unique molecular identifiers to profile microorganisms in an environmental sample. In addition, the present disclosure relates to compositions of molecular inversion probes comprising unique molecular identifiers to profile microorganisms in an environmental sample. 1. A method for profiling of microorganisms in an environmental sample , wherein the method comprisesa) extracting DNA from the environmental sample;b) denaturing the extracted DNA; wherein the MIP comprises', '(i) in the 3′ to 5′ direction,', 'a first target locus primer, wherein the first primer comprises a nucleotide sequence complementary to a first sequence in a target locus,', 'a universal backbone sequence comprising a first sequencing primer binding site and a second sequencing primer binding site, and', 'a second target locus primer, wherein the second primer comprises a nucleotide sequence complementary to a second, non-overlapping sequence in the target locus, and', '(ii) a first unique molecular identifier (UMI);', 'wherein the backbone sequence has low sequence homology to DNA in the environmental sample and has minimal ability to form secondary structures,', 'thereby generating a sample comprising denatured DNA-MIP complexes;, 'c) incubating the denatured DNA with a molecular inversion probe (MIP) under conditions that allow hybridization,'}d) after hybridization, performing an extension and ligation reaction comprising incubating the sample comprising denatured DNA-MIP complexes with nucleotides, 5′ exo-polymerase lacking strand displacement activity, and a thermostable ligase capable of ligating splinted substrates under conditions that allow extension of the 3′ end of the MIP and ligation to the 5′ end of the MIP;e) after extension and ligation, incubating the sample comprising denatured DNA- ...

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Номер записи: 5
08-01-2015 дата публикации

Method for the synthesis of a bifunctional complex

Номер: US20150011434A1
Автор: Alex Haahr Gouliaev, Anette Holtmann, Christian Klarner Sams, Eva Kampmann Olsen, Henrik Pedersen, Jakob Felding, Kim Birkebaek Jensen, Mikkel Dybro Lundorf, Per-Ola Freskgard, Sanne Birkebaek JENSEN, Soeren Nyboe Jakobsen, Thomas Franch
Принадлежит: Nuevolution AS

Disclosed is a method for obtaining a bifunctional complex comprising a display molecule part and a coding part, wherein a nascent bifunctional complex comprising a chemical reaction site and a priming site for enzymatic addition of a tag is reacted at the chemical reaction site with one or more reactants, and provided with respective tag(s) identifying the reactant(s) at the priming site is using one or more enzymes.

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Номер записи: 6
14-01-2016 дата публикации

Nanopore sequencing methods

Номер: US20160011169A1
Автор: Jonas Korlach, Stephen Turner
Принадлежит: Pacific Biosciences of California Inc

Methods are provided for sequencing of nucleic acid templates using nanopores. The rate of transport of the template nucleic acids through the nanopore is controlled using a polymerase enzyme having two slow kinetic steps. Methods are provided for sequencing hemi-natural nucleic acids such as hemi-genomic DNA, having two complementary strands, one a natural sequence and the other a synthetic sequence. The identification of modified bases can be enhanced by comparing the sequencing information from the natural sequence, which has, for example, natural base modifications, with the synthetic sequence, which typically has no base modifications. The presence and identity of a modified base can be determined by monitoring kinetics, for example the kinetics of polymer meditated nucleic acid synthesis.

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Номер записи: 7
09-01-2020 дата публикации

METHOD OF IMPROVED SEQUENCING BY STRAND IDENTIFICATION

Номер: US20200010884A1
Автор: Faham Malek, Lin Shengrong, LU Yontao, Sun Zhaohui, TANG Ling Fung, Wang Yingyu, Weng Li
Принадлежит:

In some aspects, the present disclosure provides methods for identifying sequence variants, as well as methods of determining copy number of a genetic locus in a sample. Systems and kits for performing methods of the disclosure, as well as compositions produced by or useful in methods of the disclosure are also provided. In some embodiments, methods comprise extending 3′ ends of polynucleotides by adding one or more pre-determined nucleotides. In some embodiments, methods comprise use of a strand-tagging sequence. 187.-. (canceled)88. A method of identifying complementary strands in a nucleic acid sample comprising a plurality of double-stranded polynucleotides , each double-stranded polynucleotide of the plurality comprising a first complementary strand and a second complementary strand , each having a 5′ end and a 3′ end , the method comprising:(a) modifying a polynucleotide sequence of at least one of a first complementary strand and a second complementary strand of individual double-stranded polynucleotides, wherein subsequent to the modifying, a first complementary strand and a second complementary strand originating from a common double-stranded polynucleotide are not perfectly complementary;(b) sequencing a plurality of first complementary strands and a plurality of second complementary strands, or amplification products thereof, to yield a plurality of sequencing reads; and(c) identifying from the plurality of sequencing reads, a given first complementary strand and a given second complementary strand as originating from a common double-stranded polynucleotide based on (i) sequences of the respective 3′ ends and 5′ ends and (ii) polynucleotide sequences of the corresponding complementary strands which are not perfectly complementary.89. The method of claim 88 , wherein modifying a polynucleotide sequence comprises (i) extending a 3′ end of at least one of the first complementary strand and the second complementary strand by adding one or more pre-determined ...

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Номер записи: 8
19-01-2017 дата публикации

Nucleic Acid Probe and Method of Detecting Genomic Fragments

Номер: US20170016065A1
Автор: Carl Oscar Fredrik Dahl, Olof John Ericsson
Принадлежит: Vanadis Diagnostics AB

Provided herein, among other things, is a method of processing a nucleic acid sample. In some embodiments, the method comprises a) hybridizing a sample comprising a target fragment to a nucleic acid probe comprising: i. a head sequence and a tail sequence, wherein the head and tail sequences are at the ends of a first oligonucleotide molecule; and ii. a splint sequence comprising, in order: an upstream flanking sequence that is complementary to the head sequence; a target complementary sequence that is complementary to the target fragment; and a downstream flanking sequence that is complementary to the tail sequence; thereby producing a hybridization product in which the ends of the target fragment are ligatably adjacent to the ends of the head and tail sequences in the first oligonucleotide molecule; and b) ligating the ends of the target fragment to the ends of the head and tail sequences of the first oligonucleotide molecule, thereby producing a cyclic product that comprises the target fragment and the head and tail sequences. Probes and kits for performing the method are also provided.

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Номер записи: 9
21-01-2021 дата публикации

METHOD OF PRODUCING AN IMMUNOLIGAND/PAYLOAD CONJUGATE

Номер: US20210015936A1
Автор: Beerli Roger Renzo, Grawunder Ulf
Принадлежит:

The present invention relates to a method of producing an immunoligand/payload conjugate, which method encompasses conjugating a payload to an immunoligand by means of a sequence-specific transpeptidase, or a catalytic domain thereof. 124-. (canceled)25. A method of treating a pathologic condition in a subject in need thereof , comprisingadministering to the subject in need thereof an effective amount of an immunoligand/payload conjugate,wherein the immunoligand/payload conjugate is produced by enzymatically conjugating at least one glycine-modified payload to the immunoligand with a sequence-specific sortase or a catalytic domain thereof,wherein the immunoligand is selected from the group consisting of an antibody, a modified antibody format, an antibody derivative or fragment, and an antibody mimetic, andwherein the payload is selected from the group consisting of a cytokine, a radioactive agent, an anti-inflammatory drug, a toxin, and a chemotherapeutic agent.26. The method of claim 25 , wherein the sortase is sortase A.27. The method of claim 25 , wherein the immunoligand/payload conjugate further comprises at least one linker between the immunoligand and the payload claim 25 , said linker comprising a peptide motif that is a sortase recognition motif claim 25 , and said linker being conjugated to the C-terminus of at least one peptide chain of the immunoligand.28. The method according to claim 27 , wherein the C-terminal amino acid residue of the sortase recognition motif is replaced by a glycine residue.29. The method according to claim 28 , wherein the sortase recognition motif is LPXTG (SEQ ID NO:27) or NPQTN (SEQ ID NO:28) claim 28 , wherein X represents any amino acid.30. The method of claim 25 , wherein the immunoligand/payload conjugate comprises an antibody/drug conjugate.31. The method of claim 25 , wherein the payload comprises a toxin of molecular weight ≤2500 Dalton that is cytotoxic to a mammalian cell.32Pseudomonas. The method of claim 31 , ...

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Номер записи: 10
17-01-2019 дата публикации

METHOD FOR USING HEAT-RESISTANT MISMATCH ENDONUCLEASE

Номер: US20190017037A1
Автор: Matsumura Kiyoyuki, Mukai Hiroyuki, TAKATSU Nariaki, Uemori Takashi
Принадлежит: TAKARA BIO INC.

Provided are a mismatch-specific cleavage reaction using a novel heat-resistant mismatch nuclease, a method for removing errors in a nucleic acid amplification reaction using the mismatch nuclease, a method for inhibiting the amplification of a nucleic acid having a specific base sequence during a nucleic acid amplification reaction, and a method for detecting a nucleic acid having a single-base polymorphic mutation using this inhibition method. 114-. (canceled)15. A composition comprising the following (a) to (c):(a) a DNA polymerase;(b) at least one pair of oligonucleotide primers; and(c) at least one polypeptide selected from the group consisting of the following (i) to (iii):(i) a polypeptide having an amino acid sequence of SEQ ID NO:1, 7 or 8;(ii) a polypeptide having an amino acid sequence which differs from the amino acid sequence of SEQ ID NO:1, 7 or 8 by substitution, deletion, insertion and/or addition of 1 to 10 amino acid residues, and having a mismatch endonuclease activity; and(iii) a polypeptide having an amino acid sequence which shares at least 75% amino acid sequence identity with the amino acid sequence of SEQ ID NO:1, 7 or 8, and having a mismatch endonuclease activity.16. A method of amplifying a nucleic acid , the method comprising the following steps (a) and (b):{'claim-ref': {'@idref': 'CLM-00015', 'claim 15'}, '(a) preparing a composition comprising the composition according to and a nucleic acid molecule as a template; and'}(b) reacting the composition obtained by step (a) under suitable conditions to perform nucleic acid amplification.17. The method according to claim 16 , wherein the nucleic amplification is performed by a polymerase chain reaction (PCR) method claim 16 , an isothermal nucleic acid amplification method claim 16 , or a multiple displacement amplification (MDA) method.18. A polypeptide selected from the group consisting of the following (A) to (C):(A) a polypeptide having an amino acid sequence which differs from an amino ...

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Номер записи: 11
21-01-2021 дата публикации

METHODS FOR AMPLIFICATION OF NUCLEIC ACIDS WITH ENDONUCLEASE-MEDIATED SHIFTING EQUILIBRIUM (EM-SEQ)

Номер: US20210017589A1
Автор: LIPINSKI Kamil Andrzej
Принадлежит: DNAE DIAGNOSTICS LIMITED

The embodiments and improvements relate to the design of molecular biology assays based on isothermal amplification of nucleic acids with Strand Displacement Amplification (SDA). The embodiments describe a novel method for SDA termed Endonuclease-Mediated Shifting Equilibrium Amplification (EM-SEq), which improves exponential kinetics and specificity of the reaction and enables amplification on solid surfaces. 1. A method for the strand displacement amplification of a population of double stranded nucleic acid sequences comprising:a. modifying the ends of the strands in the population such that at least one of the ends contains a low melting point region of sequence which, at a temperature of 37-80° C., is at least transiently single stranded;b. copying the population of nucleic acid molecules having low melting point ends using one or more amplification primers which hybridise to the low melting point ends, wherein the primers have a 5′ single stranded section beyond the 3′ end of the template population of nucleic acid molecules such that the 3′ end of the template is extended to form a complete recognition site for an endonuclease, and the 3′ end of the primer is extended by strand displacement to copy the template;c. using the complete recognition site for the endonuclease to nick the extended strand, thereby releasing a free 3′-OH group within the primer; and 'wherein steps b, c and d are performed isothermally, thereby resulting in the strand displacement amplification of the population of double stranded nucleic acid sequences.', 'd. extending the freed 3′-OH group by strand displacement to re-copy the template,'}2. The method according to claim 1 , step a. wherein the ends of strands in the population are modified such that at least one of the ends contains a low melting region of sequence which claim 1 , at a temperature of 37-65° C. claim 1 , is at least transiently single stranded.3. The method according to or wherein the amplification is carried out with ...

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Номер записи: 12
24-01-2019 дата публикации

Method of Preparing Cell Free Nucleic Acid Molecules by In Situ Amplification

Номер: US20190024127A1
Автор: YEH Chen-Hsiung
Принадлежит:

Methods for in situ amplification (ISA) of cfNA, such as cfDNA, in a sample are provided wherein the cfNA in the sample is not subject to a nucleic acid purification step. The methods disclosed may be used to generate an analyzable pool of cfNA present in the sample. The analyzable pool may be used with a variety of analytical techniques to characterize the nucleic acid in the sample. Methods of diagnosis, determining a therapeutic intervention and monitoring of a subject are also provided. 1. A method of in situ amplification of a cell-free nucleic acid (cfNA) the method comprising the steps of:a. providing a liquid sample containing a plurality of cfNA;b. performing at least one processing step on the sample;c. subjecting the sample to a sequential heating program and converting at least a portion of the cfNA in the sample to a modified cfNA using an enzyme mixture to add an exogenous nucleic acid sequence to at least one of the 5′ or 3′ ends of at least a portion of the cfNA in the sample to create an amplifiable cfNA pool, wherein the exogenous nucleic acid sequence contains a primer site capable of binding a primer and the exogenous nucleic acid sequence has a degenerate nucleic acid sequence flanking at least one side of the primer site; andd. amplifying the amplifiable cfNA pool to produce an analyzable pool of cfNA.wherein the cfNA in the sample is not subject to a nucleic acid purification step.2. The method of claim 1 , wherein the cfNA is selected from the group consisting of: cfDNA and cfRNA.3. (canceled)4. The method of claim 1 , wherein the cfNA is cfRNA and the cfRNA is converted to double-strand DNA prior to step (c).5. (canceled)6. The method of claim 1 , wherein at least a portion of the cfNA in the sample are ligated together.7. The method of claim 1 , wherein the cfNA in the sample has a fragment size distribution of 50 bp to 2 claim 1 ,000 bp claim 1 , 100 bp to 1 claim 1 ,000 bp claim 1 , 50 bp to 600 bp claim 1 , 100 bp to 500 bp claim 1 , 100 ...

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Номер записи: 13
24-01-2019 дата публикации

METHODS AND COMPOSITIONS FOR SEQUENCING MODIFIED NUCLEIC ACIDS

Номер: US20190024162A1
Автор: Clark Tyson A., Korlach Jonas, Turner Stephen
Принадлежит:

Methods, compositions, and systems are provided for characterization of modified nucleic acids. In certain preferred embodiments, single molecule sequencing methods are provided for identification of modified nucleotides within nucleic acid sequences. Modifications detectable by the methods provided herein include chemically modified bases, enzymatically modified bases, abasic sites, non-natural bases, secondary structures, and agents bound to a template nucleic acid. 151-. (canceled)52. A method of determining whether a cell sample comprises actively dividing cells , the method comprising:obtaining a non-amplified, double-stranded genomic DNA library from the cell sample;performing single-molecule sequencing to generate sequence reads for both strands of a plurality of individual double-stranded nucleic acids in the genomic DNA library, wherein the sequence reads comprise both base sequence data and base modification data; andanalyzing the base sequence and base modification of the plurality of individual double-stranded nucleic acids, wherein identification of individual double-stranded nucleic acids in the genomic DNA library that are hemi-modified indicates that the cell sample comprises actively dividing cells.53. The method of claim 52 , wherein the cell sample comprises one or more type of microorganism.54. The method of claim 53 , wherein the cell sample is a metagenomic sample.55. The method of claim 53 , wherein the one or more type of microorganism is selected from the group consisting of: one or more type of bacteria claim 53 , one or more type of archaean claim 53 , one or more type of protozoan claim 53 , one or more type of fungus claim 53 , or any combination thereof.56. The method of claim 53 , wherein the one or more microorganism is a pathogenic microorganism.57. The method of claim 52 , wherein the single-molecule sequencing performed is a sequencing-by-incorporation method.58. The method of claim 52 , wherein the single-molecule sequencing ...

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Номер записи: 14
24-01-2019 дата публикации

MOLECULAR IDENTIFICATION WITH SUBNANOMETER LOCALIZATION ACCURACY

Номер: US20190024165A1
Автор: Jungmann Ralf, Mir Kalim, SCHÜDER Florian, WÖHRSTEIN Johannes B.
Принадлежит:

The present invention relates to methods of determining the sequence of nucleotides in target nucleic acid molecules. Thus, the invention relates to methods of sub-unit sequencing. The methods comprise the use of identification nucleic acid detection entities which specifically hybridize to the target nucleic acid, bind identification tags and have localization tags transiently bind thereto. 1. A method of determining the sequence of nucleotides in a target nucleic acid molecule comprising the steps of:(1) providing a target nucleic acid molecule,wherein copies of the target nucleic acid molecule are immobilized on a solid substrate, (i) a specific probe nucleotide sequence,', '(ii) a localization nucleotide sequence for transient binding of a localization tag,', 'and', '(iii) an identification nucleotide sequence for stable hybridization with an identification tag specific for the specific probe nucleotide sequence (i),, '(2) providing a plurality of nucleic acid detection entities, wherein each nucleic acid detection entity is at least in part single stranded and comprises(3) providing a plurality of identification tags, is specific for a specific probe nucleotide sequence (i) of the nucleic acid detection entity, and', 'comprises a nucleotide sequence complementary to the identification nucleotide sequence (iii) of the nucleic acid detection entity,, 'wherein each identification tag'}(4) providing a plurality of localization tags, a nucleotide sequence complementary to the localization nucleotide sequence (ii) of the nucleic acid detection entity, and', 'marker(s) or label(s),, 'wherein said localization tag comprises'}(5) hybridizing and optionally ligating the nucleic acid detection entities to the single stranded target nucleic acid molecules, 'optionally, stretching and/or aligning the identification markers,', '(6) hybridizing the identification tags to the identification nucleotide sequence (iii) of the nucleic acid detection entities,'}(7) detecting the ...

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Номер записи: 15
29-01-2015 дата публикации

Ligase-assisted nucleic acid circularization and amplification

Номер: US20150031086A1
Автор: Erik Leeming Kvam, John Richard Nelson, Ryan Charles HELLER
Принадлежит: General Electric Co

Provided herein are methods for generation and amplification of a single-stranded DNA circle in a single reaction vessel from a linear DNA without any intervening purification steps. The single-stranded DNA circle is generated via a template-independent single-stranded DNA ligation. Whole-genome amplification of circulating nucleic acids extracted from blood is provided. Kits for performing the disclosed methods are also provided.

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Номер записи: 16
01-02-2018 дата публикации

ENZYME METHOD

Номер: US20180030530A1
Автор: Heron Andrew John, Moysey Ruth
Принадлежит: Oxford Nanopore Technologies Ltd.

The invention relates to a new method of characterizing 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. 143-. (canceled)44. A method of characterising a target polynucleotide , comprising:(a) contacting the target polynucleotide with a transmembrane pore and a helicase which is capable of binding to the target polynucleotide at an internal nucleotide such that the helicase controls the movement of the target polynucleotide through the pore and nucleotides in the target polynucleotide interact with the pore; and(b) measuring one or more characteristics of the target polynucleotide during one or more interactions and thereby characterising the target polynucleotide.45. A method according to claim 44 , wherein the helicase is a Hel308 helicase claim 44 , Hel308 Tga claim 44 , Hel308 Mhu or Hel308 Csy.46. A method according to claim 44 , wherein the one or more characteristics are selected from (i) the length of the target polynucleotide claim 44 , (ii) the identity of the target polynucleotide claim 44 , (iii) the sequence of the target polynucleotide claim 44 , (iv) the secondary structure of the target polynucleotide claim 44 , and (v) whether or not the target polynucleotide is modified by methylation claim 44 , by oxidation claim 44 , by damage claim 44 , with one or more proteins or with one or more labels claim 44 , tags or spacers.47. A method according to claim 44 , wherein the one or more characteristics of the target polynucleotide are measured by electrical measurement and/or optical measurement.48. A method according to claim 47 , wherein the electrical measurement is a current measurement claim 47 , an impedance measurement claim 47 , a tunnelling measurement claim 47 , or a field effect transistor (FET) measurement.49. A method ...

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Номер записи: 17
31-01-2019 дата публикации

Methods and Compositions for Rapid Assembly of Genetic Modules

Номер: US20190032047A1
Автор: MURRAY Richard, Sun Zachary Z.
Принадлежит:

Provided herein are methods and compositions for rapid assembly of genetic modules, as well as seamless transition from in vitro to in vivo testing of genetic constructs. 1. A non-naturally occurring library of genetic modules , comprising:a plurality of pre-designed promoters,a plurality of pre-designed untranslated regions,a plurality of pre-designed terminators,a plurality of pre-designed stage 1 vectors, andat least one pre-designed stage 2 vector,wherein each promoter, untranslated region, terminator, stage 1 vector and stage 2 vector are engineered to have a pair of restriction sites of a first type IIs enzyme;wherein the promoters, untranslated regions and terminators are designed to be assembled into a plurality of recombinant transcription units Tu1, Tu2 and TuN wherein N>=3, each recombinant transcription unit being present in a separate stage 1 vector and flanked by a first pair of restriction sites of the first type IIs enzyme, wherein the first pair of restriction sites for each recombinant transcription unit are pre-designed such that upon digestion by the first type IIs enzyme, compatible cohesive ends are generated to allow ligation of the recombinant transcription units into a linear DNA molecule having a predetermined order 5′-Tu1-TuN-Tu2-3′;wherein each stage 2 vector has a second pair of restriction sites of the first type IIs enzyme, wherein the second pair of restriction sites are pre-designed such that upon digestion by the first type IIs enzyme, a first and second cohesive end are generated to allow direct ligation of the first cohesive end with Tu1 at its 5′ end and direct ligation of the second cohesive end with Tu2 at its 3′ end.2. The library of claim 1 , wherein N=<9.3. The library of claim 1 , wherein the first type IIs enzyme is selected from BsaI claim 1 , Eco31I claim 1 , BspTN1 claim 1 , Bso31I claim 1 , BbsI claim 1 , BpuAI claim 1 , BpiI claim 1 , BstV21 claim 1 , BsmBI claim 1 , Esp3I claim 1 , FokI claim 1 , AlwI claim 1 , and ...

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Номер записи: 18
07-02-2019 дата публикации

IDENTIFICATION OF GENETIC MODIFICATIONS

Номер: US20190040457A1
Автор: Hall Adam R.
Принадлежит: Wake Forest University Health Sciences

Described are methods of detecting modified nucleotide bases in a DNA sample using specific DNA glycosylases to excise target modified bases. DNA molecules are then labeled using a DNA polymerase lacking 3′→5′ exo-nuclease activity and strand displacement activity. The methods can be used to detect epigenetic changes and DNA damage. Provided are methods for diagnosing a disease or condition, determining risk of a disease or condition, identifying appropriate treatment, monitoring effectiveness of treatment, and monitoring side effects of treatment in subjects based on detection of modified bases. Also provided are methods for determining environmental exposure, or an environmental exposure time, of a biological sample containing DNA. Also provided are kits, systems, and devices for performing the described methods. 1. A method of detecting a modified DNA base in a DNA sample , comprising:(a) incubating a DNA sample comprising fragmented DNA with a DNA glycosylase that excises a modified nucleotide to form an apurinic or apyrimidinic site (AP site) at the site of the modified nucleotide in the fragmented DNA;(b) treating the fragmented DNA of step (a) with a DNA polymerase and a labeled nucleotide complimentary to a nucleotide opposite the AP site thereby incorporating the labeled nucleotide at the AP site in the fragmented DNA;(c) isolating the fragmented DNA containing the labeled nucleotide; and(d) (i) detecting the position of the labeled nucleotide in the fragmented DNA to determine the location of the modified nucleotide in the DNA sample, (ii) quantitating the amount of labeled nucleotide in the fragmented DNA to determine amount of the modified nucleotide in the DNA sample, or (iii) both detecting the position and quantitating the amount of the labeled nucleotide in the fragmented DNA to determine the location and amount of the modified nucleotide in the DNA sample.2. The method of claim 1 , wherein the DNA sample is genomic DNA claim 1 , mitochondrial DNA ...

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Номер записи: 19
06-02-2020 дата публикации

SELECTIVE OXIDATION OF 5-METHYLCYTOSINE BY TET-FAMILY PROTEINS

Номер: US20200040381A1
Автор: Agarwal Suneet, Iyer Aravind, Koh Kian Peng, Rao Anjana, Tahiliani Mamta
Принадлежит:

The present invention provides for novel methods for regulating and detecting the cytosine methylation status of DNA. The invention is based upon identification of a novel and surprising catalytic activity for the family of TET proteins, namely TET1, TET2, TET3, and CXXC4. The novel activity is related to the enzymes being capable of converting the cytosine nucleotide 5-methylcytosine into 5-hydroxymethylcytosine by hydroxylation. 120.-. (canceled)21. A method comprising:(a) obtaining a sample comprising a nucleic acid sequence, wherein said nucleic acid sequence comprises a 5-hydroxymethylcytosine and a 5-methylcytosine;(b) performing a sequencing; and(c) distinguishing said 5-hydroxymethylcytosine from said 5-methylcytosine.241. The method of claim , wherein said nucleic acid sequence is a mammalian nucleic acid sequence.251. The method of claim , wherein said sample comprises genomic DNA.261. The method of claim , wherein said sample comprises an extracellular fluid.271. The method of claim , wherein said sequencing is high-throughput sequencing.281. The method of claim , wherein said sample is from a subject having or suspected of having cancer.291. The method of claim , wherein said distinguishing comprises detecting said 5-hydroxymethylcytosine.301. The method of claim , wherein said distinguishing comprises detecting said 5-methylcytosine.311. The method of claim , further comprising contacting said nucleic acid sequence with a dioxygenase or catalytically active fragment thereof to convert a methylated cytosine in said nucleic acid sequence to a modified base , wherein said dioxygenase or said catalytically active fragment thereof comprises TET1 , TET2 , TET3 , CXXC4 , a catalytically active fragment of any of these , or any combination thereof.32. The method of claim 31 , further comprising contacting said nucleic acid sequence with sodium bisulfite. This application is a continuation application under 35 U.S.C. § 120 of co-pending U.S. application Ser. No. ...

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Номер записи: 20
15-02-2018 дата публикации

NUCLEOTIDE POLYMORPHISM DETECTION METHOD

Номер: US20180044721A1
Автор: BALMFORTH Barnaby
Принадлежит: BASE4 INNOVATION LTD

A method for characterising a DNA analyte comprised of one or more polynucleotide types characteristic of a site of nucleotide polymorphism each of which includes a target region having the formula -X-Y-Z- wherein X and Z are respectively first and second characteristic flanking oligonucleotide regions and Y is one of the variants constituting the site is provided. The method is characterised by the steps of; (a) reacting a single-stranded oligonucleotide including the target region derived from at least one of the polynucleotide types with a set of unused probes comprised of (i) a single-stranded first aptamer terminating at its 3′ end in a sequence complementary to that of -X or -X-Y and (ii) one or more second single-stranded aptamers terminating at their 5′ end in a sequence complementary to that of -Z-Y or -Z (as the case may be) and labelled with detectable elements which are in an undetectable state in the presence of a ligase to create a substantially double-stranded used probe comprised of the oligonucleotide, first aptamer and one of the second aptamers; (b) wholly or in part digesting the used probe with an exonuclease or polymerase exhibiting exonuclease activity in a 3′ to 5′ direction into its constituent single nucleotides at least one of which includes a detectable element now in a detectable state and (c) thereafter detecting the detection property associated with the now detectable element thereby identifying the nature of the Y variant and therefore the allele it gives rise to. A second mirror-image method is also disclosed. Also provided are vesicles in which the method can be carried out. The method is suitable for a range of diagnostic screening applications including the detection of mutant alleles associated with genetic disorders and cancer. 1. A method for characterising a DNA analyte comprising one or more polynucleotide types characteristic of a site of nucleotide polymorphism each of which includes a target region having the formula -X-Y ...

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Номер записи: 21
14-02-2019 дата публикации

Methods for sample preparation

Номер: US20190048334A1
Автор: Joseph Dunham
Принадлежит: Seqonce Biosciences Inc, University of Southern California USC

The disclosure provides for single amplification and double amplification methods for preparing nucleic acid samples for sequencing.

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Номер записи: 22
14-02-2019 дата публикации

SELECTIVE OXIDATION OF 5-METHYLCYTOSINE BY TET-FAMILY PROTEINS

Номер: US20190048405A1
Автор: Agarwal Suneet, Iyer Aravind, Koh Kian Peng, Rao Anjana, Tahiliani Mamta
Принадлежит:

The present invention provides for novel methods for regulating and detecting the cytosine methylation status of DNA. The invention is based upon identification of a novel and surprising catalytic activity for the family of TET proteins, namely TET1, TET2, TET3, and CXXC4. The novel activity is related to the enzymes being capable of converting the cytosine nucleotide 5-methylcytosine into 5-hydroxymethylcytosine by hydroxylation. 1. A method comprising:administering to a subject, an agent that modulates activity of an enzyme that mediates conversion of a methylated cytosine residue to a modified base, wherein said subject has a cancer or is at risk of developing said cancer, wherein said modified base comprises a hydroxymethylated cytosine residue.2. The method of claim 1 , wherein said subject is a mammal.3. The method of claim 2 , wherein said methylated cytosine residue comprises a 5-methylcytosine.4. The method of claim 2 , wherein said hydroxymethylated cytosine residue is a 5-hydroxymethylcytosine.5. The method of claim 2 , wherein said cancer is leukemia.6. The method of claim 5 , wherein said leukemia is myeloid leukemia.7. The method of claim 2 , wherein said agent inhibits activity of said enzyme.8. The method of claim 7 , wherein said agent comprises a small molecule inhibitor claim 7 , a competitive inhibitor claim 7 , an antibody or antigen-binding fragment thereof claim 7 , or a nucleic acid that inhibits said enzyme.9. The method of claim 8 , wherein said agent comprises said nucleic acid that inhibits said enzyme.10. The method of claim 9 , wherein said nucleic acid that inhibits said enzyme comprises a siRNA.11. The method of claim 2 , wherein said enzyme comprises a TET family enzyme claim 2 , functional TET family derivative claim 2 , or a TET catalytically active fragment.12. The method of claim 11 , wherein said TET family enzyme comprises TET1 claim 11 , TET2 claim 11 , TET3 or CXXC4.13. The method of claim 11 , wherein said functional TET ...

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Номер записи: 23
14-02-2019 дата публикации

SELECTIVE OXIDATION OF 5-METHYLCYTOSINE BY TET-FAMILY PROTEINS

Номер: US20190048407A1
Автор: Agarwal Suneet, Iyer Aravind, Koh Kian Peng, Rao Anjana, Tahiliani Mamta
Принадлежит:

The present invention provides for novel methods for regulating and detecting the cytosine methylation status of DNA. The invention is based upon identification of a novel and surprising catalytic activity for the family of TET proteins, namely TET1, TET2, TET3, and CXXC4. The novel activity is related to the enzymes being capable of converting the cytosine nucleotide 5-methylcytosine into 5-hydroxymethylcytosine by hydroxylation. 1. A method comprising:labeling a hydroxyl group on a hydroxymethylated residue in a nucleic acid to generate a labeled hydroxymethylated residue, wherein said nucleic acid is from an extracellular fluid sample; andsequencing said nucleic acid comprising said labeled hydroxymethylated residue.2. The method of claim 1 , wherein said extracellular fluid sample is from a mammal.3. The method of claim 1 , wherein said nucleic acid is a mammalian nucleic acid.4. The method of claim 1 , wherein said labeling is covalently labeling.5. The method of claim 1 , wherein said hydroxymethylated residue is a 5-hydroxymethylcytosine.6. The method of claim 5 , wherein said labeling comprises glycosylating said 5-hydroxymethylcytosine.7. The method of claim 1 , wherein said nucleic acid further comprises a methylated cytosine residue.8. The method of claim 7 , wherein said methylated cytosine residue is a 5-methylcytosine.9. The method of claim 1 , wherein said sequencing comprises high-throughput sequencing.10. The method of claim 1 , further comprising binding said labeled hydroxymethylated residue to a support.11. The method of claim 10 , wherein said binding occurs prior to said sequencing.12. The method of claim 1 , wherein said labelling comprises associating a label with said hydroxymethylated residue.13. The method of claim 12 , wherein said label comprises a sugar.14. The method of claim 12 , wherein said label comprises a bead.15. A composition comprising a nucleic acid from an extracellular fluid sample claim 12 , wherein said nucleic acid ...

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Номер записи: 24
22-02-2018 дата публикации

MATERIALS AND METHODS FOR THE SYNTHESIS OF ERROR-MINIMIZED NUCLEIC ACID MOLECULES

Номер: US20180051280A1
Автор: Caiazza Nicky, Gibson Daniel G., Richardson Toby H.
Принадлежит:

The present invention provides materials and methods useful for error correction of nucleic acid molecules. In one embodiment of the invention, a first plurality of double-stranded nucleic acid molecules having a nucleotide mismatch are fragmented by exposure to a molecule having unidirectional mismatch endonuclease activity. The nucleic acid molecules are cut at the mismatch site or near the mismatch site, leaving a double-stranded nucleic acid molecule having a mismatch at the end or near end of the molecule. The nucleic acid molecule is then exposed to a molecule having unidirectional exonuclease activity to remove the mismatched nucleotide. The missing nucleotides can then be filled in by the action of, e.g., a molecule having DNA polymerase activity. The result is double-stranded nucleic acid molecules with a decreased frequency of nucleotide mismatches. Also provided are novel nucleic acid sequences encoding mismatch endonucleases, polypeptides encoded thereby, as well as nucleic acid constructs, transgenic cells, and various compositions thereof. 1. An isolated nucleic acid molecule comprising:a) a nucleic acid sequence hybridizing under low, moderate, or high stringency conditions to a nucleic acid sequence selected from the group consisting of SEQ ID NO: 09, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, a complement thereof or a fragment of either; orb) a nucleic acid sequence exhibiting 70% or greater identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO: 09, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, a complement thereof or a fragment of either; ord) a nucleic acid sequence encoding a polypeptide exhibiting 50% or greater identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: ...

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Номер записи: 25
21-02-2019 дата публикации

In Situ Nucleic Acid Sequencing of Expanded Biological Samples

Номер: US20190055597A1
Автор: Alon Shahar, Boyden Edward Stuart, Chen Fei, Church George, Daugharthy Evan R., Marblestone Adam Henry, Tillberg Paul Warren
Принадлежит:

The invention provides in situ nucleic acid sequencing to be conducted in biological specimens that have been physically expanded. The invention leverages the techniques for expansion microscopy (ExM) to provide new methods for in situ sequencing of nucleic acids as well as new methods for fluorescent in situ sequencing (FISSEQ) in a new process referred to herein as “expansion sequencing” (ExSEQ). 1. A method for in-situ sequencing of target nucleic acids present in a biological sample comprising the steps of:a) linking target nucleic acids present in the biological sample with a small molecule linker or a nucleic acid adaptor capable of linking to a target nucleic acid and to a swellable material;b) embedding the biological sample comprising the target nucleic acids and attached small molecule linker or nucleic acid adaptor in a swellable material wherein the small molecule linker or the nucleic acid adaptor is linked to the target nucleic acids present in the sample and to the swellable material,c) digesting proteins present in the biological sample;d) swelling the swellable material to form a first enlarged biological sample that is enlarged as compared to the biological sample;e) re-embedding the first enlarged sample in a non-swellable material;(f) modifying the target nucleic acids or the nucleic acid adaptor to form a nucleic acid adaptor useful for sequencing; and(g) sequencing the nucleic acids present in the first enlarged sample.2. The method of claim 1 , wherein biochemically modifying the target nucleic acids or the nucleic acid adapter comprises contacting the target nucleic acids or the nucleic acid adapter with reverse transcriptase.3. The method of claim 1 , wherein the sequencing step of step (g) is fluorescence in situ sequencing.4. The method of claim 1 , further comprising repeating steps (a) through (e) on the first enlarged sample to form a second enlarged sample prior to sequencing.5. The method of claim 1 , wherein nucleic acid adaptors are ...

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Номер записи: 26
01-03-2018 дата публикации

SINGLE NUCLEOTIDE DETECTION METHOD

Номер: US20180057872A1
Автор: BALMFORTH Barnaby, Frayling Cameron Alexander
Принадлежит: BASE4 INNOVATION LTD

A method of sequencing a nucleic acid such as DNA or RNA is provided. It is characterised by the steps of (1) generating a stream of single nucleoside triphosphates by progressive pyrophosphorolysis of the nucleic acid; (2) producing at least one substantially double-stranded oligonucleotide used probe by reacting in the presence of a polymerase and a ligase at least 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 sequence 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) repeating steps (3) and (4) in a cycle and (6) detecting the characteristic detectable elements released in each iteration of step (3). Suitably the detectable elements are fluorophores. The method of the present invention generates a stronger fluorescence signal from a single nucleoside triphosphate than has been described previously. Suitable probe systems are also disclosed. 116-. (canceled)17. A multi-component biological probe system characterised by comprising (a) a first single-stranded oligonucleotide labelled with one or more fluorophores in an undetectable state and (b) second and third unlabelled single-stranded oligonucleotides capable of hybridising respectively to complementary 3′ side and 5′ side flanking regions on the first oligonucleotide which are juxtaposed either side of a single nucleotide capture region. ...

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Номер записи: 27
04-03-2021 дата публикации

SYSTEMS AND METHODS FOR NON-INVASIVE PREIMPLANTATION GENETIC DIAGNOSIS

Номер: US20210062256A1
Автор: BARARIYA Dhruti Ashokbhai, MANOHARAN Arun Prasad, MUNNE-BLANCO Santiago, Wells Dagan
Принадлежит:

A system for identifying genomic features in an embryo candidate is disclosed. The system includes a genomics sequencer, a computing device and a display. The genomic sequencer is configured to obtain sequence information from concatenated genomic fragments derived from an embryo candidate. The concatenated genomic fragments each contain at least one genomic linker segment and at least one genomic fragment from the embryo candidate. The computing device is communicatively connected to the genomic sequencer and includes a sequence alignment engine and a genomic features identification engine. The sequence alignment engine is configured to subtract out sequence information related to the genomic linker segment portion of the concatenated genomic fragments and align the genomic fragment sequences to a reference genome. The genomic features identification engine is configured to identify genomic features in the aligned genomic fragment sequences. The display is communicatively connected to the computing device and configured to display a report containing the identified genomic features. 1. A method for determining copy number variation in an embryo candidate for in vitro fertilization (IVF) implantation , comprising:isolating an embryo candidate from a plurality of embryos;incubating the embryo candidate in media that is substantially free of DNA;transferring a portion of the media to an amplification vessel, wherein the portion of media includes genomic fragments shed or secreted from the embryo candidate;adding a plurality of genomic linker segments and ligase enzyme to the amplification vessel in conditions that catalyze the formation of concatenated genomic fragments containing at least one genomic linker segment and at least one genomic fragment from the isolated embryo candidate;amplifying the concatenated genomic fragments in the amplification vessel;obtaining sequence information from the amplified concatenated genomic fragments;aligning the sequence ...

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Номер записи: 28
28-02-2019 дата публикации

SINGLE NUCLEOTIDE DETECTION METHOD

Номер: US20190062816A1
Автор: BALMFORTH Barnaby
Принадлежит: Base4 Innovation Limited

A method of sequencing a nucleic acid comprising the steps of (1) generating a stream of single nucleotides by progressive pyrophosphorolysis; (2) producing at least one substantially double-stranded oligonucleotide used probe comprising (a) a first single-stranded oligonucleotide labelled with first and second regions of detectable element types and (b) second and third single-stranded oligonucleotides capable of hybridising to complementary regions on the first oligonucleotide; (2a) either (i) treating the used probe with a restriction endonuclease to cut the first oligonucleotide strand at the recognition site or (ii) treating the used probe with restriction endonuclease to cut the first oligonucleotide strand at the recognition site; (3) digesting the first oligonucleotide strand of the used probe with an enzyme ide; (4) reacting the fourth oligonucleotide with another first oligonucleotide to produce a substantially double-stranded oligonucleotide product corresponding to the used probe; (5) repeating the above steps; and (6) detecting the detectable elements. 1. A method of sequencing a nucleic acid comprising the steps of:(a) generating a stream of single nucleotides by progressive pyrophosphorolysis of the nucleic acid; (i) a first single-stranded oligonucleotide labelled with first and second regions of a characteristic detectable element in an undetectable state located respectively on the X′ and Y′ sides of a third region comprising a restriction enzyme recognition site element including a capture site and an exonuclease-blocking site on the X′ side thereof wherein either X′ is 3′ and Y′ is 5′, or X′ is 5′ and Y′ is 3′; and', '(ii) second and third single-stranded oligonucleotides that hybridize to complementary regions on the first oligonucleotide flanking the capture site;, '(b) producing at least one substantially double-stranded oligonucleotide used probe by reacting, in the presence of a polymerase and a ligase, one of the single nucleotides with a ...

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Номер записи: 29
11-03-2021 дата публикации

METHODS FOR DETERMINING BASE LOCATIONS IN A POLYNUCLEOTIDE

Номер: US20210071239A1
Автор: Akeson Mark A., Jain Miten, Olsen Hugh Edward
Принадлежит:

Disclosed are methods for polynucleotide sequencing that detect the location of selected nucleobases with greater precision. The methods can be used to determine the location and nature of modified bases in a polynucleotide, that is, non-canonical bases, or to improve accuracy of sequencing of “problem” regions of DNA sequencing such as homopolymers, GC rich areas, etc. The sequencing method exemplified is nanopore sequencing. Nanopore sequencing is used to generate a unique signal at a point in a polynucleotide sequence where an abasic site (AP site, or apurinic or apyrimidinic site) exists. As part of the method, an abasic site is specifically created enzymatically using a DNA glycosylase that recognizes a pre-determined nucleobase species and cleaves the N-glycosidic bond to release only that base, leaving an AP site in its place. 128-. (canceled)29. A method of detecting a sequence in an RNA polynucleotide molecule , the method comprising:(a) treating the RNA polynucleotide molecule with an RNA glycosylase that creates an abasic site corresponding to pseudouridine (Ψ), diydrouridine (D), inosine (I), and 7-methylguanosine (m7g) species in the polynucleotide;(b) conducting single molecule sequencing on the polynucleotide prepared in step (a) where the sequencing indicates the abasic site within the polynucleotide sequence; and(c) using the sequence from step (b) to identify the abasic site and correlating said abasic site to pseudouridine ('P), diydrouridine (D), inosine (I), and 7-methylguanosine (m7g).30. The method of claim 29 , wherein the RNA glycosylase is EC 3.2.2.22.31. The method of claim 29 , wherein the RNA glycosylase lacks beta lyase activity.32. The method of claim 31 , wherein the RNA glycosylase is engineered to lack beta lyase activity.33. The method of claim 29 , wherein conducting single molecule sequencing comprises nanopore-based sequencing comprises measuring an ionic current that identifies an abasic site.34. The method of claim 33 , ...

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Номер записи: 30
15-03-2018 дата публикации

NUCLEIC ACID SEQUENCING BY NANOPORE DETECTION OF TAG MOLECULES

Номер: US20180073071A1
Автор: Chen Roger, Davis Randall, Ju Jingyue
Принадлежит:

This disclosure provides systems and methods for sequencing nucleic acids using nucleotide analogues and translocation of tags from incorporated nucleotide analogues through a nanopore. In aspects, this disclosure is related to compositions, methods, and systems for sequencing nucleic acids using tag molecules and detection of translocation through a nanopore of tags released from incorporation of the molecule. 1. A method for sequencing a nucleic acid molecule , the method comprising:a) providing a chip comprising a plurality of individually addressable nanopores, wherein an individually addressable nanopore of said plurality of individually addressable nanopores comprises a nanopore in a membrane that is disposed adjacent to an electrode, wherein said nanopore is linked to a nucleic acid polymerase, and wherein each individually addressable nanopore is adapted to detect a tag that is released from a tagged nucleotide upon the polymerization of said tagged nucleotide;b) directing said nucleic acid molecule adjacent to or in proximity to said nanopore;c) with the aid of said polymerase, polymerizing nucleotides along said nucleic acid molecule to generate a strand that is complementary to at least a portion of said nucleic acid molecule, wherein during polymerization a tag is released from an individual nucleotide of said nucleotides, and wherein said released tag flows through or in proximity to said nanopore; andd) detecting the tag with the aid of said electrode, wherein the tag is detected subsequent to being released from said individual nucleotide.2. The method of claim 1 , wherein said detecting of (d) further comprises identifying said tag.3. The method of claim 2 , further comprising correlating said identified tag with a type of said individual nucleotide.4. The method of claim 1 , further comprising generating claim 1 , with the aid of a computer processor claim 1 , a nucleic acid sequence of the nucleic acid molecule based upon an assessment of the tags ...

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Номер записи: 31
07-03-2019 дата публикации

Epigenetic chromosome interactions

Номер: US20190071715A1
Автор: Alexandre Akoulitchev, Aroul Ramadass, Ewan Hunter
Принадлежит: Oxford BioDynamics PLC

A method of determining responsiveness to therapy for rheumatoid arthritis.

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Номер записи: 32
24-03-2022 дата публикации

Methods of Epigenetic Analysis

Номер: US20220090176A1
Автор: Agarwal Suneet, Iyer Aravind, Koh Kian Peng, Rao Anjana, Tahiliani Mamta
Принадлежит:

The present invention provides for methods of epigenetic analysis. In some cases, the methods may include obtaining a sample comprising a nucleic acid sequence. In some cases, the nucleic acid sequence may comprise one or more epigenetic marks. The methods may include performing a sequencing. The methods may include distinguishing a hydroxymethylated base from a methylated base. 1. A method for improving the generation of stable human Foxp3+ T cells , the method comprising contacting with , or delivering to , a human T cell an effective 5-methylcytosine to 5-hydroxymethylcytosine converting amount of at least one catalytically active TET family enzyme , functional TET family derivative , TET family catalytically active fragment thereof , or combination thereof.2. The method of claim 1 , wherein the human T cell is a purified human CD4+ T cell.3. The method of claim 1 , further comprising contacting with claim 1 , or delivering to claim 1 , the human T cell a composition comprising at least one cytokine claim 1 , growth factor claim 1 , activating reagent claim 1 , or combination thereof.4. The method of claim 3 , wherein said composition comprises TGF-β.5. A method for improving efficiency or rate with which an induced pluripotent stem (iPS) cell is produced from an adult somatic cell claim 3 , the method comprising contacting with claim 3 , or delivering to a somatic cell claim 3 , an effective 5-methylcytosine to 5-hydroxymethylcytosine converting amount of at least one catalytically active TET family enzyme claim 3 , functional TET family derivative claim 3 , TET catalytically active thereof claim 3 , or combination thereof.6. The method of claim 5 , wherein the catalytically active TET family enzyme is TET1 or TET2.7. The method of claim 5 , further comprising comprising contacting with claim 5 , or delivering to claim 5 , the somatic cell claim 5 , an effective amount of a TET family inhibitor.8. The method of claim 7 , wherein the TET family inhibitor is a ...

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Номер записи: 33
14-03-2019 дата публикации

REAGENTS AND METHODS FOR BIOORTHOGONAL LABELING OF BIOMOLECULES IN LIVING CELLS

Номер: US20190077776A1
Автор: Blizzard Robert, MEHL RYAN A.
Принадлежит: Oregon State University

Tetrazine non-canonical amino acids, methods for genetic encoding proteins and polypeptides using the tetrazine amino acids, proteins and polypeptides comprising the tetrazine amino acids, and compositions comprising the proteins and polypeptides having at least one post-translational modification thereof comprising in vivo reaction with the incorporated tetrazine amino acid and a second molecule. 6. The compound of claim 1 , wherein Ris selected from methyl claim 1 , ethyl claim 1 , n-propyl claim 1 , i-propyl claim 1 , n-butyl claim 1 , s-butyl claim 1 , t-butyl claim 1 , n-pentyl claim 1 , and n-hexyl.7. The compound of claim 1 , wherein Ris selected from trifluoromethyl claim 1 , 2-fluoroethyl claim 1 , and methoxy.8. The compound of claim 1 , wherein phenylene is 1 claim 1 ,4-phenylene.9. The compound of claim 1 , wherein phenylene is 1 claim 1 ,3-phenylene.1012-. (canceled)1416-. (canceled)17. The compound of selected from the group consisting of 4-(6-methyl-s-tetrazin-3-yl)phenylalanine (Tet-v2.0-methyl) claim 1 , 4-(6-ethyl-s-tetrazin-3-yl)phenylalanine (Tet-v2.0-ethyl) claim 1 , 4-(6-isopropyl-s-tetrazin-3-yl)phenylalanine (Tet-v2.0-isopropyl) claim 1 , and 4-(6-butyl-s-tetrazin-3-yl)phenylalanine (Tet-v2.0-n-butyl).18. The compound of selected from the group consisting of 3-(6-methyl-s-tetrazin-3-yl)phenylalanine (Tet-v3.0-methyl) claim 5 , 3-(6-ethyl-s-tetrazin-3-yl)phenylalanine (Tet-v3.0-ethyl) claim 5 , 3-(6-isopropyl-s-tetrazin-3-yl)phenylalanine (Tet-v3.0-isopropyl) claim 5 , 3-(6-t-butyl-s-tetrazin-3-yl)phenylalanine (Tet-v3.0-t-butyl) claim 5 , and 3-(6-n-butyl-s-tetrazin-3-yl)phenylalanine (Tet-v3.0-n-butyl).19. A method for making a protein or a polypeptide of interest claim 5 , comprising:{'claim-ref': {'@idref': 'CLM-00001', 'claim 1'}, 'incorporating a compound of into a protein or polypeptide.'}20. A method for genetically encoding a protein or a polypeptide of interest claim 5 , comprising:{'claim-ref': {'@idref': 'CLM-00001', 'claim 1'}, ' ...

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Номер записи: 34
14-03-2019 дата публикации

METHOD FOR CONTROLLED DNA FRAGMENTATION

Номер: US20190078083A1
Автор: GAIDAMAUSKAS Ervinas, LUBYS Arvydas, TAMOSEVICIUS Romas, UKANIS Mindaugas
Принадлежит:

A composition and method for controlled in vitro fragmentation of nucleic acids. A transposase forms catalytically active complexes with a modified transposon end that contains within its end sequence degenerate, apurinic/apyrimidinic sites, nicks, or nucleotide gaps, to fragment or shear a target nucleic acid sample in a controlled process. This method yields desired average nucleic acid fragment sizes. The inventive composition and method may be applied for generation of DNA fragments containing shortened transposon end sequences to facilitate subsequent reactions, for production of asymmetrically tailed DNA fragments, etc. 1. A method for fragmenting nucleic acids from an initial nucleic acid sample in an in vitro reaction , comprising:a) providing a plurality of transpososome complexes, which include (i) a plurality of transposases, (ii) a first transposon end sequence, wherein the first transposon end sequence is capable of binding to a transposase from the plurality of transposases and wherein the first transposon end sequence contains at least one nick, gap, apurinic site or apyrimidinic site, (iii) a second transposon end sequence, wherein the second transposon end sequence is capable of binding to a transposase from the plurality of transposases and wherein the second transposon end sequence contains at least one nick, gap, apurinic site or apyrimidinic site;b) contacting, in a single reaction mixture, the plurality of transpososome complexes with nucleic acids from the initial nucleic acid sample, under conditions that are suitable for transposing the first and second transposon end sequences into the nucleic acids and fragmenting the nucleic acids, where the nucleic acids includes a first nucleic acid molecule; andc) producing at least one fragmented tagged DNA molecule having a first end joined to the first transposon end sequence and a second end joined to the second transposon end sequence, by transposing the first transposon end sequences into the ...

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Номер записи: 35
14-03-2019 дата публикации

SAMPLE PREPARATION FOR NUCLEIC ACID AMPLIFICATION

Номер: US20190078139A1
Автор: Fraser Louise, Kokko-Gonzales Paula, SLATTER ANDREW
Принадлежит:

Presented are methods and compositions for preparing samples for amplification and sequencing. Particular embodiments relate to methods of obtaining nucleic acids material directly from tissues such as whole blood or FFPE samples. 116-. (canceled)17. A method of preparing a nucleic acid-containing cellular sample for library amplification comprising the following steps:(a) lysing the cells of the sample with a lysis reagent to liberate nucleic acids from within the cells of the cellular sample, thereby forming a lysate comprising liberated nucleic acids; and(b) performing tagmentation on liberated nucleic acids in the lysate without purifying the liberated acids prior to tagmentation, thereby forming tagmented nucleic acids.18. The method of claim 17 , wherein the nucleic acids are DNA.19. The method of claim 17 , wherein the sample is a blood sample claim 17 , a whole blood sample claim 17 , or a dried blood sample.20. The method of claim 17 , wherein the sample is a non-blood sample claim 17 , a tissue sample claim 17 , a tumor sample claim 17 , a biopsy sample claim 17 , an aspirate claim 17 , or an FFPE sample.21. The method of claim 17 , wherein the lysis reagent is water claim 17 , purified water claim 17 , or distilled water.22. The method of claim 17 , wherein the lysis reagent is a detergent claim 17 , a base claim 17 , an acid claim 17 , and/or an enzyme.23. The method of claim 17 , wherein step (a) comprises treating the cells of the sample or the lysate with an enzyme to disrupt the structure of the nucleic acids.24. The method of claim 23 , wherein the nucleic acids are DNA and the enzyme that disrupts the structure is proteinase K.25. The method of claim 17 , further comprising neutralizing the lysis reagent prior to the tagmentation step (b) to inactivate the lysis reagent.26. The method of claim 25 , wherein neutralizing the lysis reagent is carried out via a neutralizing agent or via heat.27. The method of claim 17 , further comprising incubating ...

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Номер записи: 36
14-03-2019 дата публикации

Methods of enriching and determining target nucleotide sequences

Номер: US20190078148A1
Автор: Zongli ZHENG
Принадлежит: Helitec Ltd

The present invention provides methods, compositions and kits for enriching and determining nucleotide sequences of a plurality of target loci from a sample comprising nucleic acids. The methods comprise one or more cycles of primer extension followed by PCR amplification of target sequences using nested target-specific primers.

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Номер записи: 37
14-03-2019 дата публикации

SEQUENCING LIBRARY, AND PREPARATION AND USE THEREOF

Номер: US20190078157A1
Автор: RUAN Jue, Wang Kaile
Принадлежит:

The present invention discloses a sequencing library comprising a nucleotide sequence. The sequence comprises a linker sequence and two target sequences. Two ends of the linker sequence are respectively linked to the target sequences and the two target sequences are direct repeat sequences. The present invention further discloses preparation and use of the sequencing library. The present invention overcomes the high error rate problem of current DNA sequencing technologies, especially in a way of very low coverage bias, and can be used to detect low frequency mutations in different kinds of samples. 1. A nucleotide sequence , comprising a linker and two target sequences , wherein two ends of the linker are respectively linked to the target sequences and the two target sequences are direct repeat sequences.2. The nucleotide sequence of claim 1 , wherein a reverse complementary region exists in the linker.3. The nucleotide sequence of claim 1 , wherein one end of at least one of the target sequences opposing the end linked with the adaptor sequence is further linked with an additional sequence claim 1 , and at least part of the region of the additional sequence is the same as part of the region of the linker.4. The nucleotide sequence of claim 1 , wherein the length of the target sequence is less than the sequencing read length of a DNA sequencing machine.5. The nucleotide sequence of claim 4 , wherein the sum of the lengths of the additional sequence and the target sequences is less than the sequencing length of the DNA sequencing machine.6. The nucleotide sequence of claim 1 , wherein the nucleotide sequence consists of a linker and target sequences linked to two ends of the linker claim 1 , and the two target sequences are direct repeat sequences.7. The nucleotide sequence of claim 6 , wherein the length of the target sequence is less than the sequencing length of a DNA sequencing machine.8. A nucleotide sequence claim 6 , comprising a linker and two target ...

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Номер записи: 38
22-03-2018 дата публикации

Single nucleotide detection method

Номер: US20180080074A1
Автор: Barnaby Balmforth, Cameron Alexander Frayling
Принадлежит: BASE4 INNOVATION LTD

A method of sequencing a nucleic acid such as DNA or RNA is provided. It is characterised by the steps of (1) generating a stream of single nucleoside triphosphates by progressive pyrophosphorolysis of the nucleic acid; (2) producing at least one substantially double-stranded oligonucleotide used probe by reacting in the presence of a polymerase and a ligase at least 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 sequence 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) repeating steps (3) and (4) in a cycle and (6) detecting the characteristic detectable elements released in each iteration of step (3). Suitably the detectable elements are fluorophores. The method of the present invention generates a stronger fluorescence signal from a single nucleoside triphosphate than has been described previously. Suitable probe systems are also disclosed.

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Номер записи: 39
23-03-2017 дата публикации

Probe set for analyzing a dna sample and method for using the same

Номер: US20170081702A1
Автор: Carl Oscar Fredrik Dahl, Filip KARLSSON, Fredrik Roos, Olof John Ericsson
Принадлежит: Vanadis Diagnostics AB

This disclosure provides, inter alia, a probe system probe system for analyzing a nucleic acid sample. In some embodiments, the probe system may comprise: a set of identifier oligonucleotides of sequence B, a set of splint oligonucleotides of formula X′-A′-B′-Z′, wherein sequence A′ is complementary to a genomic fragment and sequence B′ is complementary to at least one member of the set of identifier oligonucleotides, and one or more probe sequences comprising X and Z. Each splint oligonucleotide is capable of hybridizing to the probe sequences, a member of the set of identifier oligonucleotides and a genomic fragment, thereby producing a ligatable complex of formula X-A-B-Z. The probe system can be used to identify a chromosome aneuploidy in cell free DNA, for example.

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Номер записи: 40
25-03-2021 дата публикации

METHODS AND COMPOSITIONS FOR IDENTIFYING LIGANDS ON ARRAYS USING INDEXES AND BARCODES

Номер: US20210087613A1
Автор: Berti Lorenzo, Black Fiona E., Brodin Jeffrey Dennis, Eckhardt Allen, Fisher Jeffrey S., Leong Siew Hong, SEGALE DARREN, Teo Yin Nah, Zhang Rui
Принадлежит:

Some embodiments provided herein include methods and compositions for the detection of target ligands on an array. In some embodiments, a capture probe specifically binds to a target ligand from a sample, the location of a bead comprising the capture probe in an array is determined, and the bead is decoded to identify the capture probe and the sample. In some embodiments, a barcode is indicative of a capture probe attached to a bead; and an index is indicative of a subpopulation of beads. Some embodiments relate to sequencing target polynucleotides from several different nucleic acids samples on a bead array. 1. A method of sequencing target nucleic acids on an array , comprising: the first population of beads comprise oligonucleotides comprising first capture probes, first barcodes, and barcode primer binding sites adjacent to the first barcodes, and', 'the second population of beads comprise oligonucleotides comprising second capture probes, second barcodes, and barcode primer binding sites adjacent to the second barcodes;, '(a) obtaining a first and a second population of beads, wherein the first plurality of polynucleotides comprises first target nucleic acids, wherein the plurality of first polynucleotides is in solution, and', 'the second plurality of polynucleotides comprises second target nucleic acids, wherein the plurality of second polynucleotides is in solution;, '(b) obtaining a first and a second plurality of polynucleotides, wherein(c) hybridizing the first target nucleic acids to the first capture probes to obtain hybridized first beads, and hybridizing the second target nucleic acids to the second capture probes to obtain hybridized second beads;(d) randomly distributing the hybridized first beads and hybridized second bead on an array;(e) decoding the locations of the first and second beads on the array by sequencing the first and second barcodes; and(f) extending the first and second capture probes to obtain nucleic acid sequence data for the ...

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Номер записи: 41
29-03-2018 дата публикации

A METHOD FOR MEASURING THE PROTEASE ACTIVITY OF C3 AND C5 CONVERTASE OF THE ALTERNATIVE COMPLEMENT PATHWAY

Номер: US20180087087A1
Автор: FORBES Christen D., JOHNSON Krista K.
Принадлежит:

A method for measuring the protease activity of a convertase of the alternative complement pathway is provided. The method typically comprises immobilizing a biotinylated C3b on a solid phase coated with a biotin binding protein. Substantially homogeneous components of the alternative complement pathway may be incubated with the immobilized C3b in a serum free and gelatin free buffer, to form a convertase. The activity of the convertase is generally measured with an immunoassay. 1. A method for measuring the protease activity of C3 the steps of:a. covalently attaching biotin to C3b to produce biotinylated-C3b;b. binding biotinylated-C3b to a biotin binding protein immobilized on a solid phase;c. incubating the immobilized biotinylated-C3b, Factor D, and Factor B in a buffer to form a C3 convertase;d. transferring C3 to the buffer to cleave C3 with the convertase to form a C3a and C3b; ande. measuring the amount of C3a with an immunoassay, wherein each of the individual components bio-C3b, Factor B, Factor D, and C3 are substantially homogeneous.2. A method for measuring the protease activity of C5 convertase comprising the steps of:a. covalently attaching biotin to C3b to produce biotinylated-C3b;b. binding biotinylated-C3b to a biotin binding protein immobilized on a solid phase;c. incubating the immobilized biotinylated-C3b, Factor D, and Factor B in a buffer to form C5 convertase;d. transferring C5 to the buffer, and cleaving C5 with the convertase to form a C5a and CSb; ande. measuring the amount of the C5a with an immunoassay, wherein each of the individual components bio-C3b, Factor B, Factor D, and C5 are substantially homogeneous.3. The method of claim 1 , wherein the biotin-binding protein is selected from the group consisting of avidin claim 1 , streptavidin and neutravidin.4. (canceled)5. The method of claim 1 , wherein the buffer further comprises properdin and/or is a serum free and gelatin free buffer.6. The method of claim 1 , wherein the convertase ...

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Номер записи: 42
29-03-2018 дата публикации

Techniques for fine grained correction of count bias in massively parallel DNA sequencing

Номер: US20180089367A1
Автор: Burke John, RHEES Brian
Принадлежит:

Techniques for automated determination or correction of count bias are based on nucleic acid base content on a finer grained scale than a bin of interest in a target sequence. The techniques include obtaining a target sequence with bins where relative abundances indicate a condition and raw counts Hj of reads, from a subject, which start at each locus j. A partition indicates a fine-grained window at a position relative to a current locus and multiple strata indicating different base contents. Each locus is attributed to one stratum k(j). An expected count of each stratum, E(k), is determined based on Hj for j belonging to the stratum and a number of loci in the target belonging to the stratum. A copy number of a bin is based on a sum of E(k(j)) in the bin. Output data indicates condition of the subject based at least partly on the copy number. 1. A method executed on a processor comprising:a. obtaining first data that indicates a target sequence of nucleic acid bases at a plurality of loci, wherein the target sequence comprises a plurality of bins of loci for which a relative abundance is indicative of a condition of interest;b. obtaining second data that indicates alignment with the target sequence of reads of DNA fragments in a sample from a subject;c. determining a raw count Hj of reads that start at each locus j;d. obtaining partition data that indicates, for a first partition, a window comprising a number of bases less than a number of loci in a bin and position relative to a current locus, and a plurality of strata based on a corresponding plurality of different contents of nucleic acid bases in the window;e. attributing to each locus j in the target sequence a stratum k(j) of the plurality of strata of the first partition based on the content of nucleic acid bases in the target sequence in the window relative to the locus j;f. determining an expected count of each stratum, E(k), in the first partition based on the raw counts Hj of each locus j belonging to ...

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Номер записи: 43
05-05-2022 дата публикации

Modified enzymes

Номер: US20220135956A1
Автор: Andrew John Heron, Christopher Peter YOUD, Elizabeth Jayne Wallace, Joseph Hargreaves LLOYD, Lakmal Jayasinghe, Mark Bruce, Rebecca Victoria BOWEN, Szabolcs SOEROES
Принадлежит: Oxford Nanopore Technologies PLC

The invention relates to modified Dda helicases which can be used to control the movement of polynucleotides and are particularly useful for sequencing polynucleotides.

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Номер записи: 44
19-03-2020 дата публикации

SELECTIVE OXIDATION OF 5-METHYLCYTOSINE BY TET-FAMILY PROTEINS

Номер: US20200087715A1
Автор: Agarwal Suneet, Iyer Aravind, Koh Kian Peng, Rao Anjana, Tahiliani Mamta
Принадлежит:

The present invention provides for novel methods for regulating and detecting the cytosine methylation status of DNA. The invention is based upon identification of a novel and surprising catalytic activity for the family of TET proteins, namely TET1, TET2, TET3, and CXXC4. The novel activity is related to the enzymes being capable of converting the cytosine nucleotide 5-methylcytosine into 5-hydroxymethylcytosine by hydroxylation. 110.-. (canceled)11. A method comprising:high throughput sequencing a nucleic acid sequence; anddetecting a methylation state of a site in said nucleic acid sequence, wherein said nucleic acid sequence comprises a hydroxymethylated base.12. The method of claim 11 , wherein said nucleic acid sequence is a mammalian nucleic acid sequence.13. The method of claim 11 , wherein said nucleic acid sequence is obtained from a eukaryotic organism14. The method of claim 11 , wherein said nucleic acid sequence is not obtained from a bacteriophage.15. The method of claim 11 , wherein said nucleic acid sequence is of an extracellular fluid sample.16. The method of claim 11 , further comprising isolating said nucleic acid sequence from a sample.17. The method of claim 11 , further comprising obtaining said nucleic acid sequence.18. The method of claim 11 , further comprising associating a label with said site.19. The method of claim 11 , wherein said site comprises a genomic region.20. The method of claim 11 , wherein said nucleic acid sequence is from a subject in need thereof.21. The method of claim 11 , wherein said nucleic acid sequence is from a subject having or suspected of having cancer. This application is a continuation application under 35 U.S.C. § 120 of co-pending U.S. application Ser. No. 16/411,998 filed May 14, 2019, which is a continuation application under 35 U.S.C. § 120 of co-pending U.S. application Ser. No. 16/012,280 filed Jun. 19, 2018, which is a a continuation application under 35 U.S.C. § 120 of co-pending U.S. application Ser. ...

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Номер записи: 45
19-03-2020 дата публикации

SELECTIVE OXIDATION OF 5-METHYLCYTOSINE BY TET-FAMILY PROTEINS

Номер: US20200087716A1
Автор: Agarwal Suneet, Iyer Aravind, Koh Kian Peng, Rao Anjana, Tahiliani Mamta
Принадлежит:

The present invention provides for novel methods for regulating and detecting the cytosine methylation status of DNA. The invention is based upon identification of a novel and surprising catalytic activity for the family of TET proteins, namely TET1, TET2, TET3, and CXXC4. The novel activity is related to the enzymes being capable of converting the cytosine nucleotide 5-methylcytosine into 5-hydroxymethylcytosine by hydroxylation. 110.-. (canceled)11. A method comprising:(a) obtaining a sample, wherein said sample is at least a portion of an extracellular fluid sample that comprises a nucleic acid sequence; and(b) adding a control sample to said sample, wherein said control sample comprises a nucleic acid sequence having an epigenetically modified base.12. The method of claim 11 , wherein said epigenetically modified base is a non-natural epigenetically modified base.13. The method of claim 11 , wherein said epigenetically modified base is a hydroxymethylated cytosine or a methylated cytosine.14. The method of claim 11 , comprising denaturing said sample.15. The method of claim 11 , comprising preparing said sample for high-throughput sequencing.16. The method of claim 11 , wherein said sample is obtained from a subject having cancer or suspected of having cancer.17. The method of claim 11 , comprising contacting said sample with an antibody or antigen-binding portion thereof that specifically binds to an epigenetic modification of said sample.18. The method of claim 17 , comprising performing solid-phase purification claim 17 , wherein said antibody or antigen-binding portion thereof is immobilized on a substrate.19. The method of claim 17 , wherein said antibody or antigen-binding portion thereof is a monoclonal antibody.20. The method of claim 17 , wherein said antibody or antigen-binding portion thereof is a hydroxymethyl cytosine-specific antibody or binding fragment thereof.21. The method of claim 17 , wherein said antibody or antigen-binding portion thereof ...

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Номер записи: 46
01-04-2021 дата публикации

MULTIPLEX 5MC MARKER BARCODE COUNTING FOR METHYLATION DETECTION IN CELL FREE DNA

Номер: US20210095341A1
Автор: HE Chuan, NIE Ji
Принадлежит:

Described herein is a multiplex 5mC marker barcode counting (MMBC) to quantify 5mC markers in DNA, in which multiple 5mC markers could be targeted for capture and linear amplification. The inventors' method provides highly sensitive and specific quantification of multiple 5mC loci. Accordingly, aspects of the disclosure relate to a method for detecting methylated or unmethylated cytosines in one or more regions of target nucleic acids, the method comprising i) combining a solution comprising the target nucleic acids with a deaminating agent to convert unmethylated cytosines in the target nucleic acids to uracils; ii) next contacting the solution with at least two probes under conditions that allow for the hybridization of the two probes to one target nucleic acid region; wherein a terminal end from each probe hybridizes adjacently to the target nucleic acid region; iii) contacting the solution comprising the hybridized probes and target nucleic acids with a ligase under conditions that allow for the ligation of the terminal ends of the adjacently hybridized probes; and iv) detecting the adjacently hybridized ligated probes in the solution. 1. A method for detecting methylated or unmethylated cytosines in one or more regions of target nucleic acids , the method comprisingi) combining a solution comprising the target nucleic acids with a deaminating agent to convert unmethylated cytosines in the target nucleic acids to uracils;ii) next contacting the solution with at least two probes under conditions that allow for the hybridization of the two probes to one target nucleic acid region; wherein a terminal end from each probe hybridizes adjacently to the target nucleic acid region;iii) contacting the solution comprising the hybridized probes and target nucleic acids with a ligase under conditions that allow for the ligation of the terminal ends of the adjacently hybridized probes; andiv) detecting the adjacently hybridized ligated probes in the solution.2. The method of ...

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Номер записи: 47
05-04-2018 дата публикации

METHODS AND SYSTEMS FOR NUCLEIC ACID AMPLIFICATION

Номер: US20180094307A1
Автор: OBERSTRASS Florian
Принадлежит:

The disclosure provides methods and systems for nucleic acid amplification including isothermal nucleic acid amplification. 130.-. (canceled)31. A method for amplifying a nucleic acid sample , comprising:a) providing a support comprising a first primer;b) providing a first double-stranded nucleic acid molecule comprising a first single-stranded nucleic acid molecule and a second single-stranded nucleic acid molecule that is at least partially complementary to said first single-stranded nucleic acid molecule, wherein the first double-stranded nucleic acid molecule is derived from the nucleic acid sample;c) at least partially denaturing the first double-stranded nucleic acid molecule by binding a first invader species free from said support to at least a portion of the first or second single-stranded nucleic acid molecule, which binding exposes a segment of the first single-stranded nucleic acid molecule that is complementary to the first primer; andd) coupling the first primer to the segment of the first single-stranded nucleic acid molecule and performing a first primer extension reaction using the first primer to generate a second double-stranded nucleic acid molecule comprising the first single-stranded nucleic acid molecule and a third single-stranded nucleic acid molecule that is complementary to at least a portion of the first single-stranded nucleic acid molecule, wherein the first primer extension reaction separates the second single-stranded nucleic acid molecule from the first single-stranded nucleic acid molecule.32. The method of claim 31 , further comprising:e) at least partially denaturing the second double-stranded nucleic acid molecule by binding a second invader species free from said support to at least a portion of the first or third single-stranded nucleic acid molecule, which binding exposes a segment of the first single-stranded nucleic acid molecule that is complementary to a second primer.33. The method of claim 32 , further comprising:f) ...

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Номер записи: 48
28-03-2019 дата публикации

RECOMBINASE POLYMERASE AMPLIFICATION

Номер: US20190093088A1
Автор: Armes Niall A., Parker Matthew James David, Piepenburg Olaf
Принадлежит:

The present invention features novel, diverse, hybrid and engineered recombinase enzymes, and the utility of such proteins with associated recombination factors for carrying out DNA amplification assays. The present invention also features different recombinase ‘systems’ having distinct biochemical activities in DNA amplification assays, and differing requirements for loading factors, single-stranded DNA binding proteins (SSBs), and the quantity of crowding agent employed. 125-. (canceled)26. A composition , comprising:a T6 UvsX H66S protein.27. The composition of claim 26 , further comprising one or more additional components selected from the group consisting of a gp32 protein derived from a myoviridae phage claim 26 , a polymerase claim 26 , and a UvsY protein.28. The composition of claim 26 , further comprising at least one nucleic acid primer.29. The composition of claim 26 , further comprising a target nucleic acid.30, Acinetobacter, Aeromonas, Aeromonas, Vibrio. The composition of claim 27 , wherein the UvsY protein is derived from a myoviridae phage claim 27 , and the myoviridae phage from which the gp32 and UvsY proteins are derived is selected from the group consisting of: T4 claim 27 , T2 claim 27 , T6 claim 27 , Rb69 claim 27 , Aeh1 claim 27 , KVP40phage 133phage 65 claim 27 , cyanophage P-SSM2 claim 27 , cyanophage PSSM4 claim 27 , cyanophage S-PM2 claim 27 , Rb14 claim 27 , Rb32phage 25phage nt-1 claim 27 , phi-1 claim 27 , Rb16 claim 27 , Rb43 claim 27 , Phage 31 claim 27 , phage 44RR2.8t claim 27 , Rb49 claim 27 , phage Rb3 claim 27 , and phage LZ2.31. The composition of claim 27 , wherein the gp32 protein is Rb69 gp32.32. The composition of claim 26 , further comprising a crowding agent.33. The composition of claim 32 , wherein the crowding agent is selected from the group comprising: polyethylene glycol claim 32 , polyethylene oxide claim 32 , polystyrene claim 32 , Ficoll claim 32 , dextran claim 32 , PVP claim 32 , and albumin.34. The composition ...

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Номер записи: 49
28-03-2019 дата публикации

METHODS OF IDENTIFYING MULTIPLE EPITOPES IN CELLS

Номер: US20190093146A1
Автор: Nolan Garry P.
Принадлежит: Roche Sequencing Solutions, Inc.

The invention provides methods, compositions, kits and devices for the detection of target molecules. In some embodiments, the invention allows for multiplexed target molecule detection. 1. A method for identifying whether a plurality of targets are present in a plurality of cells comprising:a) binding to the targets in the plurality of cells a plurality of tags, wherein a tag comprises a unique binding agent (UBA) that is specific for one of the targets;b) adding one or more linker oligonucleotide andc) assembling cell originating barcodes (COB) by subsequently adding multiple assayable polymer subunit (APS) oligonucleotides to each of the bound tags in the plurality of cells in an ordered manner during successive rounds of split pool synthesis wherein the APS oligonucleotides in each round anneal to the one or more linker oligonucleotide adjacently to the APS from a previous round and are covalently linked to the adjacently annealed APS to create unique codes that represent the identities of individual cells in which the tags are bound, and wherein the method does not include a step of isolating each cell in the plurality of cells.2. The method of claim 1 , wherein the UBA comprises an antibody.3. The method of claim 1 , wherein the UBA is a nucleic acid.4. The method of claim 3 , wherein the nucleic acid is an aptamer.5. The method of claim 1 , wherein an epitope specific barcode (ESB) is attached to the UBA.6. The method of claim 5 , wherein the ESB is a nucleic acid.7. The method of claim 1 , wherein the linker added is a single common linker.8. The method of claim 1 , wherein multiple linkers are added.9. The method of claim 1 , wherein the adjacent APSs annealed to the one or more linkers are linked by ligation.10. The method of claim 9 , wherein the ligation is a gap-filling ligation.11. The method of claim 1 , wherein the adjacent APSs are linked by Click chemistry.12. The method of claim 1 , wherein the common linker comprises APS-annealing regions ...

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Номер записи: 50
19-04-2018 дата публикации

PYROPHOSPHOROLYTIC SEQUENCING

Номер: US20180105874A1
Автор: Meuleman Wouter
Принадлежит:

A method for determining the sequence of a target nucleic acid, including steps of contacting a target nucleic acid with a polymerase to sequentially remove nucleotide triphosphates from the target nucleic acid, wherein the nucleotide triphosphates that are removed have a variety of different base moieties; and distinguishing the different base moieties for the nucleotide triphosphates that are removed. Also provided is a apparatus including a nanopore positioned in a fluid impermeable barrier to form a passage through which a nucleotide triphosphate can pass from a first fluid reservoir to a second fluid reservoir, and a reaction mix in the first fluid reservoir that includes a polymerase, target nucleic acid having two strands, and pyrophosphorolytic concentration of pyrophosphate. 124-. (canceled)25. A method for determining the sequence of a target nucleic acid , comprising:(a) providing a target nucleic acid having two strands;(b) contacting the target nucleic acid with a polymerase under conditions to sequentially remove nucleotides from the first of the two strands by pyrophosphorolysis, thereby sequentially producing nucleotide triphosphates having a variety of different base moieties; and(c) distinguishing the different base moieties for the sequentially produced nucleotide triphosphates, wherein the distinguishing of the different base moieties for the sequentially produced nucleotide triphosphates comprises passing the nucleotide triphosphates through a nanopore and distinguishing the nucleotide triphosphates by detecting variations in the ionic current flowing through the nanopore, thereby determining the sequence of the target nucleic acid.26. The method of claim 25 , wherein the polymerase is attached to the nanopore.27. The method of claim 25 , wherein the nanopore comprises a protein nanopore that is embedded in a membrane.28. The method of claim 27 , wherein the second of the two strands of the target nucleic acid is attached to the membrane.29. The ...

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Номер записи: 51
02-04-2020 дата публикации

Multiplex End-Tagging Amplification of Nucleic Acids

Номер: US20200102598A1
Автор: CHANG Chi-han, Tan Longzhi, Xie Xiaoliang Sunney, Xing Dong
Принадлежит:

The present disclosure provides a method for assembly of genomic DNA using multiplex end-tagging amplification of genomic fragments. 1. A method of DNA amplification comprisingcontacting genomic DNA with a library of transposomes with each transposome of the library having two transposases and two transposon DNA, wherein each transposon DNA includes a transposase binding site and a primer binding site sequence, wherein the primer binding site sequence is different from the primer binding site of other members of the transposome library,wherein the library of transposomes bind to target locations along the genomic DNA and the transposase cleaves the genomic DNA into a plurality of double stranded genomic DNA fragments representing a genomic DNA fragment library, with each double stranded genomic DNA fragment includes a unique and/or different primer binding site sequence on each end of the genomic DNA fragment,filling a gap between the transposon DNA and the genomic DNA fragment to form a library of double stranded genomic DNA fragment extension products having unique and/or different primer binding site sequences at each end, andamplifying the double stranded genomic DNA fragment extension products to produce amplicons.2. The method of further including sequencing the amplicons.3. The method of wherein each transposome within the library of transposomes includes two different primer binding site sequences.4. The method of wherein each transposome within the library of transposomes includes two identical primer binding site sequences on each transposon of the transposome claim 1 , which are different from primer binding site sequences in other transposomes of the library of transposomes.5. The method of wherein the genomic DNA is whole genomic DNA obtained from a single cell.6. The method of wherein the transposase is Tn5 transposase claim 1 , Mu transposase claim 1 , Tn7 transposase or IS5 transposase.7. The method of wherein the transposon DNA includes a double- ...

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Номер записи: 52
03-05-2018 дата публикации

PYRIMIDINE DERIVATIVES

Номер: US20180118721A1
Автор: Heinrich Timo, PEHL Ulrich
Принадлежит: Merck Patent GmBH

Compounds of Formula I or II 2. Compound according to Formula I according to claim 1 , wherein —NHRis methylamino.3. Compound according to Formula I according to claim 1 , wherein —NHRis cyclopropylamino.4. Compound according to Formula I or II according to claim 1 , wherein Xand Xtogether with the N to which they are attached form a heterocycle according to Formula 2 claim 1 , wherein Q is CRR.5. Compound according to Formula I or II according to claim 1 , wherein Xand Xtogether with the N to which they are attached form a heterocycle according to Formula 3 claim 1 , wherein U is CRRand T is CRR.6. Compound according to Formula I or II according to claim 1 , wherein Xand Xtogether with the N to which they are attached form a heterocycle according to Formula 1 claim 1 , wherein at least one of R claim 1 , R claim 1 , R claim 1 , R claim 1 , R claim 1 , and Ris selected from —O—CH claim 1 , —O—CH—CH claim 1 , —O—(Calkyl) claim 1 , —O-ALK1 claim 1 , —CH—O—CH claim 1 , —(CH)—O—(CH)CH claim 1 , —CH—S—CH claim 1 , —OH claim 1 , —CH—OH claim 1 , —(CH)—OH claim 1 , —CF claim 1 , —CH—Br claim 1 , —(CH)—Br claim 1 , —F claim 1 , —Cl claim 1 , substituted or unsubstituted phenyl claim 1 , substituted or unsubstituted benzyl claim 1 , chloro-benzyl claim 1 , 2-chlorobenzyl claim 1 , 3-chlorobenzyl claim 1 , 4-chlorobenzyl claim 1 , methoxy-benzyl claim 1 , 2-methoxy-benzyl claim 1 , 4-methoxy-benzyl claim 1 , methyl-benzyl claim 1 , 2-methyl-benzyl claim 1 , 3-methyl-benzyl claim 1 , 1-methyl-1-phenyl-ethyl claim 1 , phenethyl claim 1 , diphenyl-hydroxy-methyl (—C(OH)(CH)) claim 1 , benzofuranyl claim 1 , 2-benzofuranyl claim 1 , thiophenyl claim 1 , thiophen-3-yl claim 1 , substituted or unsubstituted methyl claim 1 , substituted or unsubstituted ethyl claim 1 , substituted or unsubstituted isopropyl claim 1 , substituted or unsubstituted isobutyl claim 1 , substituted or unsubstituted cyclopentyl claim 1 , —CH—C(O)—O—CH claim 1 , —C(O)—NH claim 1 , —C(O)—NH—(CH) claim 1 , —C ...

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Номер записи: 53
03-05-2018 дата публикации

Selective oxidation of 5-methylcytosine by tet-family proteins

Номер: US20180119113A1
Автор: Anjana Rao, Aravind Iyer, Kian Peng Koh, Mamta Tahiliani, Suneet Agarwal
Принадлежит: Childrens Medical Center Corp, US Department of Health and Human Services

The present invention provides for novel methods for regulating and detecting the cytosine methylation status of DNA. The invention is based upon identification of a novel and surprising catalytic activity for the family of TET proteins, namely TET1, TET2, TET3, and CXXC4. The novel activity is related to the enzymes being capable of converting the cytosine nucleotide 5-methylcytosine into 5-hydroxymethylcytosine by hydroxylation.

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Номер записи: 54
03-05-2018 дата публикации

MACROMOLECULE DELIVERY TO NANOWELLS

Номер: US20180119205A1
Автор: Gray Phillip N.
Принадлежит:

Provided herein is technology relating to depositing and/or placing a macromolecule at a desired site for an assay and particularly, but not exclusively, to methods and systems for transporting a macromolecule such as a protein, a nucleic acid, or a protein:nucleic acid complex to an assay site, such as the bottom of a nanopore, a nanowell, or a zero mode waveguide. 1. A system for transporting a macromolecule , the system comprising:a) a transport guide comprising an actin filament or a microtubule;b) a molecular motor for transporting the macromolecule along the transport guide; andc) a linking domain for linking the macromolecule to the molecular motor.2. The system of further comprising a phospholinked nucleotide.3. The system of further comprising a zero mode waveguide.4. The system of further comprising an anchor to maintain the macromolecule at a site. The present Application is a divisional of U.S. application Ser. No. 14/369,642 filed Jun. 27, 2014, which is a national phase application under 35 U.S.C. § 371 of PCT International Application No. PCT/US2012/072075, filed on Dec. 28, 2012, which claims priority to U.S. Provisional Application Ser. No. 61/581,508 filed Dec. 29, 2011, the entirety of which is incorporated by reference herein.DEVELOPMENTThis invention was made with government support under HDTRA1-10-C-0080 awarded by Defense Threat Reduction Agency. The government has certain rights in the invention.Provided herein is technology relating to depositing and/or placing a macromolecule at a desired site for an assay and particularly, but not exclusively, to methods and systems for transporting a macromolecule such as a protein, a nucleic acid, or a protein:nucleic acid complex to an assay site, such as the bottom of a nanopore, a nanowell, or a zero mode waveguide.The massive parallelization of biological assays and realization of single-molecule resolution have yielded profound advances in the ways that biological systems are characterized and ...

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Номер записи: 55
03-05-2018 дата публикации

Measuring a level of a 5-hydroxymethylcytosine in a sample from a subject having a cancer or suspected of having cancer

Номер: US20180120304A1
Автор: Anjana Rao, Aravind Iyer, Kian Peng Koh, Mamta Tahiliani, Suneet Agarwal
Принадлежит: Childrens Medical Center Corp, US Department of Health and Human Services

Provided herein are methods and kits for measuring a level of a 5-hydroxymethylcytosine in a nucleotide sequence from a subject, wherein the subject is a subject having a cancer or suspected of having cancer.

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Номер записи: 56
25-08-2022 дата публикации

METHODS, COMPOSITIONS AND KITS FOR SMALL RNA CAPTURE, DETECTION AND QUANTIFICATION

Номер: US20220267830A1
Автор: Dong Shoulian, Liu Chunmei, WONG Linda
Принадлежит:

Methods, compositions and kits for capturing, detecting and quantifying mature small RNAs are provided herein. Embodiments of the methods comprise tailing both the 5′ and 3′ ends of mature small RNA by ligating a 5′ ligation adaptor to the 5′ end and polyadenylating the 3′ end. Other embodiments comprise reverse transcribing the adaptor ligated, polyadenylated mature small RNA with a universal reverse transcription primer and amplifying the cDNA with universal primers. 1. A method for detecting a mature small RNA , the method comprising:providing a sample comprising a mature small RNA;polyadenylating the 3′ end of the mature small RNA and ligating a single-stranded adaptor to the 5′ end of the mature small RNA in the presence of single strand RNA ligase, whereby an RNA ligation product is formed, wherein the adaptor comprises a universal forward primer portion;reverse transcribing the RNA ligation product using a reverse transcription (RT) primer, thereby forming a cDNA product of the RNA ligation product, wherein the RT primer comprises a poly(T) portion and a tail portion, wherein the tail portion comprises a universal reverse primer portion;amplifying the cDNA product using a first forward and reverse primer pair to form an amplification product, wherein the first forward primer can hybridize to the universal forward primer portion or its complement, and the first reverse primer can hybridize to the universal reverse primer portion or its complement; anddetecting the amplification product corresponding to the mature small RNA via quantitative real-time polymerase chain reaction (qPCR).232-. (canceled)33. A kit for synthesizing and amplifying a mature small RNA cDNA , the kit comprising:a single-stranded adaptor comprising a 3′ terminal —OH group and a universal forward primer portion;a reverse transcription (RT) primer, wherein the RT primer comprises a poly(T) portion and a tail portion and wherein the tail portion comprises a universal reverse primer portion;a ...

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Номер записи: 57
27-05-2021 дата публикации

METHODS FOR DNA TARGETS DETECTION DIRECTLY IN CRUDE SAMPLES THROUGH POLYMERASE CHAIN REACTION AND GENOTYPING VIA HIGH RESOLUTION MELTING ANALYSIS

Номер: US20210155973A1
Автор: Avian Alice, Foschi Nicola, Ippodrino Rudy, Marini Bruna, Mauro Elisabetta, Mocenigo Marco
Принадлежит: ULISSE BIOMED S.P.A.

A method id disclosed for DNA targets detection directly in crude samples through Polymerase Chain Reaction (PCR) and genotyping via High Resolution Melting (HRM) analysis. The method comprises diluting the crude sample, treating the diluted crude sample by performing a ramp of increasing and then decreasing temperature, performing PCR amplification and then an HRM analysis. The method further comprises monitoring, during the HRM analysis, the change in the signal emission resulting from the temperature-induced denaturation of the double-stranded amplicons into two single-stranded DNA, due to the release of an intercalating molecule or compound. The detection of DNA targets in the crude sample is performed through a reader analysing the signal variation, obtaining the result of the analysis through a graphic interface connected to the reader. 1. A method for DNA targets detection directly in crude samples through Polymerase Chain Reaction (PCR) and genotyping via High Resolution Melting (HRM) analysis , said method comprising:providing a crude sample to be subjected to detection analysis;diluting said crude sample using a processing buffer comprising a protein-digesting enzyme, wherein the crude sample is diluted up to a protein concentration ranging from 0.1 μg/μl to 10 μg/μl;treating the diluted crude sample by performing a ramp of increasing and then decreasing temperature;providing a PCR reaction mixture comprising two or more pairs of amplification primers for amplifying in a multiplex approach two or more target nucleic acids and an amplification buffer comprising an intercalating molecule or compound incorporated into the double-stranded amplicon and emitting a detectable signal;performing PCR amplification using said PCR reaction mixture and said treated diluted crude sample;performing, at the end of the PCR amplification, an HRM analysis on the PCR reaction mixture and said treated diluted crude sample previously subjected to PCR amplification;wherein the ...

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Номер записи: 58
10-05-2018 дата публикации

HIGH-THROUGHPUT SEQUENCING OF POLYNUCLEOTIDES

Номер: US20180127804A1
Автор: Dean Erik Jedediah, HOLMES Victor, REEVES Christopher, SHAPLAND Elaine
Принадлежит:

Provided herein are methods, compositions, and kits for simultaneously sequencing polynucleotides from a plurality of samples in a single sequencing run. In an embodiment, the present invention improves efficiency of the next-generation sequencing process, in part, by reducing reaction volumes to a sub-microliter range and generating and using a set of novel barcode sequences to tag a plurality of polynucleotides. In addition, the sample preparation processes have been simplified to save time and cost, while providing high-quality sequence coverage for all samples. 1. A method of preparing a plurality of polynucleotides for simultaneous sequencing , the method comprising: (a) amplifying the input polynucleotide by rolling circle amplification (RCA) in an RCA solution to generate a target polynucleotide;', '(b) diluting the RCA solution comprising the target polynucleotide by a standard dilution factor;', '(c) generating a reaction mixture having a volume of about 0.005 μL to about 2 μL and comprising tagged polynucleotide fragments by contacting the diluted RCA solution comprising the target polynucleotide with transposases pre-loaded with transposon end sequences to fragment and tag the target polynucleotide;', '(d) removing the transposases from the tagged polynucleotide fragments, thereby generating a reaction solution; and', '(e) performing a polymerase chain reaction (PCR) with the reaction solution comprising the tagged polynucleotide fragments, wherein the PCR utilizes adapter primers comprising barcode sequences that are capable of hybridizing to the tagged polynucleotide fragments to generate barcoded polynucleotide fragments., 'for each input polynucleotide of a plurality of input polynucleotides,'}2. The method of claim 1 , further comprising:(f) combining the barcoded polynucleotide fragments generated for each input polynucleotide of the plurality of input polynucleotides;(g) sequencing the combined barcoded polynucleotide fragments in step (f) in a ...

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Номер записи: 59
11-05-2017 дата публикации

Single nucleotide detection method

Номер: US20170130265A1
Автор: Bamaby BALMFORTH, Cameron Alexander Frayling
Принадлежит: BASE4 INNOVATION LTD

A method of sequencing a nucleic acid such as DNA or RNA is provided. It is characterised by the steps of (1) generating a stream of single nucleoside triphosphates by progressive pyrophosphorolysis of the nucleic acid; (2) producing at least one substantially double-stranded oligonucleotide used probe by reacting in the presence of a polymerase and a ligase at least 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 sequence 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) repeating steps (3) and (4) in a cycle and (6) detecting the characteristic detectable elements released in each iteration of step (3). Suitably the detectable elements are fluorophores. The method of the present invention generates a stronger fluorescence signal from a single nucleoside triphosphate than has been described previously. Suitable probe systems are also disclosed.

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Номер записи: 60
23-04-2020 дата публикации

CHARGE-TAGGED NUCLEOTIDES AND METHODS OF USE THEREOF

Номер: US20200123193A1
Автор: Gravina Silvia, Mandell Jeffrey, Peisajovich Sergio, PUGLIESE Kaitlin, Teo Yin Nah, YANG Xiangyuan
Принадлежит:

Provided is a method including detecting an incorporation of a labelled nucleotide into a nascent polynucleotide strand complementary to a template polynucleotide strand by a polymerase, wherein the polymerase is tethered to a solid support conductive channel by a tether and the labelled nucleotides is a compound of Formula I: 155-. (canceled)57. The method of claim 56 , wherein the charge is between about −100e and about +100e.58. The method of claim 57 , wherein the charge density is between about −100e per cubic nanometer and about +100e per cubic nanometer.59. The method of claim 56 , wherein the charge is between about −200e and about +200e.60. The method of claim 59 , wherein the charge density is between about −200e per cubic nanometer and about +200e per cubic nanometer.61. (canceled)62. The method of claim 56 , wherein A was formed by a reaction comprising a linking reaction and the linking reaction is selecting from an azide-alkyne copper-assisted click reaction claim 56 , a tetrazine-trans-cyclooctene ligation claim 56 , an azide-dibenzocyclooctyne group copper-free click reaction claim 56 , and a thiol-maleimide conjugation.63. The method of claim 56 , further comprising successively incorporating a plurality of labelled nucleotides wherein the charge of each of the plurality of labelled nucleotides differs from the charge of any other of the plurality of labelled nucleotides when the Y of the each and the Y of the any other differ from each other.64. The method of further comprising identifying the Y of one or more labelled polynucleotide incorporated into the nascent polynucleotide strand based on the charge detected by the conductive channel.65. The method of any claim 56 , wherein Xis (—O—CH—CH—)wherein a is an integer from 1 to 24.66. The method of claim 65 , wherein a is 24.67. The method of claim 65 , wherein a is 16.68. The method of claim 65 , wherein a is 12.69. The method of claim 65 , wherein a is 8.70. The method of claim 65 , wherein a is 4 ...

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Номер записи: 61
19-05-2016 дата публикации

Method of producing an immunoligand/payload conjugate

Номер: US20160136298A1
Автор: Roger Renzo BEERLI, Ulf Grawunder
Принадлежит: NBE Therapeutics AG

A method of producing an immunoligand/payload conjugate can encompass conjugating a payload to an immunoligand by means of a sequence-specific transpeptidase, or a catalytic domain thereof.

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Номер записи: 62
30-04-2020 дата публикации

METHODS FOR TARGETED NUCLEIC ACID SEQUENCE ENRICHMENT WITH APPLICATIONS TO ERROR CORRECTED NUCLEIC ACID SEQUENCING

Номер: US20200131561A1
Автор: Hipp Michael, Kennedy Scott R., Nachmanson Daniela, Risques Rosa Ana, SALK Jesse J., Schmidt Elizabeth
Принадлежит:

The present technology relates generally to methods and compositions for targeted nucleic acid sequence enrichment, as well as uses of such enrichment for error-corrected nucleic acid sequencing applications. In some embodiments, highly accurate, error corrected and massively parallel sequencing of nucleic acid material is possible using a combination of uniquely labeled strands in a double-stranded nucleic acid complex in such a way that each strand can be informatically related to its complementary strand, but also distinguished from it following sequencing of each strand or an amplified product derived therefrom. In various embodiments, this information can be used for the purpose of error correction of the determined sequence. 1. A method comprising:providing double-stranded nucleic acid material comprising one or more double-stranded nucleic acid molecules, wherein each double-stranded nucleic acid molecule comprises a single molecule identifier sequence on each strand and an adapter on at least one of the 5′ and/or 3′ ends of the nucleic acid molecule, and wherein, for each nucleic acid molecule, a first adapter sequence is associated with a first strand and a second adapter sequence is associated with a second strand of the nucleic acid molecule;amplifying the nucleic acid material;separating the amplified nucleic acid material into a first sample and a second sample;amplifying the first strand in the first sample through use of a primer specific to the first adapter sequence to provide a first nucleic acid product;amplifying the second strand in the second sample through use of a primer specific to the second adapter sequence to provide a second nucleic acid product;sequencing each of the first nucleic acid product and second nucleic acid product; andcomparing the sequence of the first nucleic acid product to the sequence of the second nucleic acid product.2. The method of claim 1 , wherein the nucleic acid material is or comprises at least one of double- ...

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Номер записи: 63
30-04-2020 дата публикации

METHODS AND COMPOSITIONS FOR IDENTIFYING LIGANDS ON ARRAYS USING INDEXES AND BARCODES

Номер: US20200131570A1
Автор: Berti Lorenzo, Black Fiona E., Brodin Jeffrey Dennis, Fisher Jeffrey
Принадлежит:

Some embodiments provided herein include methods and compositions for the detection of target ligands on an array. In some embodiments, a capture probe specifically binds to a target ligand from a sample, the location of a bead comprising the capture probe in an array is determined, and the bead is decoded to identify the capture probe and the sample. In some embodiments, a barcode is indicative of a capture probe attached to a bead; and an index is indicative of a subpopulation of beads. In some embodiments, the barcode and the index are determined by sequencing. 1. A method of detecting a plurality of target nucleic acids , comprising: a capture probe which specifically binds to a target nucleic acid,', 'a first polynucleotide comprising a barcode indicative of the capture probe, and a barcode primer binding site 3′ of the barcode, and', 'a second polynucleotide comprising an index and an index primer binding site 3′ of the index, wherein the indexes of the first subpopulation are different from the indexes of the second subpopulation;, '(a) obtaining first and second subpopulations of beads, wherein each bead comprises(b) contacting first target nucleic acids to the capture probes of the first subpopulation of beads, and contacting second target nucleic acids to the capture probes of the second subpopulation of beads;(c) distributing the first and second subpopulations of beads comprising the specifically bound first and second target nucleic acids on a substrate;(d) extending the capture probes specifically bound to the first and second target nucleic acids and detecting the extended capture probes; and(e) decoding the locations of beads comprising the detected capture probes on the substrate.2. (canceled)3. The method of claim 1 , wherein the first polynucleotide comprises the capture probe.4. The method of claim 1 , wherein the capture probes of the first and the second subpopulations of beads each comprise different nucleotide sequences from one another.5. ( ...

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Номер записи: 64
10-06-2021 дата публикации

ENZYME METHOD

Номер: US20210172011A1
Автор: Heron Andrew John, Moysey Ruth
Принадлежит: Oxford Nanopore Technologies Ltd.

The invention relates to a new method of characterizing a target polynucleotide. The method uses a pore and a Hel308 helicase or a molecular 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. 143.-. (canceled)44. A method of characterising a target polynucleotide , comprising:(a) contacting the target polynucleotide with a transmembrane pore such that a portion of the target polynucleotide is captured by a transmembrane pore, wherein the pore is present in a membrane;(b) contacting the target polynucleotide with a Hel308 helicase such that the helicase binds to the target polynucleotide, thereby controlling the movement of the target polynucleotide through the transmembrane pore; and(c) measuring one or more characteristics of the target polynucleotide as the target polynucleotide moves through the transmembrane pore, thereby characterising the target polynucleotide.45. The method according to claim 44 , wherein the one or more characteristics are selected from (i) the length of the target polynucleotide claim 44 , (ii) the identity of the target polynucleotide claim 44 , (iii) the sequence of the target polynucleotide claim 44 , (iv) the secondary structure of the target polynucleotide and (v) whether or not the target polynucleotide is modified.46. The method according to claim 45 , wherein the target polynucleotide is modified by methylation claim 45 , by oxidation claim 45 , by damage claim 45 , with one or more proteins claim 45 , or with one or more labels claim 45 , tags claim 45 , or spacers.47. The method according to claim 44 , wherein at least a portion of the target polynucleotide is double stranded.48. The method according to claim 44 , wherein the transmembrane pore is a protein pore or a solid state pore.49Mycobacterium smegmatisNeisseria. The method according to claim 48 , wherein the transmembrane protein pore ...

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Номер записи: 65
24-05-2018 дата публикации

Method, Lysis Solution and Kit for Selectively Depleting Animal Nucleic Acids in a Sample

Номер: US20180142231A1
Автор: KUBICEK Johann, Sander Antje-Katrin, Singer Thorsten
Принадлежит: QIAGEN GmbH

The present invention relates to a method for selectively depleting animal nucleic acids from non-animal nucleic acids in a sample, which comprises animal cells and at least one further type of cells, selected from microbial cells and plant cells or a combination thereof, to a lysis solution A to be used in and to a kit to carry out said method as well as to the use of a particular silica membrane as both, a filtration matrix for separating essentially intact microbial and/or plant cells from lysed animal cells and an adsorption matrix for nucleic acids, in particular in a method according to the present invention. 115-. (canceled)16. A method for selectively depleting animal nucleic acids from non-animal nucleic acids in a sample , the sample comprising animal cells and at least one further type of cells selected from microbial cells and plant cells or a combination thereof , the method comprising the steps of:i) selectively lysing the animal cells by contacting the sample with a lysis solution to obtain a mixture comprising intact microbial cells and/or plant cells as well as the animal nucleic acids released from the lysed animal cells into solution,ii) digesting both DNA and RNA of the animal nucleic acids in the presence of the intact microbial and/or plant cells, andiii) separating the microbial and/or plant cells from the liquid part of the sample, including the digested animal nucleic acids.17. The method according to claim 16 , wherein in step iii) both DNA and RNA of the animal nucleic acids are digested in one step.18. The method according to claim 16 , wherein in step iii) both DNA and RNA of the animal nucleic acids are digested in one step with an endonuclease that degrades both DNA and RNA.19. The method according to claim 16 , wherein the lysis solution is an essentially chaotrope-free and comprises at least one surfactant in an amount from 0.01 to 15% (w/v) based on the whole lysis solution.20. The method according to claim 19 , wherein the ...

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Номер записи: 66
15-09-2022 дата публикации

METHOD FOR IN SITU DETERMINATION OF NUCLEIC ACID PROXIMITY

Номер: US20220290224A1
Автор: DUDCHENKO Olga, Lander Eric, Lieberman Aiden Erez, Rao Suhas, STAMENOVA Elena
Принадлежит:

Disclosed is an in situ method for detecting spatial proximity relationships between nucleic acid sequences, such as DNA, in a cell. The method includes: providing a sample of one or more cells comprising nucleic acids; fragmenting the nucleic acids present in the cells that leaves 5′ overhanging ends; filling in the overhanging ends with at least one labeled nucleotide; joining the filled in end of the fragmented nucleic acids that are in close physical proximity to create one or more end joined nucleic acid fragments having a junction; isolating the one or more end joined nucleic acid fragments using the labeled nucleotide; and determining the sequence at the junction of the one or more end joined nucleic acid fragments. 1117-. (canceled)118. A method for altering one or more chromatin loops anchored on a pair of loop anchors of one or more specific genomic regions of a chromosome in one or more cells of a cell type from a sample , wherein the sample is from an organ or a tissue or primary cells or cultured cells , said method comprising:introducing one or more CRISPR systems into the one or more cells of the cell type from the sample, wherein the one or more CRISPR systems target a region within or around a loop anchor on one or more specific genomic regions of a chromosome in the one or more cells of the cell type, wherein the one or more specific genomic regions of the chromosome in the one or more cells of the cell type comprise one or more chromatin loops, wherein the one or more chromatin loops are identified using a chromosome conformation capture technology, and wherein the one or more CRISPR systems are CRISPR RNA-guided DNA endonuclease systems,wherein said introducing one or more CRISPR systems into the one or more cells of the cell type introduces a sequence into the loop anchor of the one or more specific genomic regions of the chromosome in the one or more cells of the cell type, orwherein said introducing one or more CRISPR systems into the one or ...

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Номер записи: 67
31-05-2018 дата публикации

METHODS, COMPOSITIONS, AND KITS FOR NUCLEIC ACID ANALYSIS

Номер: US20180148756A1
Автор: Lucero Michael Y., So Austin
Принадлежит:

Aspects of the invention relate to methods and kits for assessing cancer. Some aspects of the invention relate to methods and kits for preparing a sample library for sequencing. Some aspects of the invention relate to methods and kits for allele detection. Some aspects of the invention relate to high efficiency ligation methods and kits. Some aspects of the invention relate to sensitive detection of amplicons. 1316-. (canceled)317. A method , comprising:a. ligating a first single-stranded adaptor to a single-stranded nucleic acid fragment, thereby generating a single-stranded nucleic acid fragment ligated to said first singled-stranded adaptor, wherein an efficiency of said ligating is at least 10%, 30%, 50%, 70%, or 90%;b. hybridizing a target-selective oligonucleotide to said single-stranded nucleic acid fragment ligated to said first single-stranded adaptor to create a hybridization product, wherein said target-selective oligonucleotide comprises (i) a sequence that is complementary to said single-stranded nucleic acid fragment; and (ii) a second single-stranded adaptor sequence located at a first end of said target-selective oligonucleotide; andc. extending said target-selective oligonucleotide in said hybridization product to create an extension product.318. The method of claim 317 , wherein said efficiency of said ligating is at least 30%.319. The method of claim 317 , wherein said efficiency of said ligating is at least 50%.320. The method of claim 317 , wherein said ligating comprises ligating said first single-stranded adaptor to said single-stranded nucleic acid fragment using an ATP-dependent ligase.321. The method of claim 317 , wherein said ligating comprises ligating said first single-stranded adaptor to said single-stranded nucleic acid fragment using an RNA ligase.322. The method of claim 317 , wherein said ligating comprises ligating a 3′ end of said first single-stranded adaptor to a 5′ end of said single stranded nucleic acid fragment.323. The ...

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Номер записи: 68
17-06-2021 дата публикации

CHARGE-TAGGED NUCLEOTIDES AND METHODS OF USE THEREOF

Номер: US20210179657A1
Автор: BACIGALUPO Maria Candelaria Rogert, Gravina Silvia, Mandell Jeffrey, Peisajovich Sergio, PUGLIESE Kaitlin, Teo Yin Nah, YANG Xiangyuan
Принадлежит:

Provided is a method including detecting an incorporation of a labelled nucleotide into a nascent polynucleotide strand complementary to a template polynucleotide strand by a polymerase, wherein the polymerase is tethered to a solid support conductive channel by a tether and the labelled nucleotides is a compound of Formula I: 170-. (canceled)72. The method of claim 71 , wherein the q number of B has a charge and the charge is between about −100e and about +100e.73. The method of claim 72 , wherein the q number of B has a charge density and the charge density is between about −100e per cubic nanometer and about +100e per cubic nanometer.74. The method of claim 71 , wherein the q number of B has a charge and the charge is between about −200e and about +200e.75. The method of claim 74 , wherein the q number of B has a charge density and the charge density is between about −200e per cubic nanometer and about +200e per cubic nanometer.76. The method of claim 71 , wherein the q number of B comprises a polynucleotide.77. The method of claim 76 , wherein the polynucleotide is selected from a branched polynucleotide and one or more hairpin loops.78. The method of claim 77 , wherein the polynucleotide comprises between two and five hairpin loops.79. The method of claim 71 , wherein the q number of B comprises a polypeptide.80. The method of claim 79 , wherein the polypeptide is selected from a branched polypeptide claim 79 , coiled polypeptide claim 79 , and coiled-coil polypeptide.81. The method of claim 71 , wherein B comprises an amino acid claim 71 , and one or more of the q number of B comprise methyllysine claim 71 , dimethyllysine claim 71 , or trimethyllysine.83. The method of claim 71 , further comprising successively incorporating a plurality of labelled nucleotides wherein the charge of each of the plurality of labelled nucleotides differs from the charge of any other of the plurality of labelled nucleotides when the Y of the each and the Y of the any other differ ...

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Номер записи: 69
31-05-2018 дата публикации

Regeneratable Biosensor and Methods of Use Thereof

Номер: US20180149656A1
Автор: Hesham Azizgolshani, Jonathan Coppeta, Madeline Cooper, Stephanie Angione, Thomas Mulhern
Принадлежит: Charles Stark Draper Laboratory Inc

A multiplex-able, regeneratable nucleic-acid linked immunoassay method and system for the detection of a single specific, or multiple, soluble analytes in solution and regeneratable biosensor devices for same are described.

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Номер записи: 70
07-06-2018 дата публикации

SURFACE-BASED TAGMENTATION

Номер: US20180155709A1
Автор: Gormley Niall Anthony, Ioannou Avgousta, Jackson Rosamond, Morrell Natalie
Принадлежит:

Presented herein are methods and compositions surface-based tagmentation. In particular embodiments, methods of preparing an immobilized library of fragmented and tagged DNA molecules on a solid surface are presented. In particular embodiments, the solid surface comprises immobilized transposomes in a dried format, suitable for reconstitution upon contact with liquid, such as a liquid sample. 1. A method of preparing a solid support for DNA amplification comprising:(a) providing a solid support having transposome complexes immobilized thereon;(b) applying a target nucleic acid to the solid support under conditions suitable for tagmentation, thereby immobilizing fragments of the target nucleic acid to the solid support;(c) washing the solid support to remove any unbound nucleic acids; and(d) amplifying the immobilized fragments.2. The method of claim 1 , wherein the solid support comprises a sample tube.3. The method of claim 1 , wherein the solid support comprises a membrane.4. The method of any of - claim 1 , wherein the solid support is coated with streptavidin and the transposome complexes comprise biotin.5. The method of claim 1 , wherein the solid support comprises dried tagmentation reagents configured to be reconstituted to form a tagmentation reaction mixture upon contact with a liquid sample.6. The method of claim 5 , wherein the liquid sample comprises a crude cell lysate.7. The method of claim 5 , wherein the liquid sample comprises purified genomic DNA.8. The method of claim 3 , wherein the membrane comprises filter paper.9. A lateral flow device for tagmentation comprising a solid support comprising:i. a sample deposition region;ii. a buffer region; andiii. a tagmentation region comprising immobilized transposome complexes;wherein the solid support is configured for sample migration via capillary action from the sample deposition region to the tagmentation region.10. The lateral flow device of claim 9 , wherein the buffer region comprises lysis reagents ...

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Номер записи: 71
07-06-2018 дата публикации

Compositions, systems, and methods for sequencing polynucleotides using tethers anchored to polymerases adjacent to nanopores

Номер: US20180155774A1
Автор: Jeffrey G. MANDELL, Kevin L. Gunderson
Принадлежит: Illumina Inc

A composition includes a nanopore including first and second sides and an aperture, nucleotides each including an elongated tag, and a first polynucleotide that is complementary to a second polynucleotide. A polymerase can be disposed adjacent to the first side of the nanopore and configured to add nucleotides to the first polynucleotide based on a sequence of the second polynucleotide. A permanent tether can include a head region anchored to the polymerase, a tail region, and an elongated body disposed therebetween that occurs in the aperture of the nanopore. A first moiety can be disposed on the elongated body that binds to the elongated tag of a first nucleotide upon which the polymerase is acting. A reporter region can be disposed on the elongated body that indicates when the first nucleotide is complementary or is not complementary to a next nucleotide in the sequence of the second polynucleotide.

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Номер записи: 72
14-05-2020 дата публикации

METHODS OF PRODUCING NUCLEIC ACID LIBRARIES

Номер: US20200149098A1
Автор: Green Richard E.
Принадлежит:

Provided are methods of producing nucleic acid libraries. In certain aspects, the methods include combining target nucleic acids (e.g., 5′ phosphorylated nucleic acids) and an oligonucleotide pool. Oligonucleotides of the oligonucleotide pool may include complementarity regions of varying length and nucleotide sequence, and a complementarity region identification sequence. In such aspects, the combining is under conditions in which oligonucleotides of the oligonucleotide pool hybridize to nucleic acids of the target nucleic acids (e.g., 5′ phosphorylated nucleic acids) having overhang regions that are complementary in sequence and have corresponding lengths with respect to the complementarity regions of the oligonucleotides. Compositions and kits that find use, e.g., in practicing the methods of the present disclosure are also provided. 1196.-. (canceled)197. A method for producing a nucleic acid library , comprising: a nucleic acid composition comprising target nucleic acids, wherein some or all of the target nucleic acids comprise an overhang; and', some or all of the oligonucleotides comprise (i) a complementarity region capable of hybridizing to an overhang in a target nucleic acid, and (ii) a complementarity region identification polynucleotide,', 'the oligonucleotides in the pool comprising complementarity regions include complementarity regions of different lengths, and', 'the complementarity region identification polynucleotide is specific to one or more features of the complementarity region, wherein the one or more features comprise length of the complementarity region;, 'an oligonucleotide pool comprising oligonucleotides, wherein], 'combiningunder conditions in which complementarity regions in the oligonucleotides hybridize to overhangs in the target nucleic acids having a corresponding length, thereby forming hybridization products for a nucleic acid library.198. The method of claim 197 , wherein the one or more features of the complementarity region ...

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Номер записи: 73
24-06-2021 дата публикации

Simplified polynucleotide sequence detection method

Номер: US20210189478A1
Автор: Ana SILVA-WEATHERLEY, Barnaby Balmforth, Magdalena STOLAREK-JANUSZKIEWICZ, Paulina POWALOWSKA
Принадлежит: BIOFIDELITY LTD

Methods of detecting target polynucleotide sequences may include introducing one or more nucleic acid analytes to a first reaction mixture comprising a single-stranded probe oligonucleotide A 0 , a pyrophosphorolysing enzyme, and a ligase. The analyte may anneal to the single-stranded probe oligonucleotide A 0 to create a first intermediate product which is at least partially double-stranded, where the 3′ end of A 0 forms a double-stranded complex with the analyte and where A 0 is pyrophosphorylsed in the 3′-5′ direction from the 3′ end to create at least a partially digested strand A 1 . A 1 may undergo ligation to form oligonucleotide A 2 . The methods may also include detecting a signal derived from the formed oligonucleotides, and inferring therefrom the presence or absence of the target polynucleotide sequence in the analyte.

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Номер записи: 74
14-06-2018 дата публикации

Methods and devices for high fidelity polynucleotide synthesis

Номер: US20180163200A1
Автор: George Church, Joseph Jacobson, Larry Li-Yang Chu
Принадлежит: Gen9 Inc

Disclosed are methods for synthesizing and/or assembling at least one polynucleotide product having a predefined sequence from a plurality of different oligonucleotides. In exemplary embodiments, the methods involve synthesis and/or amplification of different oligonucleotides immobilized on a solid support, release of synthesized/amplified oligonucleotides in solution to form droplets, recognition and removal of error-containing oligonucleotides, moving or combining two droplets to allow hybridization and/or ligation between two different oligonucleotides, and further chain extension reaction following hybridization and/or ligation to hierarchically generate desired length of polynucleotide products.

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Номер записи: 75
14-06-2018 дата публикации

METHODS FOR PRODUCING A PAIRED TAG FROM A NUCLEIC ACID SEQUENCE AND METHODS OF USE THEREOF

Номер: US20180163253A1
Автор: Malek Joel, McKernan Kevin, SMITH Douglas
Принадлежит:

Methods for producing a paired tag from a nucleic acid sequence are provided in which the paired tag comprises the 5′ end tag and 3′ end tag of the nucleic acid sequence. In one embodiment, the nucleic acid sequence comprises two restriction endonuclease recognition sites specific for a restriction endonuclease that cleaves the nucleic acid sequence distally to the restriction endonuclease recognition sites. In another embodiment, the nucleic acid sequence further comprises restriction endonuclease recognition sites specific for a rare cutting restriction endonuclease. Methods of using paired tags are also provided. In one embodiment, paired tags are used to characterize a nucleic acid sequence. In a particular embodiment, the nucleic acid sequence is a genome. In one embodiment, the characterization of a nucleic acid sequence is karyotyping. Alternatively, in another embodiment, the characterization of a nucleic acid sequence is mapping of the sequence. In a further embodiment, a method is provided for identifying nucleic acid sequences that encode at least two interacting proteins. 13-. (canceled)4. A method for producing a paired tag from a first nucleic acid sequence fragment , without cloning , comprising the steps of:a) joining the 5′ and 3′ ends of a first nucleic acid sequence fragment to at least one adapter;b) cleaving the adapter(s), thereby producing a second nucleic acid sequence fragment with compatible ends;c) circularizing the second nucleic acid sequence fragment such that a 5′ end of the first nucleic acid sequence fragment is joined to a 3′ end of the first nucleic acid sequence fragment via a linker derived from the adapter(s), thereby producing a circular nucleic acid molecule;d) cleaving the circular nucleic acid molecule, thereby producing a paired tag wherein a 5′ end tag of the first nucleic acid sequence fragment is joined to a 3′ end tag of the first nucleic acid sequence fragment via the linker.5. The method of claim 4 , wherein:a) the ...

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Номер записи: 76
30-05-2019 дата публикации

METHOD, COMPOSITION AND SENSOR FOR ANALYTE DETECTION

Номер: US20190162730A1
Автор: Behrendt Jonathan
Принадлежит:

A method of testing a liquid sample for the presence of an analyte, the method comprising the steps of: forming a mixture by contacting the sample with a composition comprising an oxidase for formation of hydrogen peroxide from the analyte, a fluorescent indicator precursor capable of forming a fluorescent indicator in the presence of an oxygen radical and an iron compound wherein the iron compound is dissolved in the mixture; irradiating the mixture; and measuring fluorescence from the fluorescent indicator. The method may be carried out using a device in which the mixture in a channel or chamber () of a microfluidic device is irradiated by light from light source () and emission from the fluorescent indicator is detected by photodetector (). 1. A method of testing a liquid sample for the presence of an analyte , the method comprising the steps of:forming a mixture by contacting the sample with a composition comprising an oxidase for formation of hydrogen peroxide from the analyte, a fluorescent indicator precursor capable of forming a fluorescent indicator in the presence of an oxygen radical and an iron compound wherein the iron compound is dissolved in the mixture;irradiating the mixture; andmeasuring fluorescence from the fluorescent indicator.2. A method according to wherein hydrogen peroxide is formed from the analyte by an oxidase-catalysed reaction of the analyte.3. A method according to wherein the analyte is glucose and the oxidase is glucose oxidase.4. A method according to wherein the analyte is cholesterol and the oxidase is cholesterol oxidase.5. A method according to wherein the analyte undergoes one or more preliminary reactions to form a compound capable of oxidase-catalysed production of hydrogen peroxide.6. A method according to wherein the fluorescent indicator precursor is a fluorescein or a salt thereof.7. A method according to wherein the mixture does not comprise a quencher capable of quenching emission from the fluorescent indicator.8. A ...

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Номер записи: 77
21-05-2020 дата публикации

Methods and compositions for whole transcriptome amplification

Номер: US20200157600A1
Автор: Christina Fan, Craig Betts, Glenn Fu, Gretchen Yinbon Lam
Принадлежит: Cellular Research Inc

The disclosure provides for methods, compositions, systems, devices, and kits for whole transcriptome amplification using stochastic barcodes.

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Номер записи: 78
11-09-2014 дата публикации

TREATMENT OF COAGULOPATHY WITH HYPERFIBRINOLYSIS

Номер: US20140256640A1
Автор: Foley Jonathan Herbert, Nesheim Michael Ernest, Petersen Karl-Uwe
Принадлежит: Paion Deutschland GmbH

The present invention relates to the use of thrombomodulin analogues for the manufacture of a medicament for the treatment of coagulopathy with hyperfibrinolysis, such as haemophilia disorders. These thrombomodulin analogs exhibit at therapeutically effective dosages an antifibrinolytic effect. Novel protein modifications together with methods for their identification are disclosed.

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Номер записи: 79
21-06-2018 дата публикации

Helicase Suppression of Non-Template Amplification

Номер: US20180171399A1
Автор: Evans, JR. Thomas C., Tanner Nathan
Принадлежит: NEW ENGLAND BIOLABS, INC.

Provided herein is a method for reducing amplification of non-template molecules in a nucleic acid sample. In certain embodiments, the method involves adding a helicase to a reaction mixture for non-helicase-dependent amplification of target nucleic acid. 1. A reaction mixture comprising:a) a nucleic acid sample comprising a template;b) nucleotides;c) four or more primers;d) a polymerase; ande) a helicase;wherein the reaction mixture does not contain a single-stranded DNA binding protein (SSBP) and wherein the reaction mixture is capable of amplifying the template when placed under isothermal or polymerase chain reaction conditions.2. The reaction mixture of claim 1 , wherein the helicase is a thermostable helicase.3. The reaction mixture of claim 2 , wherein the helicase is a PcrA/UvrD/Rep helicase.4Thermoanaerobacter tengcongensisThermus thermophilusAquifex aeolicus. The reaction mixture of claim 3 , wherein the helicase is selected from the group consisting of a (Tte) helicase claim 3 , (Tth) helicase and (Aq793) helicase.5. The reaction mixture of claim 1 , wherein the polymerase is a strand-displacing polymerase.6. The reaction mixture of claim 5 , wherein the polymerase is selected from the group consisting of a Bst polymerase claim 5 , a polD polymerase claim 5 , a 9° N polymerase and phi29 polymerase.7. The reaction mixture of claim 1 , wherein the polymerase is a thermostable polymerase.8. The reaction mixture of claim 1 , wherein the template is RNA and the polymerase is a reverse transcriptase.9. The reaction mixture of claim 1 , wherein the template is genomic DNA.10. A method for reducing amplification of non-template molecules from a nucleic acid sample claim 1 , comprising:a) incubating a reaction mixture comprising a nucleic acid sample comprising a template, nucleotides, at least four primers, a polymerase, and a helicase under amplification conditions, andb) amplifying the template; wherein the amplification reaction is not helicase dependent but ...

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Номер записи: 80
22-06-2017 дата публикации

DNA Glycosylase/Lyase and AP Endonuclease Substrates

Номер: US20170175173A1
Автор: Armes Niall A., Piepenburg Olaf
Принадлежит:

A new class of nucleic acid substrates for AP endonucleases and members of the glycosylase/lyase family of enzymes is described. Representatives of each family, the enzymes Nfo and fpg, respectively, cleave nucleic acid backbones at positions in which a base has been replaced by a linker to which a variety of label moieties may be attached. The use of these synthetic substrates embedded within oligonucleotides is of utility in a number of applications. 135.-. (canceled)36. An oligonucleotide probe comprising an oligonucleotide between 30 and 60 nucleotides in length , wherein the oligonucleotide contains a dR-O—[C]n nucleotide that lacks a base and has a sugar with a carbon at a 1′ position , and wherein the carbon at the 1′ position is covalently linked through an oxygen atom to a carbon atom of a linker containing n carbon atoms , wherein said oligonucleotide contains a fluorophore-quencher pair separated by 10 nucleotides or less , and wherein the dR-O—[C]n nucleotide is conjugated with either the fluorophore or the quencher.37. The probe of claim 36 , wherein n is 3-6.38. The probe of claim 37 , wherein n is 6.39. The probe of claim 36 , wherein the probe is blocked at its 3′-end to prevent polymerase extension.40. The probe of claim 36 , wherein the fluorophore and quencher are separated by 4-6 bases.41. The probe of claim 36 , wherein the fluorophore or quencher not conjugated to the dR-O—[C]n nucleotide is conjugated at one end of the probe.42. The probe of claim 41 , wherein the conjugated end of the probe is the 5′-end.43. A kit claim 41 , comprising(i) an oligonucleotide containing a dR-O—[C]n nucleotide that lacks a base and has a sugar with a carbon at a 1′ position, and wherein the carbon at the 1′ position is covalently linked through an oxygen atom to a carbon atom of a linker containing n carbon atoms; and(ii) a nuclease selected from the group consisting of AP endonuclease, DNA glycosylase/lyase and DNA glycosylase.44. The kit according to claim 43 ...

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Номер записи: 81
05-07-2018 дата публикации

Kinetic exclusion amplification of nucleic acid libraries

Номер: US20180187252A1
Автор: Claire Bevis-Mott, Jonathan Boutell, Peter Mcinerney, Shaun Hunter
Принадлежит: Illumina Cambridge Ltd, Illumina Inc

An example method includes reacting a first solution and a different, second solution on a flow cell by flowing the first solution over amplification sites on the flow cell and subsequently flowing the second solution over the amplification sites. The first solution includes target nucleic acids and a first reagent mixture that comprises nucleoside triphosphates and replication enzymes. The target nucleic acids in the first solution transport to and bind to the amplification sites at a transport rate. The first reagent mixture amplifies the target nucleic acids that are bound to the amplification sites to produce clonal populations of amplicons originating from corresponding target nucleic acids. The amplicons are produced at an amplification rate that exceeds the transport rate. The second solution includes a second reagent mixture and lacks the target nucleic acids. The second solution is to increase a number of the amplicons at the amplification sites.

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Номер записи: 82
18-06-2020 дата публикации

IN VITRO RECOMBINATION METHOD

Номер: US20200190539A1
Автор: Gibson Daniel Glenn, Smith Hamilton O., Young Lei
Принадлежит:

The present invention relates, e.g., to in vitro method, using isolated protein reagents, for joining two double stranded (ds) DNA molecules of interest, wherein the distal region of the first DNA molecule and the proximal region of the second DNA molecule share a region of sequence identity, comprising contacting the two DNA molecules in a reaction mixture with (a) a non-processive ′ exonuclease; (b) a single stranded DNA binding protein (SSB) which accelerates nucleic acid annealing; (c) a non strand-displacing DNA polymerase; and (d) a ligase, under conditions effective to join the two DNA molecules to form an intact double stranded DNA molecule, in which a single copy of the region of sequence identity is retained. The method allows the joining of a number of DNA fragments, in a predetermined order and orientation, without the use of restriction enzymes. 1. An in vitro method , using isolated proteins , for joining two double strand (ds) DNA molecules of interest , comprising:providing a first dsDNA molecule and a second dsDNA molecule which share a region of sequence identity at a terminal end on each DNA molecule; and (a) a purified 5′ exonuclease;', '(b) a purified DNA polymerase; and', '(c) a purified ligase, under conditions whereby:, 'contacting the two dsDNA molecules witha 3′ single-stranded overhang is generated in each molecule by the exonuclease without the use of a restriction enzyme;the two single-stranded overhangs anneal to form a gapped molecule;the gaps are filled in by the polymerase; andnicks are sealed by the ligase, thereby joining the molecules and forming a substantially intact double stranded DNA molecule, in which a single copy of the region of sequence identity is retained, wherein none of the enzymatic reactions is actively terminated prior to beginning another of the reactions.2. The method of claim 1 , wherein the 3′ single stranded overhangs comprise the region of sequence identity.3. The method of claim 1 , wherein the proteins of ...

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Номер записи: 83
29-07-2021 дата публикации

METHODS OF EPIGENETIC ANALYSIS

Номер: US20210230679A1
Автор: Agarwal Suneet, Iyer Aravind, Koh Kian Peng, Rao Anjana, Tahiliani Mamta
Принадлежит:

The present invention provides for methods of epigenetic analysis. In some cases, the methods may include obtaining a sample comprising a nucleic acid sequence. In some cases, the nucleic acid sequence may comprise one or more epigenetic marks. The methods may include performing a sequencing. The methods may include distinguishing a hydroxymethylated base from a methylated base. 110.-. (canceled)11. A method , comprising:contacting a polynucleotide with an enzyme or a catalytically active fragment thereof that performs an oxidation on an epigenetic modification to form a contacted polynucleotide; andsequencing the contacted polynucleotide.12. The method of claim 11 , wherein the enzyme or the catalytically active fragment performs a conversion selected from the group consisting of:oxidizing 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC);oxidizing 5mC to an acid beyond hydroxymethylation;oxidizing 5mC to an aldehyde beyond hydroxymethylation;oxidizing 5hmC to an acid beyond hydroxymethylation;oxidizing 5hmC to an aldehyde beyond hydroxymethylation; and any combination thereof.13. The method of claim 11 , further comprising isolating the polynucleotide from a sample.14. The method of claim 11 , wherein the polynucleotide is isolated from an extracellular fluid.15. The method of claim 11 , wherein the enzyme or the catalytically active fragment thereof is isolated prior to contacting.16. The method of claim 11 , wherein the polynucleotide is contacted with the enzyme.17. The method of claim 11 , wherein the polynucleotide is contacted with the catalytically active fragment of the enzyme.18. The method of claim 11 , wherein the enzyme or the catalytically active fragment thereof comprises a catalytically active TET family enzyme or a functional fragment thereof.19. The method of claim 11 , wherein the sequencing comprises determining a methylation state of the contacted polynucleotide.20. The method of claim 11 , wherein the sequencing comprises an imaging.21. ...

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Номер записи: 84
19-07-2018 дата публикации

NANOPORE SEQUENCING METHODS

Номер: US20180201993A1
Автор: Korlach Jonas, Turner Stephen
Принадлежит:

Methods are provided for sequencing of nucleic acid templates using nanopores. A polymerase-nucleic acid complex having a strand displacing polymerase is provided. The nucleic acid in the polymerase-nucleic acid complex is a circular nucleic acid template. Components required for nucleic acid synthesis are provided whereby the polymerase produces a nascent strand that passes through the nanopore while the nascent strand is produced. Current through the nanopore over time is measured allowing for the determination of the sequence of the nascent strand as it translates through the pore using the measured current over time. Because the polymerase can proceed around the circular template multiple times, multiple reads corresponding to the same sequence in the circular template can be obtained in one sequencing process. Where modified bases are present in the template, the method can be used to determine the presence and position of the modified bases using the kinetics of the polymerase. 1. A method for sequencing a nucleic acid using a nanopore comprising:providing a substrate having an upper solution above the substrate and a lower solution below the substrate, the substrate comprising a nanopore connecting the upper solution and lower solution, the nanopore sized to pass a single stranded nucleic acid;providing a voltage across the nanopore to produce a measurable current flow through the nanopore;providing a polymerase-nucleic acid complex comprising a strand displacing polymerase and a circular nucleic acid template and providing the components required for nucleic acid synthesis whereby the polymerase produces a nascent strand that passes through the nanopore while the nascent strand is produced;measuring the current through the nanopore over time as the nascent strand is translated through the nanopore;determining a sequence of the nascent strand as it translates through the pore using the measured current over time, whereby the polymerase proceeds around the ...

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Номер записи: 85
26-07-2018 дата публикации

COMPOSITIONS AND METHODS OF RNA ANALYSIS

Номер: US20180208967A1
Автор: Credle Joel, Larman Harry Benjamin
Принадлежит:

The present disclosure relates to compositions and methods of RNA analysis. In particular, the present disclosure provides a method of RNA analysis that includes obtaining a sample, applying one or more multi-partite probes to the sample, where each of the one or more multi-partite probes includes at least two sub-probes, annealing at least one of the applied one or more multi-partite probes to at least one target nucleic acid within the sample, and ligating the at least two sub-probes associated with the at least one annealed multi-partite probe to create a target nucleic acid proxy that can be detected. 1. A method , comprising:obtaining a sample;applying one or more multi-partite probes to the sample, wherein each of the one or more multi-partite probes includes at least two sub-probes;annealing at least one of the applied one or more multi-partite probes to at least one target nucleic acid within the sample; andligating the at least two sub-probes associated with the at least one annealed multi-partite probe to create a target nucleic acid proxy that can be detected.2. The method of claim 1 , further comprising:releasing the target nucleic acid proxy from the target nucleic acid; andamplifying the target nucleic acid proxy.3. The method of claim 1 , wherein the at least one target nucleic acid is RNA.4. The method of claim 1 , wherein the sub-probes comprise appended primer binding sites to facilitate subsequent amplification of the target nucleic acid proxy.5. The method of claim 1 , wherein the at least two sub-probes are ligated with an enzyme claim 1 , a chemical reaction claim 1 , or a photoreaction.6. The method of claim 5 , wherein the enzyme is a ligase.7. The method of claim 6 , wherein the ligase is selected from the group consisting of a T4 RNA Ligase 2 (Rnl2) claim 6 , T4 DNA ligase claim 6 , a Chlorella virus DNA Ligase (PBCV-1 DNA Ligase) claim 6 , a Rnl2 derivative claim 6 , PBCV-1 derivative claim 6 , and any combination thereof.8. The method of ...

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Номер записи: 86
27-07-2017 дата публикации

METHOD FOR RELATIVE QUANTIFICATION OF NUCLEIC ACID SEQUENCE, EXPRESSION, OR COPY CHANGES, USING COMBINED NUCLEASE, LIGATION, AND POLYMERASE REACTIONS

Номер: US20170212103A1
Автор: BARANY Francis, Mir Alain, SPIER Eugene
Принадлежит:

The present invention is directed to methods for identifying the presence of one or more target nucleotide sequences in a sample that involve a nuclease-ligation reaction. In some embodiments, the ligation products formed in the nuclease-ligation process of the present invention are subsequently amplified using a polymerase chain reaction. The ligated product sequences or extension products thereof are detected, and the presence of one or more target nucleotide sequences in the sample is identified based on the detection 1. A method for identifying a presence of one or more target nucleotide sequences in a sample , said method comprising:providing a sample potentially containing the target nucleotide sequence;providing one or more oligonucleotide probe sets, each set comprising (a) a first oligonucleotide probe having a target-specific portion, and (b) a second oligonucleotide probe having 5′ non-target specific flap portion and a target-specific portion containing one or more thiophosphate-modified nucleotide bases, wherein the first and second oligonucleotide probes of a probe set are configured to hybridize on the target nucleotide sequence;contacting the sample and the one or more oligonucleotide probe sets under conditions effective for first and second oligonucleotide probes of a probe set to hybridize in a base specific manner to their corresponding target nucleotide sequences, if present in the sample;cleaving the 5′ non-target specific flap portion of the second oligonucleotide probe with an enzyme having 5′ nuclease activity, thereby liberating a 5′ phosphate at a first nucleotide base of the target-specific portion of the second oligonucleotide;ligating first and second oligonucleotide probes of the one or more oligonucleotide probe sets together to form ligated product sequences containing the target-specific portions with the one or more thiophosphate-modified nucleotide bases;detecting ligated product sequences in the sample; andidentifying the ...

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Номер записи: 87
04-07-2019 дата публикации

SINGLE NUCLEOTIDE DETECTION METHOD AND ASSOCIATED PROBES

Номер: US20190203273A1
Автор: BALMFORTH Barnaby, Frayling Cameron Alexander
Принадлежит: Base4 Innovation Limited

A method of sequencing a nucleic acid comprising the steps of (1) generating a stream of single nucleoside triphosphates; (2) producing at least one substantially double-stranded primary oligonucleotide used probe comprising (a) a first single-stranded oligonucleotide including a first restriction endonuclease nicking-site, a single nucleotide capture site, and oligonucleotide flanking regions juxtaposed either side of the capture site and (b) second and third single-stranded oligonucleotides; (3) nicking the first oligonucleotide strand of the used primary probe; (4) separating the first oligonucleotide components; (5) producing at least one substantially double-stranded secondary used probe by reacting with a corresponding secondary probe comprising (c) a complementary fourth oligonucleotide and optionally (d) a single-stranded fifth oligonucleotide; (6) nicking the fourth oligonucleotide strand of the used secondary probe to create separate fourth oligonucleotide components having fluorophores and a single-stranded sixth oligonucleotide; and (7) detecting the fluorophores released in step (6). 1. A method of sequencing a nucleic acid comprising the steps of:(1) generating a stream of single nucleoside triphosphates by progressive enzymatic digestion of the nucleic acid; (a) a first single-stranded oligonucleotide including a first restriction endonuclease nicking-site, a single nucleotide capture site for capturing the single nucleoside triphosphate, and oligonucleotide flanking regions juxtaposed either side of the capture site and', '(b) second and third single-stranded oligonucleotides capable of hybridizing to the first oligonucleotide flanking regions;, '2) producing at least one substantially double-stranded primary oligonucleotide used probe by reacting, in the presence of a polymerase and a ligase, at least one of the single nucleoside triphosphates with a corresponding primary probe comprising(3) nicking the first oligonucleotide strand of the used ...

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Номер записи: 88
05-08-2021 дата публикации

METHODS FOR MULTIPLEX PCR

Номер: US20210238657A1
Автор: LALIBERTE JULIE, Makarov Vladimir
Принадлежит:

Methods for performing multiplex PCR-based enrichment of a target substrate are provided. Systems and methods for generating a sequencing library are also provided. 1. A method of multiplex PCR amplification of a specific target locus on a nucleic acid substrate for preparing a targeted next generation sequencing library comprising the steps of:(i) combining a plurality of target-specific primers with the nucleic acid substrate to yield a single polymerase chain reaction (PCR) reaction mixture, wherein the plurality of target-specific primers comprise a first forward primer, a second forward primer, a first reverse primer and a second reverse primer, wherein each of the first and second forward and reverse primers comprise a 3′ complementary sequence that is fully complementary to a sequence of the specific target locus and a 5′ noncomplementary sequence that is not complementary to a sequence of the nucleic acid substrate, wherein the 3′ complementary sequence for each of the first and second forward and reverse primers is different, wherein the 3′ complementary sequence is between 10 and 40 bases in length, wherein the nucleic acid substrate is human genomic DNA, and wherein the specific target locus is a gene known to have clinical relevance in oncology(ii) subjecting the PCR reaction mixture to a multiplex polymerase chain reaction thereby generating at least three amplicons within the specific target locus, wherein the at least three amplicons comprise a first amplicon produced by the first forward primer and the first reverse primer, a second amplicon produced by the second forward primer and the second reverse primer, and a third amplicon produced by the second forward primer and the first reverse primer, wherein the third amplicon is shorter in length than the first and second amplicons, wherein at least a portion of the 5′ noncomplementary sequence of the second forward primer and the first reverse primer is the same such that each strand of the third ...

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Номер записи: 89
05-08-2021 дата публикации

Increasing capture efficiency of spatial assays

Номер: US20210238680A1
Автор: Felice Alessio BAVA
Принадлежит: 10X Genomics Inc

This disclosure relates to methods for increasing capture efficiency of a spatial array using rolling circle amplification of a padlock probe that hybridizes to a capture probe. Also provided are methods for using such spatial arrays to detect a biological analyte in a biological sample.

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Номер записи: 90
02-08-2018 дата публикации

ENZYME METHOD

Номер: US20180216175A9
Автор: Heron Andrew John, Moysey Ruth
Принадлежит: Oxford Nanopore Technologies Ltd.

The invention relates to a new method of characterizing 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. 143-. (canceled)44. A method of characterising a target polynucleotide , comprising:(a) contacting the target polynucleotide with a transmembrane pore and a helicase which is capable of binding to the target polynucleotide at an internal nucleotide such that the helicase controls the movement of the target polynucleotide through the pore and nucleotides in the target polynucleotide interact with the pore; and(b) measuring one or more characteristics of the target polynucleotide during one or more interactions and thereby characterising the target polynucleotide.45. A method according to claim 44 , wherein the helicase is a Hel308 helicase claim 44 , Hel308 Tga claim 44 , Hel308 Mhu or Hel308 Csy.46. A method according to claim 44 , wherein the one or more characteristics are selected from (i) the length of the target polynucleotide claim 44 , (ii) the identity of the target polynucleotide claim 44 , (iii) the sequence of the target polynucleotide claim 44 , (iv) the secondary structure of the target polynucleotide claim 44 , and (v) whether or not the target polynucleotide is modified by methylation claim 44 , by oxidation claim 44 , by damage claim 44 , with one or more proteins or with one or more labels claim 44 , tags or spacers.47. A method according to claim 44 , wherein the one or more characteristics of the target polynucleotide are measured by electrical measurement and/or optical measurement.48. A method according to claim 47 , wherein the electrical measurement is a current measurement claim 47 , an impedance measurement claim 47 , a tunnelling measurement claim 47 , or a field effect transistor (FET) measurement.49. A method ...

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Номер записи: 91
02-07-2020 дата публикации

Methods for nucleic acid assembly and high throughput sequencing

Номер: US20200208139A1
Автор: Daniel Schindler, Ishtiaq SAAEM, Li-Yun A. Kung, Michael E. Hudson, Stephen Archer
Принадлежит: Gen9 Inc

Methods and apparatus of some aspects of the invention relate to the synthesis of high fidelity polynucleotides. In particular, aspects of the invention relate to concurrent enzymatic removal of amplification sequences and ligation of processed oligonucleotides into nucleic acid assemblies. According to some embodiments, the invention provides a method for producing a target nucleic acid having a predefined sequence. In some embodiments, the method comprises the step of providing a plurality of oligonucleotides, wherein each oligonucleotides comprises (i) an internal sequence identical to a different portion of a sequence of a target nucleic acid, (ii) a 5′ sequence flanking the 5′ end of the internal sequence and a 3′ flanking sequence flanking the 3′ end of the internal sequence, each of the flanking sequence comprising a primer recognition site for a primer pair and a restriction enzyme recognition site.

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Номер записи: 92
12-08-2021 дата публикации

Methods of Epigenetic Analysis

Номер: US20210246494A1
Автор: Agarwal Suneet, Iyer Aravind, Koh Kian Peng, Rao Anjana, Tahiliani Mamta
Принадлежит:

The present invention provides for methods of epigenetic analysis. In some cases, the methods may include obtaining a sample comprising a nucleic acid sequence. In some cases, the nucleic acid sequence may comprise one or more epigenetic marks. The methods may include performing a sequencing. The methods may include distinguishing a hydroxymethylated base from a methylated base. 110.-. (canceled)11. A kit , comprising:a methylcytosine dioxygenase;a DNA glucosyltransferase; andpackaging materials and instructions therein to use said kit.12. The kit of claim 11 , wherein said methylcytosine dioxygenase is a ten-eleven translocation (TET) enzyme or fragment thereof.13. The kit of claim 12 , wherein said TET enzyme or fragment thereof comprises TET1 claim 12 , TET2 claim 12 , TET3 claim 12 , CXXC4 claim 12 , or a combination thereof.14. The kit of claim 11 , wherein said DNA glucosyltransferase is a DNA glucosyltransferase encoded by a bacteriophage of the T even family.15. The kit of claim 11 , wherein said DNA glucosyltransferase is a beta-glucosyltransferase.16. The kit of claim 11 , wherein said DNA glucosyltransferase is an alpha-glucosyltransferase171. The kit of claim claim 11 , further comprising a glucose derivative substrate.18. The kit of claim 11 , further comprising a DNA methyltransferase.19. The kit of claim 18 , wherein said DNA methyltransferase is DNMT1.20. The kit of claim 11 , further comprising purification columns claim 11 , buffers claim 11 , or a substrate solution.21. The kit of claim 11 , further comprising nucleic acid sequencing reagents.22. A kit claim 11 , comprising:a methylcytosine dioxygenase;a glucose derivative substrate; andpackaging materials and instructions therein to use said kit.23. The kit of claim 22 , wherein said methylcytosine dioxygenase is a ten eleven translocation (TET) enzyme or fragment thereof.24. The kit of claim 23 , wherein said TET enzyme or fragment thereof comprises TET1 claim 23 , TET2 claim 23 , TET3 claim 23 ...

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Номер записи: 93
12-08-2021 дата публикации

HUMAN IDENTIFICATION USING A PANEL OF SNPS

Номер: US20210246498A9
Автор: Lagace Robert
Принадлежит:

The present invention provides methods, compositions, kits, systems and apparatus that are useful for multiplex PCR of one or more nucleic acids which belong to a panel of single nucleotide polymorphisms (SNPs) useful to identify a human. In particular, various target-specific primers are provided that allow for the selective amplification of one or more target sequences in the panel. In some aspects, amplified target sequences obtained using the disclosed methods, kits, systems and apparatuses can be used in various downstream processes including nucleic acid sequencing and used to identify a human. 1. A method comprising:a) amplifying a plurality of different target sequences within a sample comprising:b) amplifying within a single amplification reaction mixture a plurality of different target sequences wherein the target sequences are a panel of at least fifty single nucleotide polymorphism (SNP) regions selected from Table A, wherein the amplifying includes contacting at least some portion of the sample with a plurality of target-specific primers and a polymerase under amplification conditions, thereby producing an amplified plurality of target sequences wherein at least two of the different amplified target sequences are less than 50% complementary to each other and wherein at least one of the plurality of amplified target sequences includes a cleavable group, wherein the cleavable group is uracil;c) cleaving a cleavable group of at least one amplified target sequence of the amplified plurality of target sequences;d) ligating at least one adapter to at least one amplified target sequence in a blunt-ended ligation reaction, thereby producing one or more adapter-ligated amplified target sequences, ande) reamplifying at least one of the adapter-ligated amplified target sequences, wherein at least one target-specific primer is substantially complementary to at least a portion of a corresponding target sequence in the sample.2. The method of claim 1 , wherein one or ...

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Номер записи: 94
09-08-2018 дата публикации

Multiplex Amplification Methods

Номер: US20180223333A1
Автор: Eugeni A. Namsaraev
Принадлежит: Affymetrix Inc

Compositions and methods for amplifying selected polynucleotides, including DNA and RNA, particularly in multiplex amplification reactions using common primers amplification. Generally, methods of the invention employ multiple steps such as template-specific hybridization, a linear amplification, partial degradation of nucleic acid, and ligation. At the end of the process the sequences of selected polynucleotides are flanked by the common sequences which can be used for exponential amplification using common primers. In some aspects the polynucleotides are associated with a barcode and the presence of the barcode is detected to measure the amount of the polynucleotide.

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Номер записи: 95
09-08-2018 дата публикации

LIGASE-ASSISTED NUCLEIC ACID CIRCULARIZATION AND AMPLIFICATION

Номер: US20180223349A1
Автор: Heller Ryan Charles, Kvam Erik Leeming, Nelson John Richard
Принадлежит:

Provided herein are methods for generation and amplification of a single-stranded DNA circle in a single reaction vessel from a linear DNA without any intervening purification steps. The single-stranded DNA circle is generated via a template-independent single-stranded DNA ligation. Whole-genome amplification of linear chromosomal DNA in a single tube using ligation-assisted DNA amplification is also provided. 1. A method for nucleic acid amplification , the method comprising:(a) providing a linear chromosomal DNA;(b) incubating the linear chromosomal DNA with a ligase that is capable of template-independent, intra-molecular ligation of a single-stranded DNA sequence to generate a single-stranded DNA circle; and(c) amplifying the single-stranded DNA circle via rolling circle amplification using a random primer mixture to form an amplified DNA product,wherein the random primer mixture comprises oligonucleotide sequences comprising at least one nucleotide analogue, andwherein all the steps of the method are performed in a single reaction vessel without any intervening isolation or purification steps.2. The method of claim 1 , wherein the at least one nucleotide analogue comprises a 2-amino-deoxyadenosine.3. The method of claim 1 , wherein the at least one nucleotide analogue comprises a 2-thio-deoxythymidine.4. The method of claim 1 , wherein the random primer mixture comprises selective binding complementary oligonucleotides.5. The method of claim 4 , wherein each member of the selective binding complementary oligonucleotides comprises at least one nucleotide comprising a 2-amino-deoxyadenosine or at least one nucleotide comprising a 2-thio-deoxythymidine.6. The method of claim 1 , wherein the random primer mixture comprises oligonucleotide sequences comprising a phosphorothioate modified nucleotide claim 1 , a LNA nucleotide claim 1 , a nucleotide comprising a 2-amino-deoxyadenosine claim 1 , a nucleotide comprising a 2-thio-deoxythymidine claim 1 , or combinations ...

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Номер записи: 96
09-08-2018 дата публикации

CYCLIC SINGLE MOLECULE SEQUENCING PROCESS

Номер: US20180223355A1
Автор: Rigler Rudolf
Принадлежит:

The invention relates to a process for parallel high throughput sequencing of nucleic acid molecules, in particular in the single molecule format. 1. A process for sequencing an individual nucleic acid , comprising the following steps:(a) providing a nucleic acid-synthesizing enzyme molecule, a circular nucleic acid template molecule, a primer annealed to said template, or capable of annealing to said template and fluorescence-labelled nucleotide building blocks,(b) generating a nucleic acid molecule complementary to the sequence of the circular nucleic acid template having incorporated said nucleic building blocks in a primer elongation catalyzed by a nucleic acid-synthesizing enzyme molecule,(c) contacting said generated nucleic acid molecule with a nucleic acid-degrading enzyme molecule and cleaving off individual nucleotide building blocks from said generated nucleic acid molecule in a nuclease digestion catalyzed by said nucleic acid-de- grading enzyme molecule, and(d) determining the base sequence of said circular nucleic acid template molecule on the basis of the time-dependent fluorescence change, caused when nucleotide building blocks are incorporated during primer elongation and/or cleaved off during nuclease digestion,wherein the nucleic acid-synthesizing enzyme molecule is immobilized on a support by high-affinity interactions between partners of a specific binding pair, or adsorption, or covalent immobilization.2. The process of claim 1 , wherein the nucleic acid-degrading enzyme molecule is present in free form.3. The process of claim 1 , wherein the individual base sequence of a plurality of individual circular nucleic acid template molecules is determined.4. The process of claim 1 , wherein the base sequence of an individual circular nucleic acid template molecule is determined at least in 2 cycles claim 1 , each comprising elongation and digestion.5. The process of claim 1 , wherein the process comprises the following steps:(a) providing at least ...

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Номер записи: 97
09-07-2020 дата публикации

COMPOSITIONS AND METHODS FOR DETECTION OF GENOMIC VARIATIONS

Номер: US20200216881A1
Автор: BEN ZVI ROZEVSKY Yana, GILBOA Tal, Meller Amit, PATKIN Michelle
Принадлежит:

The present invention concerns compositions, kits and methods for genetic variation detection and classification. These methods combine the specificity of a ligation reaction with the single-molecule sensitivity of nanopore biosensors. This invention can achieve clinical detection of many diseases, including bacterial infections and cancer entities, by identification of specific genetic alterations. 1. A method for detection of a nucleic acid variant in a sample , comprising:a. providing a sample containing nucleic acids;b. contacting said sample with at least one nanopore-sensible barcode linked to nucleic acids complementary to a first sequence, said first sequence comprising a first region of a nucleic acid template adjacent to said variant; and at least one selection marker linked to nucleic acids complementary to a second sequence, said second sequence comprising a second region contiguous to said first sequence in said nucleic acid template, wherein said first or second sequence further comprises said variant at an end of said first or second sequence;c. contacting said sample with a ligase;d. isolating a composition containing said selection marker; ande. passing said composition through at least one nanopore configured to sense said barcode;thereby detecting a nucleic acid variant in a sample.2. The method of claim 1 , wherein said nucleic acid variant is selected from the group consisting of: a single nucleotide variation claim 1 , an insertion claim 1 , a deletion and a combination thereof.3. (canceled)4. The method of claim 1 , wherein said nucleic acid variant is at an end of said first sequence not linked to said barcode or an end of said second sequence not linked to said selection marker and said first and second sequences are contiguous at said variant.5. The method of claim 1 , wherein said barcode is linked to a 5′ end of said nucleic acids and said selection marker is linked to a 3′ end of said nucleic acids or said barcode is linked to a 3′ end ...

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Номер записи: 98
09-07-2020 дата публикации

NANOPORE SEQUENCING COMPLEXES

Номер: US20200216887A1
Автор: CRAIG Timothy Kellogg, Dipetro Matt, HARRIS Corissa, Jensen Liv E., Porter Marshall, TZITZILONIS Christos, Yang Alexander H., Yang Charlotte
Принадлежит: GENIA TECHNOLOGIES, INC.

A method is provided for preparing nanopore sequencing complexes in membranes for sequencing of polymers, e.g., polynucleotides and polypeptides. The nanopore sequencing complex is formed by the sequential linking of an enzyme to a nanopore that is inserted in a membrane, and of a polymer to the enzyme. Alternatively, the nanopore sequencing complex is formed by linking a preformed enzyme-polymer complex to a nanopore that is inserted in a membrane. The enzyme polymer complex is interchangeable. 115-. (canceled)16. A method for preparing a nanopore sequencing complex , the method comprising:(a) inserting a nanopore into a membrane;(b) contacting the nanopore with a sequencing enzyme;(c) attaching the enzyme to the nanopore to form an enzyme-nanopore complex; and(d) binding a polymer to the enzyme-nanopore complex to provide a nanopore sequencing complex.17. The method of claim 16 , wherein the sequencing enzyme of is a polymerase claim 16 , an exonuclease claim 16 , a helicase claim 16 , or an unfoldase.18. The method of claim 17 , wherein the polymerase is a DNA polymerase claim 17 , a reverse transcriptase claim 17 , or an RNA polymerase.19. The method of claim 17 , wherein the polymerase is a wild-type or variant thereof.20. The method of claim 19 , wherein the variant polymerase has increased enzyme activity claim 19 , fidelity claim 19 , processivity claim 19 , elongation rate claim 19 , stability claim 19 , or solubility.21. The method of claim 16 , wherein the nanopore is selected from a monomeric nanopore claim 16 , a homo-oligomeric nanopore claim 16 , and a hetero-oligomeric nanopore.22. The method of claim 16 , wherein the nanopore comprises an a-hemolysin nanopore (αHL) claim 16 , and outer membrane protein G nanopore (OmpG) claim 16 , or a variant or modified variant thereof.23. A method for sequencing a nucleic acid sample claim 16 , comprising:{'b': '1', '(a) preparing a nanopore sequencing complex in a lipid bilayer according to claim ;'}(b) ...

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Номер записи: 99
16-08-2018 дата публикации

OLIGONUCLEOTIDES AND METHODS FOR THE PREPARATION OF RNA LIBRARIES

Номер: US20180230516A1
Автор: BRAMLETT Kelli, BURNETT Christopher, GU Jian
Принадлежит:

Disclosed are compositions and methods for the preparation of RNA libraries for sequencing, gene expression profiling, microarray and other uses and for simplification of the library preparation process. The disclosure provides blocking oligonucleotides which bind to byproduct nucleic acid molecules formed during the ligation of adapters to nucleic acid segments prior to sequencing and inhibit or block amplification of the byproduct nucleic acid molecules in subsequent amplification reactions. Methods for library preparation using blocking oligonucleotides are also provided. 1. A composition comprising: a blocking oligonucleotide hybridized to an adaptor-dimer , wherein (i) the adaptor-dimer comprises a single-stranded nucleic acid molecule having one strand of a first adaptor joined to one strand of a second adaptor , and wherein (ii) the blocking oligonucleotide is a fusion molecule comprising a first segment that hybridizes with the first portion of the adaptor-dimer and a second segment that hybridizes with the second portion of the adaptor-dimer , wherein the first segment of the blocking oligonucleotide comprises 2-14 consecutive modified nucleotides , and wherein the blocking oligonucleotide comprises at least one inosine base which is located within the internal region of the blocking oligonucleotide.2. The composition of claim 1 , wherein the modified nucleotides comprise 2′-O-methyl modified nucleotides claim 1 , 2′-deoxy-2′-fluoro modified nucleotides claim 1 , 2′-deoxy-modified nucleotides claim 1 , 2′-alkyl-modified nucleotides claim 1 , nucleotides comprising a 5′-phosphorothioate group claim 1 , 2′-amino-modified nucleotides claim 1 , morpholino nucleotides claim 1 , or phosphoramidates.3. The composition of claim 1 , wherein the blocking oligonucleotide comprises at least 13 consecutive 2′-O-methyl modified nucleotides.4. The composition of claim 1 , wherein the 3′ terminal end of the blocking oligonucleotide comprises a blocking moiety that blocks ...

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Номер записи: 100