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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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Форма поиска

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 698. Отображено 100.
18-10-2012 дата публикации

Method for detecting a circularized dna, and use of said method for detecting mutations

Номер: US20120264630A1
Автор: Ismaïl Cisse, Ulrich Bockelmann, Virgile Viasnoff
Принадлежит: Individual

The present invention relates to a method for detecting a circularized single-stranded DNA by means of the isothermal hyperbranched rolling circle amplification technique, in which the primers used comprise a detectable barcode sequence and, optionally, a spacer which blocks polymerization by DNA polymerase. The present invention also relates to the use of said method for detecting a genetic polymorphism of one or more base pair(s).

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Номер записи: 1
19-09-2013 дата публикации

Compositions and methods for sequencing nucleic acids

Номер: US20130244886A1
Автор: Jason Richard Betley, Jonathan Mark Boutell, Niall Anthony Gormley, Roberto Rigatti
Принадлежит: Illumina Inc

Disclosed herein are compositions and methods for sequencing nucleic acids.

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Номер записи: 2
03-01-2019 дата публикации

RNase H-Based Assays Utilizing Modified RNA Monomers

Номер: US20190002865A1
Автор: BEHLKE Mark Aaron, DOBOSY Joseph, Rose Scott, WALDER Joseph Alan
Принадлежит:

The present invention pertains to novel oligonucleotide compounds for use in various biological assays, such as nucleic acid amplification, ligation and sequencing reactions. The novel oligonucleotides comprise a ribonucleic acid domain and a blocking group at or near the 3′ end of the oligonucleotide. These compounds offer an added level of specificity previously unseen. Methods for performing nucleic acid amplification, ligation and sequencing are also provided. Additionally, kits containing the oligonucleotides are also disclosed herein. 1. A kit for the ligation of the donor and accepter ends of a single oligonucleotide in the presence of a target nucleic acid sequence comprising:(a) an oligonucleotide in which either its donor end, its accepter end, or both its donor and accepter ends comprise a cleavage domain and a blocking group preventing ligation of its 5′ phosphate group to its 3′ OH group;(b) a cleaving agent;(c) a ligation agent; and(d) optionally, an instruction manual for ligating the accepter and donor ends of the oligonucleotide in the presence of a target nucleic acid sequence.2. The kit of claim 1 , wherein the cleavage domain is an RNase H cleavable domain.3. The kit of claim 2 , wherein the cleavage domain comprises a single RNA residue.4. The kit of claim 2 , wherein the cleavage domain lacks an RNA residue.5. The kit of claim 4 , wherein the cleavage domain comprises one or more 2′-modified nucleosides.6. The kit of claim 1 , wherein the cleaving agent is an RNase H enzyme.7. The kit of claim 6 , wherein the RNase H enzyme is an RNase H2 enzyme.8. The kit of claim 1 , wherein the ligation agent is a DNA ligase enzyme.9. The kit of claim 8 , wherein the DNA ligase enzyme is a thermostable DNA ligase.10. The kit of claim 9 , wherein the thermostable DNA ligase is a hot-start DNA ligase having reduced activity at lower temperatures.11. The kit of claim 10 , wherein the DNA ligase inherently has lower activity at reduced temperatures or is ...

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Номер записи: 3
14-01-2021 дата публикации

Labelled nucleotides

Номер: US20210009623A1
Автор: John Milton, Silke Ruediger, Xiaohai Liu, Xiaolin Wu
Принадлежит: Illumina Cambridge Ltd

The invention provides a nucleotide or nucleoside having a base attached to a detectable label via a cleavable linker, characterised in that the cleavable linker contains a moiety selected from the group comprising: Formula (I) (wherein X is selected from the group comprising O, S, NH and NQ wherein Q is a C 1-10 substituted or unsubstituted alkyl group, Y is selected from the group comprising O, S, NH and N(allyl), T is hydrogen or a C 1-10 substituted or unsubstituted alkyl group and * indicates where the moiety is connected to the remainder of the nucleotide or nucleoside).

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Номер записи: 4
11-01-2018 дата публикации

METHODS AND SYSTEMS FOR SEQUENCING LONG NUCLEIC ACIDS

Номер: US20180010180A1
Автор: Matsuzaki Hajime, Mei Rui, Zhou Wei
Принадлежит:

The present invention provides methods and systems for sequencing long nucleic acid fragment. The present invention also provides a method of sequencing a target polynucleotide with fewer probes. Further, the present invention provides a method of sequencing a target polynucleotide with longer reads. Locus-specific, ligation-assisted sequencing/genotyping method and ligation-captured sequencing method are also provided in the present invention. The methods of the present invention allow low-cost, high-throughput and accurate sequencing of nucleic acids. 1. A method for sequencing a target nucleic acid , comprising:sequencing one or more bases of a target nucleic acid by extending a first probe hybridized to the target nucleic acid to generate a first extension product, thereby obtaining a first sequence read to determine the sequence of the target nucleic acid by performing a sequencing reaction, wherein the target nucleic acid is from a plurality of target nucleic acid fragments.2. The method of claim 1 , prior to said sequencing claim 1 , further comprising:removing a first cap from the first probe by cleaving a cleavage site at a 3′ end of the first probe, wherein the first probe comprises:(i) a capture probe from a plurality of capture probes; and 'wherein the capture probe and the first solution are ligated.', '(ii) a first solution probe from a plurality of first solution probes, each of the plurality of first solution probes comprising the first cap on the 3′ end linked via the cleavage site;'}3. The method of claim 2 , prior to said removing claim 2 , further comprising:adding a second cap to any of the plurality of capture probes not ligated to any of the plurality of first solution probes.4. The method of claim 3 , prior to said adding claim 3 , further comprising:ligating the capture probe and the first solution probe, thereby forming the first probe.5. The method of claim 4 , prior to said ligating claim 4 , further comprising:hybridizing the target ...

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Номер записи: 5
14-01-2021 дата публикации

METHOD AND APPARATUS TO NORMALIZE QUANTITATIVE READOUTS IN SINGLE-CELL EXPERIMENTS

Номер: US20210010061A1
Автор: Mendez Pedro, Ruff David
Принадлежит:

Provided herein are methods and systems for detection of nucleic acids for single cell samples. As part of the detection, a unique step of normalization of different single cell samples is included. One embodiment of the method includes i) selecting one or more target nucleic acid sequence of interest in an individual cell, where the target nucleic acid sequence is complementary to a nucleic acid in a cell; ii) providing a sample having a plurality of individual single cells and encapsulating one or more individual cell(s); iii) providing a sample normalization component to one or more encapsulated cell, where the normalization component comprises an exogenous nucleic acid having a known sequence; iv) providing nucleic acid primers for the target nucleic acid and the exogenous nucleic acid; v) providing a protease to each encapsulated cell and incubating the encapsulated cell with the protease in the drop to produce a cell lysate; vi) performing a nucleic acid amplification reaction to form an amplification product from the nucleic acid of a single cell, where the amplification product comprise amplicons of one or more target nucleic acid sequence and an amplicon for the exogenous nucleic acid; and vii) comparing the amplification products from the target amplicons and the exogenous nucleic acid amplicons and determining the copy number or sequence of the target nucleic acid in a single cell. 1. A method for nucleic acid detection for single cell samples , the method comprising , independent of order presented , the following steps:i) selecting one or more target nucleic acid sequence of interest in an individual cell, wherein the target nucleic acid sequence is complementary to a nucleic acid in a cell;ii) providing a sample having a plurality of individual single cells and encapsulating one or more individual cell(s);iii) providing a sample normalization component to one or more encapsulated cell, wherein the normalization component comprises an exogenous nucleic ...

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Номер записи: 6
03-02-2022 дата публикации

Orthogonal deblocking of nucleotides

Номер: US20220033898A1
Автор: Elaine H. Trepagnier, Tarun Khurana
Принадлежит: Illumina Inc

A method including steps of (a) providing an array of sites, wherein each site comprises a mixture of different nucleic acid templates; (b) extending primers hybridized to the different nucleic acid templates at each of the sites with different nucleotide analogs having different reversible blocking moieties, respectively, thereby producing different primer extension products at each site; (c) detecting the different primer extension products to distinguish the different nucleotide analogs at each site; and (d) removing the different reversible blocking moieties from the primer extension products at each of the sites using a first treatment that is selective for a first of the different reversible blocking moieties and a second treatment that is selective for a second of the different reversible blocking moieties.

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Номер записи: 7
16-01-2020 дата публикации

MODIFIED NUCLEOTIDES

Номер: US20200017908A1
Автор: Barnes Colin Lloyd, Brennan Joseph, Liu Xiaohai, Milton John, Ruediger Silke, Smith Mark Edward Brennan, Wu Xiaolin
Принадлежит:

The invention provides modified nucleotide or nucleoside molecule comprising a purine or pyrimidine base and a ribose or deoxyribose sugar moiety having a removable 3′-OH blocking group covalently attached thereto, such that the 3′ carbon atom has attached a group of the structure —O—Z wherein Z is any of —C(R′)2-O—R″, —C(R′)2-N(R″)2, —C(R′)2-N(H)R″, —C(R′)2-S—R″ and —C(R′)2-F, wherein each R″ is or is part of a removable protecting group; each R′ is independently a hydrogen atom, an alkyl, substituted alkyl, arylalkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclic, acyl, cyano, alkoxy, aryloxy, heteroaryloxy or amido group, or a detectable label attached through a linking group; or (R′)2 represents an alkylidene group of formula=C(R′″)2 wherein each R′″ may be the same or different and is selected from the group comprising hydrogen and halogen atoms and alkyl groups; and wherein said molecule may be reacted to yield an intermediate in which each R″ is exchanged for H or, where Z is —C(R′)2-F, the F is exchanged for OH, SH or NH2, preferably OH, which intermediate dissociates under aqueous conditions to afford a molecule with a free 3′OH; with the proviso that where Z is —C(R′)2-S—R″, both R′ groups are not H. 1. (canceled)2. (canceled)3. A modified nucleoside triphosphate molecule comprising a base and a deoxyribose sugar moiety , wherein the 3′ carbon atom of the sugar moiety has covalently attached thereto a group of the structure:{'br': None, '—O—Z'}wherein Z is a removable protecting group comprising an azido group.4. The molecule of claim 3 , wherein the removable protecting group is azidomethyl.5. The molecule of claim 4 , wherein the base is a purine claim 4 , a pyrimidine or a deazapurine.6. The molecule of claim 4 , wherein the base is adenine claim 4 , 7-deazaadenine claim 4 , guanine claim 4 , or 7-deazaguanine.7. The molecule of claim 4 , wherein the base is cytosine claim 4 , thymine or uracil.8. The molecule of claim 4 , wherein the base is linked ...

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Номер записи: 8
24-01-2019 дата публикации

HIGH INTENSITY LABELED REACTANT COMPOSITIONS AND METHODS FOR SEQUENCING

Номер: US20190024168A1
Автор: DODD David, Huang Haidong, Lackey Jeremy, Reed Brian, Rothberg Jonathan M., Shi Xinghua
Принадлежит: Quantum-Si incorporated

Compositions useful for the detection of single molecules in a sample are provided. In some aspects, the disclosure provides a nucleic acid connected to a nucleotide and two or more luminescent labels. In some embodiments, the nucleic acids described herein comprise one or more structural features that provide enhanced fluorescence intensity. In some aspects, methods of sequencing using the labeled nucleotides of the disclosure are provided. 1. A labeled nucleotide comprising a nucleotide connected to two or more luminescent labels via a linker , wherein each luminescent label is at least 5 angstroms separated from any other luminescent label.2. The labeled nucleotide of claim 1 , wherein each luminescent label comprises a center of mass that is at least 5 angstroms separated from the center of mass of any other luminescent label.3. The labeled nucleotide of claim 1 , wherein one or more luminescent labels are attached to the linker via a spacer molecule that comprises at least 8 contiguous atoms between the luminescent label and an attachment site on the linker.4. The labeled nucleotide of claim 3 , wherein the spacer molecule comprises fewer than 50 claim 3 , fewer than 40 claim 3 , fewer than 30 claim 3 , or fewer than 20 contiguous atoms between the luminescent label and the attachment site on the linker.5. (canceled)6. The labeled nucleotide of claim 1 , wherein the linker is an oligomer that comprises at least 10 monomeric units.7. The labeled nucleotide of claim 6 , wherein the oligomer comprises fewer than 150 claim 6 , fewer than 100 claim 6 , or fewer than 50 monomeric units.8. The labeled nucleotide of claim 6 , wherein one or more luminescent labels are attached to the linker at an attachment site that is at least 5 monomeric units separated from any other attachment site.9. The labeled nucleotide of claim 1 , wherein the linker is a nucleic acid.1013-. (canceled)14. A labeled nucleotide comprising a nucleotide connected to two or more luminescent labels ...

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Номер записи: 9
04-02-2016 дата публикации

Modified nucleotides

Номер: US20160032378A1
Автор: Colin Barnes, John Milton, Joseph Brennan, Mark Smith, Silke Ruediger, Xiaohai Liu, Xiaolin Wu
Принадлежит: Illumina Cambridge Ltd

The invention provides modified nucleotide or nucleoside molecule comprising a purine or pyrimidine base and a ribose or deoxyribose sugar moiety having a removable 3′-OH blocking group covalently attached thereto, such that the 3′ carbon atom has attached a group of the structure —O—Z wherein Z is any of —C(R′)2-O—R″, —C(R′)2-N(R″)2, —C(R′)2-N(H)R″, —C(R′)2-S—R″ and —C(R′)2-F, wherein each R″ is or is part of a removable protecting group; each R′ is independently a hydrogen atom, an alkyl, substituted alkyl, arylalkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclic, acyl, cyano, alkoxy, aryloxy, heteroaryloxy or amido group, or a detectable label attached through a linking group; or (R′)2 represents an alkylidene group of formula ═C(R′″)2 wherein each R′″ may be the same or different and is selected from the group comprising hydrogen and halogen atoms and alkyl groups; and wherein said molecule may be reacted to yield an intermediate in which each R″ is exchanged for H or, where Z is —C(R′)2-F, the F is exchanged for OH, SH or NH2, preferably OH, which intermediate dissociates under aqueous conditions to afford a molecule with a free 3′OH; with the proviso that where Z is —C(R′)2-S—R″, both R′ groups are not H.

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Номер записи: 10
31-01-2019 дата публикации

METHOD OF DESIGNING PRIMERS, METHOD OF DETECTING SINGLE NUCLEOTIDE POLYMORPHISMS (SNPs), METHOD OF DISTINGUISHING SNPs, AND RELATED PRIMERS, DETECTABLE OLIGONUCLEOTIDES, AND KITS

Номер: US20190032133A1
Автор: Erickson Brian J., Huang Shihai X., Su Hong X.
Принадлежит: Abbott Molecular, Inc.

A method of designing a primer for detecting a single nucleotide polymorphism (SNP), a method of detecting an SNP, a method of distinguishing SNPs, primers, detectable oligonucleotides, and kits. 1. A method of detecting at least one mutation (X) of the codon encoding valine at amino acid position 600 (V600X) in exon 15 of the BRAF gene in a sample of nucleic acid from a human , which method comprises:(a) performing an amplification reaction with the sample of nucleic acid, wherein the amplification reaction comprises a primer, the last three nucleotides at the 3′ terminus of which encodes X and wherein the fourth nucleotide from the 3′ terminus contains a base other than adenine (A), wherein, if X is present, the primer anneals to X,wherein, if the sample of nucleic acid is mRNA, step (a) further comprises obtaining cDNA reverse-transcribed from the mRNA or reverse-transcribing cDNA from the mRNA before performing the amplification reaction,whereupon, if X is present, the amplification reaction produces an amplification product comprising X, and(b) detecting, and optionally quantitating, the amplification product comprising X,wherein, if X is encoded by more than one codon, the amplification reaction comprises a primer for each codon,whereupon a V600X mutation in exon 15 of the BRAF gene in a sample of nucleic acid from a human is detected.2. The method of claim 1 , wherein the amplification reaction further comprises at least one peptide nucleic acid (PNA) clamp claim 1 , wherein at least one PNA clamp blocks the amplification from wild-type target claim 1 , and wherein claim 1 , if the amplification reaction comprises one or more other PNA clamps claim 1 , the PNA clamps a detectable oligonucleotide and/or a primer by binding an unwanted target and preventing a primer from amplifying from an unwanted target.3. The method of claim 1 , wherein detecting the amplification product comprising X comprises detecting a labeled primer or contacting the amplification ...

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Номер записи: 11
03-03-2022 дата публикации

Polymerase Chain Reaction Primers and Probes for Mycobacterium Tuberculosis

Номер: US20220064715A1
Автор: ALLAND David, Chakravorty Soumitesh
Принадлежит: Rutgers, The State University of New Jersey

The present invention relates to novel primers and sloppy molecular beacon and molecular beacon probes for amplifying segments from different genes in for identifying the presence of M.tb DNA and/or resistance to anti-tuberculosis drugs. 128-. (canceled)29. An isolated nucleic acid probe comprising a probe sequence that is at least 85% identical to SEQ ID No: 56.30. The nucleic acid probe of claim 29 , comprising the sequence of SEQ ID No: 56.31. The nucleic acid probe of claim 29 , wherein the probe is labeled.32. The nucleic acid probe of claim 31 , wherein the probe is labeled with a fluorophore and a quencher at its two ends respectively.33. The nucleic acid probe of wherein the fluorophore is fluorescein claim 32 , cyanine 5 claim 32 , or TexasRed or TAMRA.34. The nucleic acid probe of claim 32 , wherein the quencher is BHQ®1 claim 32 , BHQ®2 claim 32 , or DABCYL.35M. tuberculosis. An oligonucleotide set for amplifying a portion of an katG gene claim 32 , comprising claim 32 , a first pair of forward and reverse primers specific for a first portion of the katG gene claim 32 , wherein each primer has a sequence that is at least 85% identical to an oligonucleotide sequence selected from SEQ ID Nos: 7-11.36. The oligonucleotide set of claim 35 , wherein the first pair of the primers are selected from the group consisting of SEQ ID Nos: 7-11.37. A kit comprising an isolated nucleic acid probe of and a packaging material.38. The kit of further comprising a first pair of forward and reverse primers specific for a first portion of a katG gene claim 37 , wherein each primer has a sequence that is at least 85% identical to an oligonucleotide sequence selected from SEQ ID Nos: 7-11.39. The kit of further comprising a DNA polymerase claim 38 , extension nucleotides claim 38 , and a buffer.40M. tuberculosis. A method for detecting drug resistance in claim 38 , comprising amplifying a first nucleic acid target sequence with a first primer pair to generate a first amplicon ...

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Номер записи: 12
08-05-2014 дата публикации

Methods and compositions for nucleic acid amplification

Номер: US20140127700A1
Автор: Dmitry LYAKHOV, James D. Carlson, Lyle J. Arnold, Jr., Michael M. Becker, Norman C. Nelson, Steven T. Brentano
Принадлежит: Gen Probe Inc

Compositions that are used in nucleic acid amplification in vitro are disclosed, which include a target specific universal (TSU) promoter primer or promoter provider oligonucleotide that includes a target specific (TS) sequence that hybridizes specifically to a target sequence that is amplified and a universal (U) sequence that is introduced into the sequence that is amplified, by using a primer for the universal sequence. Methods of nucleic acid amplification in vitro are disclosed that use one or more TSU oligonucleotides to attached a U sequence to a target nucleic acid in a target capture step and then use a primer for a U sequence in subsequent amplification steps performed in substantially isothermal conditions to make amplification products that contain a U sequence that indicates the presence of the target nucleic acid in a sample.

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Номер записи: 13
14-02-2019 дата публикации

ELIMINATION OF PRIMER-PRIMER INTERACTIONS DURING PRIMER EXTENSION

Номер: US20190048410A1
Автор: Godwin Brian
Принадлежит:

The invention comprises a method of amplifying nucleic acids by primer extension with reduced formation of primer-primer byproducts. 1. A method of synthesizing nucleic acid strands by primer extension with reduced primer-printer interaction comprising: contacting a target nucleic strand with a nucleic acid polymerase and a primer comprising at least one modified nucleotide that stalls nucleotide incorporation by the polymerase.2. The method of claim 1 , wherein the primer is single-stranded oligonucleotide consisting of two arms complementary to a target sequence separated by a linker sequence non-complementary to the target sequence.3. The method of claim 1 , wherein the modified nucleotide is selected from a group consisting of uracil claim 1 , ate abasic modified nucleotide and a pyrimidine dimer.4. A method of amplifying a target nucleic acid in a sample with reduced primer-primer interaction comprising:a. a primer extension step, wherein the sample is contacted with a nucleic acid polymerase and a first primer complementary to the target nucleic acid comprising at least one modified nucleotide that stalls nucleotide incorporation by the polymerase, to generate a primer extension product; andb. an exponential amplification step wherein the sample is contacted with a second primer complementary to the primer extension product.5. The method of claim 4 , wherein the primer extension product is ligated to a double stranded adaptor prior to step b.6. The method of claim 4 , wherein the adaptor and the first primer comprise binding sites for universal amplification primers and the second primer is a universal primer.7. The method of claim 4 , wherein the primer is a single-stranded oligonucleotide consisting of two arms complementary to a target sequence separated by a linker sequence non-complementary to the target sequence.8. The method of claim 4 , wherein the exponential. amplification step utilizes a polymerase tolerant of the modified nucleotide.9. The method ...

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Номер записи: 14
08-03-2018 дата публикации

DNA SEQUENCING WITH REAGENT RECYCLING ON WIREGRID

Номер: US20180067052A1
Автор: LUB JOHAN, Van Der Zaag Pieter Jan, Wimberger-Friedl Reinhold
Принадлежит:

The present invention relates to DNA sequencing with reagent cycling on the wiregrid. The sequencing approach suggested with which allows to use a single fluid with no washing steps. Based on strong optical confinement and of excitation light and of cleavage light, the sequencing reaction can be read-out without washing the surface. Stepwise sequencing is achieved by using nucleotides with optically cleavable blocking moieties. After read-out the built in nucleotide is deblocked by cleavage light through the same substrate. This ensures that only bound nucleotides will be unblocked. 1. A device for optically controlling an iterative stepwise reaction to determine a sequence of a nucleic acid by synthesis , the device comprising:a substrate for binding at least one molecule on a first surface of the substrate; andan optical arrangement,{'sub': 'Ex1', 'wherein the optical arrangement is configured to direct excitation light of at least a first excitation wavelength λto the substrate to excite a fluorescent label of a first nucleotide,'}wherein the first nucleotide is incorporated into the molecule bound on the first surface of the substrate,wherein the optical arrangement is configured to receive and detect fluorescent light emitted by the fluorescent label of the first nucleotide,{'sub': 'CL', 'wherein the optical arrangement is configured to direct cleavage light of a cleavage wavelength λto the substrate to optically induce a cleavage reaction at the first nucleotide to cleave a blocking moiety and the fluorescent label away from the first nucleotide,'}wherein the substrate is configured to provide for an evanescent wave of the excitation light at the first surface of the substrate.2. The device according to claim 1 , the device further comprising:a solution with a plurality of nucleotides and an enzyme, wherein the nucleotides of the solution respectively comprise a respective blocking moiety,wherein the respective blocking moiety is configured to block a ...

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Номер записи: 15
15-03-2018 дата публикации

PCR Method

Номер: US20180073053A1
Автор: Christopher John MATTOCKS, Daniel Leonard WARD
Принадлежит: Salisbury NHS Foundation Trust

A method for generating amplicon constructs of a target sequence is disclosed, the method comprising providing a target sequence; an oligonucleotide probe, comprising a universal sequence and further comprising, at or towards its 5′ end, a target specific sequence capable of hybridising to the reverse complement of a sequence at, or flanking one of the 3′ ends of the target sequence; a universal primer, comprising at its 3′ end a sequence capable of hybridising to the universal sequence of the oligonucleotide probe and performing a Polymerase Chain Reaction (PCR).

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Номер записи: 16
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: 17
26-03-2015 дата публикации

Methods and Compositions for Size-Controlled Homopolymer Tailing of Substrate Polynucleotides by a Nucleic Acid Polymerase

Номер: US20150087027A1
Автор: Laurie KURIHARA, Vladimir Makarov
Принадлежит: Swift Biosciences Inc

The present invention is directed to methods and compositions for adding tails of specific lengths to a substrate polynucleotide. The invention also contemplates methods and compositions for immobilization of tailed substrates to a solid support. The disclosure contemplates that the attenuator molecule is any biomolecule that associates with a tail sequence added to a substrate polynucleotide and controls the addition of a tail sequence to the 3′ end of the substrate polynucleotide. The sequence that is added to the substrate polynucleotide is referred to herein as a tail sequence, or simply a tail, and the process of adding a nucleotide to a substrate polynucleotide is referred to herein as tailing.

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Номер записи: 18
25-03-2021 дата публикации

Primer and probe set for diagnosis, detection or screening of colorectal cancer

Номер: US20210087636A1
Автор: Guodong Zhao
Принадлежит: Suzhou Versabio Technologies Inc

Disclosed is a primer and probe set for diagnosis, detection or screening of colorectal cancer. A forward primer of SEPT9 is selected from any one of SEQ ID NOs: 1-25, a reverse primer of SEPT9 is selected from any one of SEQ ID NOs: 26-51, the blocker primer of SEPT9 is selected from any one of SEQ ID NOs: 52-67, and the probe of SEPT9 is selected from any one of SEQ ID NOs: 68-90. A forward primer of SDC2 is selected from any one of SEQ ID NOs: 91-117, a reverse primer of SDC2 is selected from any one of SEQ ID NOs: 118-143, the blocker primer of SDC2 is selected from any one of SEQ ID NOs: 144-156, and the probe of SDC2 is selected from any one of SEQ ID NOs: 157-174.

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Номер записи: 19
21-03-2019 дата публикации

Massive parallel method for decoding dna and rna

Номер: US20190085016A1
Автор: Jingyue Ju, John Robert Edwards, Yasuhiro Itagaki, Zengmin Li
Принадлежит: Columbia University of New York

This invention provides methods for attaching a nucleic acid to a solid surface and for sequencing nucleic acid by detecting the identity of each nucleotide analogue after the nucleotide analogue is incorporated into a growing strand of DNA in a polymerase reaction. The invention also provides nucleotide analogues which comprise unique labels attached to the nucleotide analogue through a cleavable linker, and a cleavable chemical group to cap the —OH group at the 3′-position of the deoxyribose.

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Номер записи: 20
30-03-2017 дата публикации

Massive parallel method for decoding dna and rna

Номер: US20170088575A1
Автор: Jingyue Ju, John Robert Edwards, Yasuhiro Itagaki, Zengmin Li
Принадлежит: Columbia University of New York

This invention provides methods for attaching a nucleic acid to a solid surface and for sequencing nucleic acid by detecting the identity of each nucleotide analogue after the nucleotide analogue is incorporated into a growing strand of DNA in a polymerase reaction. The invention also provides nucleotide analogues which comprise unique labels attached to the nucleotide analogue through a cleavable linker, and a cleavable chemical group to cap the —OH group at the 3′-position of the deoxyribose.

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Номер записи: 21
30-03-2017 дата публикации

Massive parallel method for decoding dna and rna

Номер: US20170088891A1
Автор: Jingyue Ju, John Robert Edwards, Yasuhiro Itagaki, Zengmin Li
Принадлежит: Columbia University of New York

This invention provides methods for attaching a nucleic acid to a solid surface and for sequencing nucleic acid by detecting the identity of each nucleotide analogue after the nucleotide analogue is incorporated into a growing strand of DNA in a polymerase reaction. The invention also provides nucleotide analogues which comprise unique labels attached to the nucleotide analogue through a cleavable linker, and a cleavable chemical group to cap the —OH group at the 3′-position of the deoxyribose.

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Номер записи: 22
19-03-2020 дата публикации

SYSTEMS AND METHODS FOR IDENTIFYING AND DISTINGUISHING GENETIC SAMPLES

Номер: US20200087717A1
Автор: KAIN Robert Charles, Shen Richard
Принадлежит:

Method and systems for identifying and distinguishing subjects using a biochip are described. Biochips comprising subject specific features comprising multiple non-overlapping probes are disclosed. 1. A biochip comprising: one or more sets of probes , wherein each set of said one or more sets of probes comprises a plurality of probes , wherein each of said plurality of probes comprises one or more subject-specific features and wherein each set of said one or more sets of probes binds to a target nucleic acid from a different subject of a plurality of different subjects.2. The biochip of claim 1 , wherein each of said plurality of probes within a set of probes are identical.3. The biochip of claim 1 , wherein each of said plurality of probes within a set of probes are different.4. The biochip of claim 1 , wherein each set of said plurality of probes comprises a plurality of unique probes.5. The biochip of claim 4 , wherein each set of said plurality of probes comprises an average representation of said plurality of unique probes.6. The biochip of claim 5 , wherein said average representation of said plurality of unique probes is controlled by limiting the total number of probes within each set of said one or more sets of probes claim 5 , by mixing said plurality of unique probes at a predefined ratio claim 5 , or a combination of both.7. The biochip of claim 4 , wherein each of said one or more sets of probes comprises about 2-1000 unique probes.8. The biochip of claim 5 , wherein said average representation comprises about 2-1000 representations of each of said plurality of unique probes within said set of probes.9. The biochip of claim 1 , wherein subject-specific features within each set of probes are identical.10. The biochip of claim 1 , wherein each set of said one or more sets of probes comprises a different subject-specific feature.11. The biochip of claim 1 , wherein each set of said one or more sets of probes is individually addressable.12. The biochip of ...

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Номер записи: 23
19-03-2020 дата публикации

ULTRA SENSITIVE PROBES FOR DETECTION OF NUCLEIC ACID

Номер: US20200087726A1
Автор: LEI Xiaojun, Li Qiang, Yuan Yuan, Zhang Yi
Принадлежит:

The present disclosure provides a composition with ultra sensitivity for detection of nucleic acid and the method of use thereof. The composition comprises a target probe capable of hybridizing to a target nucleic acid, at least one first bridge probe, at least one second bridge probe, and a label probe. The target probe includes a pre-bridge region having a first tail nucleotide sequence. The first bridge probe includes sequentially a first head region having a first head nucleotide sequence, a first gap region having a gap nucleotide sequence, and a first tail region having the first tail nucleotide sequence. The second bridge probe includes sequentially a second head region having a second head nucleotide sequence complementary to the first head nucleotide sequence, a second gap region having the gap nucleotide sequence, and a second tail region having a second tail nucleotide sequence complementary to the first tail nucleotide sequence. The label probe is capable of hybridizing to the first and the second gap nucleotide region. 1. A composition comprising:a) a target probe capable of hybridizing to a target nucleic acid, said target probe comprising a pre-bridge region having a first tail nucleotide sequence; (i) a first head region having a first head nucleotide sequence;', '(ii) a first gap region having a gap nucleotide sequence;', '(iii) a first tail region having the first tail nucleotide sequence;, 'b) at least one first bridge probe, comprising sequentially'} (i) a second head region having a second head nucleotide sequence complementary to the first head nucleotide sequence;', '(ii) a second gap region having the gap nucleotide sequence;', '(iii) a second tail region having a second tail nucleotide sequence complementary to the first tail nucleotide sequence; and, 'c) at least one second bridge probe, comprising sequentially'}d) a label probe capable of hybridizing to the first and the second gap nucleotide region, said label probe comprising a label.2. ...

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Номер записи: 24
05-04-2018 дата публикации

COMPOSITIONS AND METHODS FOR AMPLIFYING A NUCLEIC ACID SEQUENCE IN A SAMPLE

Номер: US20180094310A1
Автор: JUDICE STEPHEN A., Shaffer Daniel
Принадлежит: ENVIROLOGIX INC.

The present invention features compositions and methods for quantifying detection of a target oligonucleotide in a sample in real time. These methods are compatible with target oligonucleotides amplified using a NEAR reaction. 1. A method of detecting a specific product in an amplification reaction , the method comprising:(a) contacting a test sample comprising a target nucleic acid molecule under substantially isothermal conditions with a polymerase, two or more primer oligonucleotides, each of which specifically binds to a complementary sequence on the target nucleic acid molecule, and a nicking enzyme, wherein each of the primer oligonucleotides comprises one or more 2′ modified nucleotides positioned at the 3′ end of the sequence complementary to the target nucleic acid molecule;(b) generating amplicons comprising at least a portion of the target nucleic acid molecule sequence; and(c) contacting the amplicon with a detectable polynucleotide probe and detecting a signal specific for oligonucleotide probe hybridization to the amplicon, wherein the signal indicates detection of the target nucleic acid molecule in the test sample or an amplicon thereof.2. The method of claim 1 , wherein the test sample comprises a pathogen.3. The method of claim 2 , wherein the pathogen is a virus claim 2 , bacteria claim 2 , yeast or fungus.4. The method of claim 1 , wherein the test sample is a biological sample.5. The method of claim 4 , wherein the biological sample is a biological fluid claim 4 , cell claim 4 , or tissue sample.6. The method of claim 5 , wherein the biological fluid is urine claim 5 , semen claim 5 , vaginal secretion claim 5 , or stool.7. The method of claim 1 , wherein the detectable polynucleotide probe is incorporated into a lateral flow device.8. The method of claim 1 , wherein the method is carried out at a point of care.9. The method of claim 1 , wherein the detecting is carried out at the endpoint of the amplification reaction.10. A method of detecting ...

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Номер записи: 25
28-03-2019 дата публикации

Massive parallel method for decoding dna and rna

Номер: US20190092805A1
Автор: Jingyue Ju, John Robert Edwards, Yasuhiro Itagaki, Zengmin Li
Принадлежит: Columbia University of New York

This invention provides methods for attaching a nucleic acid to a solid surface and for sequencing nucleic acid by detecting the identity of each nucleotide analogue after the nucleotide analogue is incorporated into a growing strand of DNA in a polymerase reaction. The invention also provides nucleotide analogues which comprise unique labels attached to the nucleotide analogue through a cleavable linker, and a cleavable chemical group to cap the —OH group at the 3′-position of the deoxyribose.

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Номер записи: 26
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: 27
28-03-2019 дата публикации

Methods of lowering the error rate of massively parallel dna sequencing using duplex consensus sequencing

Номер: US20190093160A1
Автор: Jesse Salk, Lawrence A. Loeb, Michael Schmitt
Принадлежит: University of Washington Center for Commercialization

Next Generation DNA sequencing promises to revolutionize clinical medicine and basic research. However, while this technology has the capacity to generate hundreds of billions of nucleotides of DNA sequence in a single experiment, the error rate of approximately 1% results in hundreds of millions of sequencing mistakes. These scattered errors can be tolerated in some applications but become extremely problematic when “deep sequencing” genetically heterogeneous mixtures, such as tumors or mixed microbial populations. To overcome limitations in sequencing accuracy, a method Duplex Consensus Sequencing (DCS) is provided. This approach greatly reduces errors by independently tagging and sequencing each of the two strands of a DNA duplex. As the two strands are complementary, true mutations are found at the same position in both strands. In contrast, PCR or sequencing errors will result in errors in only one strand. This method uniquely capitalizes on the redundant information stored in double-stranded DNA, thus overcoming technical limitations of prior methods utilizing data from only one of the two strands.

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Номер записи: 28
28-03-2019 дата публикации

METHODS OF LOWERING THE ERROR RATE OF MASSIVELY PARALLEL DNA SEQUENCING USING DUPLEX CONSENSUS SEQUENCING

Номер: US20190093161A1
Автор: LOEB Lawrence A., SALK Jesse, SCHMITT Michael
Принадлежит:

Next Generation DNA sequencing promises to revolutionize clinical medicine and basic research. However, while this technology has the capacity to generate hundreds of billions of nucleotides of DNA sequence in a single experiment, the error rate of approximately 1% results in hundreds of millions of sequencing mistakes. These scattered errors can be tolerated in some applications but become extremely problematic when “deep sequencing” genetically heterogeneous mixtures, such as tumors or mixed microbial populations. To overcome limitations in sequencing accuracy, a method Duplex Consensus Sequencing (DCS) is provided. This approach greatly reduces errors by independently tagging and sequencing each of the two strands of a DNA duplex. As the two strands are complementary, true mutations are found at the same position in both strands. In contrast, PCR or sequencing errors will result in errors in only one strand. This method uniquely capitalizes on the redundant information stored in double-stranded DNA, thus overcoming technical limitations of prior methods utilizing data from only one of the two strands. 136-. (canceled)37. A method of generating a high accuracy sequence read of a double-stranded target nucleic acid molecule comprising:amplifying each original strand of the double-stranded target nucleic acid molecule resulting in each original strand generating a distinct yet related set of amplified target nucleic acid products;sequencing the amplified target nucleic acid products generated from each original strand;confirming the presence of at least one sequence read of an amplified target nucleic acid product generated from each of the original strands;comparing the at least one sequence read obtained from the amplified target nucleic acid products generated from one original strand with the at least one sequence read obtained from the amplified target nucleic acid products generated from the other original strand; andgenerating a consensus sequence of the ...

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Номер записи: 29
28-03-2019 дата публикации

METHODS OF LOWERING THE ERROR RATE OF MASSIVELY PARALLEL DNA SEQUENCING USING DUPLEX CONSENSUS SEQUENCING

Номер: US20190093162A1
Автор: LOEB Lawrence A., SALK Jesse, SCHMITT Michael
Принадлежит:

Next Generation DNA sequencing promises to revolutionize clinical medicine and basic research. However, while this technology has the capacity to generate hundreds of billions of nucleotides of DNA sequence in a single experiment, the error rate of approximately 1% results in hundreds of millions of sequencing mistakes. These scattered errors can be tolerated in some applications but become extremely problematic when “deep sequencing” genetically heterogeneous mixtures, such as tumors or mixed microbial populations. To overcome limitations in sequencing accuracy, a method Duplex Consensus Sequencing (DCS) is provided. This approach greatly reduces errors by independently tagging and sequencing each of the two strands of a DNA duplex. As the two strands are complementary, true mutations are found at the same position in both strands. In contrast, PCR or sequencing errors will result in errors in only one strand. This method uniquely capitalizes on the redundant information stored in double-stranded DNA, thus overcoming technical limitations of prior methods utilizing data from only one of the two strands. 136-. (canceled)37. A method for generating high accuracy DNA sequence reads of selectively enriched deoxyribonucleic acid (DNA) molecules , comprising:(a) ligating a plurality of double-stranded DNA molecules with a set of duplex adapters, wherein each duplex adapter of the set of duplex adapters differently tags complementary strands of each parent double-stranded DNA molecule of the plurality of double-stranded DNA molecules to provide tagged strands;(b) for each genetic locus in a set of one or more genetic loci among a larger set of genetic sequences in a sample, selectively enriching the tagged strands for a subset of the tagged strands that map to the genetic locus, to provide enriched tagged strands;(c) sequencing at least a portion of the enriched tagged strands to generate a plurality of raw sequence reads; and (i) grouping the plurality of raw sequence ...

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Номер записи: 30
02-04-2020 дата публикации

NUCLEIC ACID SEQUENCING-BY-SYNTHESIS (SBS) METHODS THAT COMBINE SBS CYCLE STEPS

Номер: US20200102609A1
Автор: Barker David, Glezer Eli, SPAVENTA Andrew
Принадлежит:

The present disclosure provides improved nucleic acid sequencing-by-synthesis (SBS) methods, related kits and reagents, and systems for performing such methods using such kits and reagents. 1. A method to determine the identity of a nucleotide residue in an extension product , comprising:(a) combining in a sequencing reaction mixture a plurality of identical primed template DNA molecules, a DNA polymerase, at least 4 distinguishable, blocked deoxyribonucleotide triphosphate (dNTP) analogue species, at least 3 of which distinguishable, blocked dNTP analogue species are labeled such that each distinguishable, blocked dNTP analogue species in the sequencing reaction mixture can be distinguished from the other distinguishable, blocked dNTP analogue species, thereby incorporating a distinguishable, blocked dNTP analogue species into at least one of said plurality of identical primed template DNA molecules and forming a distinguishable, blocked extension product;(b) after step (a), adding to the sequencing reaction mixture at least 4 unlabeled, blocked dNTP analogue species, thereby forming an unlabeled, blocked extension product; and(c) during step (a) and/or (b), determining the identity of the distinguishable, blocked dNTP analogue incorporated into the labeled, blocked extension product in step (a).2. A method to determine the identity of a nucleotide residue in an extension product , comprising:(a) combining in a sequencing reaction mixture a plurality of identical primed template DNA molecules, a DNA polymerase, a labeled, 3′-blocked dGTP analogue, a labeled, 3′-blocked dATP analogue, a labeled, 3′-blocked dCTP analogue, and a labeled, 3′-blocked dTTP analogue and/or a labeled, 3′-blocked dUTP analogue, thereby incorporating a labeled, 3′-blocked deoxyribonucleotide triphosphate (dNTP) analogue species into at least one of said plurality of identical primed template DNA molecules and forming a labeled, blocked extension product;(b) after step (a), adding to said ...

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Номер записи: 31
11-04-2019 дата публикации

DNA SEQUENCING

Номер: US20190106744A1
Автор: Eshoo Mark W.
Принадлежит:

Provided herein is technology relating to sequencing nucleic acids and particularly, but not exclusively, to methods, compositions, systems, and kits for sequencing a nucleic acid using a degenerate two-base code. 1. A composition comprising a first nucleotide and a second nucleotide wherein the first nucleotide is labeled with a first label and the second nucleotide is labeled with said first label.2. The composition of wherein the label is a fluorescent moiety.3. The composition of further comprising a third nucleotide and a fourth nucleotide claim 1 , wherein the third nucleotide is labeled with a second label and the fourth nucleotide is labeled with said second label.4. The composition of wherein the first nucleotide is an A claim 13 , the second nucleotide is a G claim 13 , the third nucleotide is a C claim 13 , and the fourth nucleotide is a T.5. The composition of wherein the first nucleotide is an A claim 3 , the second nucleotide is a C claim 3 , the third nucleotide is a G claim 3 , and the fourth nucleotide is a T.6. The composition of wherein the first nucleotide is a C claim 3 , the second nucleotide is a G claim 3 , the third nucleotide is an A claim 3 , and the fourth nucleotide is a T.7. The composition of further comprising a target nucleic acid claim 1 , a sequencing primer claim 1 , and a polymerase.8. The composition of further comprising a nucleic acid comprising the first nucleotide and the second nucleotide.9. A system for sequencing a nucleic acid claim 1 , the system comprising:a) a sequencing apparatus; andb) a functionality to differentiate a first nucleotide and a second nucleotide from a third nucleotide and a fourth nucleotide.10. The system of further comprising an output functionality to provide a degenerate two-base nucleotide sequence of the nucleic acid.11. The system of further comprising a functionality to merge a first degenerate two-base nucleotide sequence of the nucleic acid and a second degenerate two-base nucleotide ...

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Номер записи: 32
09-04-2020 дата публикации

LABELLED NUCLEOTIDES

Номер: US20200109448A1
Автор: Balasubramanian Shankar, Barnes Colin Lloyd, Liu Xiaohai, Milton John
Принадлежит:

Nucleosides and nucleotides are disclosed that are linked to detectable labels via a cleavable linker group. 125.-. (canceled)26. A method for determining the sequence of a target single stranded polynucleotide , comprising:a) providing four different types of nucleotides, wherein each of the nucleotides comprises: a 3′-hydroxyl group and a base that is linked to a detectable label via a cleavable linker; wherein the detectable label and the cleavable linker block the incorporation of a further nucleotide into a growing polynucleotide strand, wherein the detectable label linked to each nucleotide can be distinguished upon detection from the detectable label used for the other three nucleotides;b) incorporating one nucleotide from step a) into a complementary strand of the target single stranded polynucleotide;c) removing non-incorporated nucleotides;d) detecting the detectable label of the incorporated nucleotide of step b), thereby determining the type of nucleotide incorporated; ande) removing the detectable label of the incorporated nucleotide of step b).27. The method of claim 26 , further comprising: repeating steps a)-e) one or more times claim 26 , thereby determining the sequence of the target single stranded polynucleotide.28. The method of claim 26 , wherein the target single-stranded polynucleotide is immobilized on a solid support.29. The method of claim 26 , wherein each of the nucleotides for incorporation is a deoxyribonucleotide triphosphate.30. The method of claim 29 , wherein the base is a purine or a pyrimidine.31. The method of claim 30 , wherein the base is a deazapurine.32. The method of claim 26 , wherein the detectable label is a fluorophore.33. The method of claim 26 , wherein the linker is an acid labile linker or a photolabile linker.34. The method of claim 33 , wherein the linker comprises an azido moiety or a disulfide linkage.35. The method of claim 26 , wherein the incorporation of the nucleotide is accomplished by a terminal ...

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Номер записи: 33
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: 34
16-04-2020 дата публикации

LABELLED NUCLEOTIDES

Номер: US20200115747A1
Автор: Balasubramanian Shankar, Barnes Colin Lloyd, Liu Xiaohai, Milton John, Wu Xiaolin
Принадлежит:

Nucleosides and nucleotides are disclosed that are linked to detectable labels via a cleavable linker group. 1. A nucleotide or nucleoside molecule , having a base that is linked to a detectable label via a cleavable linker.2. The molecule of claim 1 , wherein the base is a purine claim 1 , or a pyrimidine.3. The molecule of or claim 1 , wherein the base is a deazapurine.4. The molecule of any of to claim 1 , having a ribose or deoxyribose sugar moiety.5. The molecule of claim 4 , wherein the ribose or deoxyribose sugar comprises a protecting group attached via the 2′ or 3′ oxygen atom.6. The molecule of any of to claim 4 , that i s a deoxyribonucleotide triphosphate.7. The molecule of any of to claim 4 , wherein the detectable label is a fluorophore.8. The molecule of any of to claim 4 , wherein the linker is acid labile claim 4 , photolabile or contains a disulphide linkage.9. A method of labeling a nucleic acid molecule claim 4 , the method comprising incorporating into -the nucleic acid molecule a nucleotide or nucleoside molecule claim 4 , wherein the nucleotide or nucleoside molecule has a base that is linked to a detectable label via a cleavable linker.10. The method of claim 9 , wherein said incorporating is accomplished via a terminal transferase claim 9 , polymerase or a reverse transcriptase.11. The method of or claim 9 , wherein the base is a deazapurine.12. The method of any of to claim 9 , wherein the nucleotide or nucleoside molecule has a ribose or deoxyribose sugar moiety claim 9 , and wherein the ribose or deoxyribose sugar comprises a protecting group attached via the 2′ or 3′ oxygen atom and which can be modified or removed to expose a 3′ OH group.13. The method of any of to claim 9 , wherein the nucleotide is a deoxyribonucleotide triphosphate.14. The method of any of to claim 9 , wherein the label is a fluorophore.15. The method of any of to 14 claims 9 , wherein the linker is acid labile claims 9 , photolabile claims 9 , or contains a ...

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Номер записи: 35
10-05-2018 дата публикации

DETECTION OF TARGET NUCLEIC ACID SEQUENCE BY PTO CLEAVAGE AND EXTENSION-DEPENDENT SIGNALING OLIGONUCLEOTIDE HYBRIDIZATION ASSAY

Номер: US20180127812A1
Автор: Chun Jong Yoon, Lee Young Jo
Принадлежит:

The present invention relates to the detection of a target nucleic acid sequence by a PCE-SH (PTO Cleavage and Extension-Dependent Signaling Oligonucleotide Hybridization) assay. The present invention does not use probes to be hybridized with target nucleic acid sequences for providing target signals. Interestingly, the present invention uses probes (signaling oligonucleotides) to be hybridized with the extended strand formed in a target-dependent manner in which the extended strand is synthesized using the CTO artificially selected as templates. 1. A kit for detecting a target nucleic acid sequence from a DNA or a mixture of nucleic acids by a PCE-SH (PTO Cleavage and Extension-Dependent Signaling Oligonucleotide Hybridization) assay , comprising:(a) a probing and targeting oligonucleotide (PTO); wherein the PTO comprises (i) a 3′-targeting portion comprising a hybridizing nucleotide sequence complementary to the target nucleic acid sequence and (ii) a 5′-tagging portion comprising a nucleotide sequence non-complementary to the target nucleic acid sequence; the 5′-tagging portion, the 3′-targeting portion or a junction site between the 5′-tagging portion and the 3′-targeting portion has a cleavage site for an enzyme having a 5′ nuclease activity;(b) an upstream oligonucleotide comprising a hybridizing nucleotide sequence complementary to the target nucleic acid sequence; wherein the upstream oligonucleotide is located upstream of the PTO;(c) a capturing and templating oligonucleotide (CTO); wherein the CTO comprises in a 3′ to 5′ direction (i) a capturing portion comprising a nucleotide sequence complementary to the 5′-tagging portion or a part of the 5′-tagging portion of the PTO and (ii) a templating portion comprising a nucleotide sequence non-complementary to the 5′-tagging portion and the 3′-targeting portion of the PTO; wherein the CTO is not labeled;(d) a signaling oligonucleotide (SO) having at least one label; wherein the SO comprises a complementary ...

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Номер записи: 36
08-09-2022 дата публикации

METHODS AND PROBES FOR PERFORMING PCR WITH MELT ANALYSIS FOR INCREASED MULTIPLEXING

Номер: US20220282307A1
Автор: WHITMAN Doug
Принадлежит: Luminex Corporation

Methods and compositions for the detection and quantification of nucleic acids are provided. In certain embodiments, methods involve the use of cleavable probes capable of forming double-stranded structures, such as hairpin structures, which probes can be distinguished from one another on the basis of reporter signal, melt properties, or both. 1. A method for detecting the presence of a target nucleic acid comprising:(a) contacting a sample with a first primer comprising a first nucleotide sequence complementary to a first portion of the target nucleotide sequence and a probe, the probe comprising, from 5′ to 3′, (i) a target-specific region comprising a second nucleotide sequence, wherein the second nucleotide sequence is complementary to a second portion of the target nucleotide sequence, wherein the second portion of the target nucleotide sequence is located downstream of the first portion, wherein the second nucleotide sequence is labelled with a first quencher and a fluorophore, and wherein the first quencher is located 5′ relative to the fluorophore; (ii) a polymerase extension-blocking moiety; (iii) a melt-signature region comprising a third nucleotide sequence, wherein the third nucleotide sequence is not complementary to the target nucleotide sequence, and wherein the third nucleotide sequence includes a first non-naturally occurring nucleotide, wherein the first non-naturally occurring nucleotide is an isoC nucleotide or an isoG nucleotide; (iii) a loop region; and (iv) a melt-signature complementary region comprising a fourth nucleotide sequence, wherein the forth nucleotide sequence is complementary to a portion of the third nucleotide sequence;(b) hybridizing the melt-signature region with the melt-signature complementary region to form a first hairpin structure having a first melting temperature;(c) extending the melt-signature complementary region along the melt-signature region using a polymerase to form a second hairpin structure having a second ...

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Номер записи: 37
24-05-2018 дата публикации

METHODS OF LOWERING THE ERROR RATE OF MASSIVELY PARALLEL DNA SEQUENCING USING DUPLEX CONSENSUS SEQUENCING

Номер: US20180142293A1
Автор: LOEB Lawrence A., SALK Jesse, SCHMITT Michael
Принадлежит:

Next Generation DNA sequencing promises to revolutionize clinical medicine and basic research. However, while this technology has the capacity to generate hundreds of billions of nucleotides of DNA sequence in a single experiment, the error rate of approximately 1% results in hundreds of millions of sequencing mistakes. These scattered errors can be tolerated in some applications but become extremely problematic when “deep sequencing” genetically heterogeneous mixtures, such as tumors or mixed microbial populations. To overcome limitations in sequencing accuracy, a method Duplex Consensus Sequencing (DCS) is provided. This approach greatly reduces errors by independently tagging and sequencing each of the two strands of a DNA duplex. As the two strands are complementary, true mutations are found at the same position in both strands. In contrast, PCR or sequencing errors will result in errors in only one strand. 136-. (canceled)37. A method of generating an error corrected sequence read of a double-stranded target nucleic acid molecule , comprising: (a) a degenerate or semi-degenerate single molecule identifier (SMI) sequence that alone or in combination with the target nucleic acid fragment ends uniquely labels the double stranded target nucleic acid molecule, and', '(b) a nucleotide sequence that distinguishes each strand of the adapter-target nucleic acid complex such that each strand of the adapter-target nucleic acid complex has a distinctly identifiable nucleotide sequence relative to its complementary strand;, 'ligating the double-stranded target nucleic acid molecule to at least one adapter molecule, to form an adapter-target nucleic acid complex, wherein the at least one adapter molecule comprises—'}amplifying each strand of the adapter-target nucleic acid complex to produce a plurality of first strand adapter-target nucleic acid complex amplicons and a plurality of second strand adapter-target nucleic acid complex amplicons;sequencing the adapter-target ...

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Номер записи: 38
21-05-2020 дата публикации

PROBE FOR DETECTION OF SNPS

Номер: US20200157601A1
Автор: Mergemeier Steffen
Принадлежит: CONGEN BIOTECHNOLOGIE GMBH

The invention relates to a hydrolysis probe for use in a real-time PCR for the detection of a single nucleotide polymorphism (SNP) in a nucleic acid target sequence and methods and kits employing said probe. 1. A hydrolysis probe for use in a real-time PCR for detection of a SNP (Single Nucleotide Polymorphism) in a nucleic acid target sequence , said probe comprisinga) an oligonucleotide sequence complementary to a region of the nucleic acid target sequence comprising said SNP, andb) a pair of interactive labels, at least one label being signal-generating and the labels being effectively positioned on the oligonucleotide to quench generation of a detectable signal, said labels being separated by a nuclease susceptible cleavage site,c) wherein said probe is blocked at its 3′-end terminus to prohibit incorporation of said probe into a primer extension product, andwherein said probe comprises one or more mismatches to the target sequence.2. The hydrolysis probe according to claim 1 , wherein said one or more mismatches to the target sequence are C-A claim 1 , C-T or A-A mismatches.3. The hydrolysis probe according to claim 1 , wherein said oligonucleotide sequence of said probe (probe sequence) comprises two adjacent portions claim 1 , said portions being of equal length or differing by one nucleotide in length claim 1 , whereina) a base complementary to said SNP is located within a first portion of the probe sequence from a 5′-end, andb) said one or more mismatches are located within a second portion of the probe,orc) the base complementary to said SNP is located within the second portion of the probe, andd) said one or more mismatches are located within the first portion of the probe.4. The hydrolysis probe according to claim 3 , whereina) the base complementary to said SNP is located within a central part of said first portion, wherein said central part consists essentially of a sequence comprising half of the nucleotides of the first portion, andb) said one or ...

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Номер записи: 39
21-05-2020 дата публикации

Enrichment of targeted genomic regions for multiplexed parallel analysis

Номер: US20200157602A1
Автор: Acilleas ACHILLEOS, Elena KYPRI, George KOUMBARIS, Kyriakos TSANGARAS, Marios loannides, Petros MINA, Philippos PATSALIS
Принадлежит: Nipd Genetics Public Co Ltd

The invention provides improved methods for enriching targeted genomic regions of interest to be analyzed by multiplexed parallel sequencing. The methods of the invention utilize a pool of TArget Capture Sequences (TACS), wherein the pool comprises a plurality of TACS families, each member of a family binding to the same target sequence but with different start and/or stop positions on the sequence (i.e., staggered binding of the family members to the target sequence) to thereby enrich for target sequences of interest, followed by massive parallel sequencing and statistical analysis of the enriched population. The methods of the invention can be used for a variety of clinical purposes, including non-invasive prenatal testing for chromosomal abnormalities, for example using a maternal blood sample or a sample of fetal cells, assessment of maternal and paternal carrier status for genetic disorders and detection of tumor biomarkers (e.g., liquid biopsy). Kits for carrying out the methods of the invention are also provided.

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Номер записи: 40
06-06-2019 дата публикации

ONE-STEP REVERSE TRANSCRIPTION TEMPLATE-SWITCHING PCR

Номер: US20190169679A1
Автор: Shiroguchi Katsuyuki
Принадлежит:

The present invention provides technology for carrying out one-step reverse transcription template-switching PCR more quickly, more easily, and with high specificity. The present invention provides a nucleic acid amplification method for amplifying at least a partial region of RNA using a modified oligonucleotide primer, said nucleic acid amplification method being characterized by the following: a nucleic acid amplification reaction comprises a reverse transcription step a) in which RNA is used as a template, a template switching step b) in which a template-switching oligonucleotide is added to cDNA synthesized in step a), and a DNA amplification step c) in which DNA amplification is carried out by PCR in which the template-switch cDNA synthesized in step b) is used as a template; the steps a) to c) are performed in a single stage in the same reaction system; as a result of being modified, some or all of the primer function of the modified oligonucleotide primer is blocked in the reverse transcription step a); and blocking of the primer function is cancelled in the DNA amplification step c). 1. A method of amplifying at least a part of a region of a target RNA , the method comprising the steps of:a) mixing the target RNA, a reagent required for reverse transcription, a reagent required for template switching, and a reagent required for a polymerase chain reaction and subjecting the mixture to a condition under which reverse transcription occurs to provide a cDNA comprising a nucleic acid sequence corresponding to the target RNA and a template switching oligonucleotide; and 'wherein the reagent required for a polymerase chain reaction comprises a modified oligonucleotide primer designed to have a primer function that is partially or completely blocked in step a) and designed to have blocking of the primer function cleared in step b).', 'b) subjecting the cDNA obtained in step a) to a condition under which a polymerase chain reaction occurs to amplify at least a part ...

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Номер записи: 41
28-05-2020 дата публикации

DIGITAL AMPLIFICATION WITH PRIMERS OF LIMITED NUCLEOTIDE COMPOSITION

Номер: US20200165668A1
Автор: Chen Xin, WANG Rong, WANG Youxiang, Yang Zhijie, Zhao Yu
Принадлежит: ATILA BIOSYSTEMS INCORPORATED

The invention provides methods of digital amplification using primers of limited nucleotide composition. Limited nucleotide composition means that the primers are underrepresented in at least one nucleotide type. Such primers have much reduced capacity to prime from each other or to extend initiated by mispriming from other than at their intended primer binding sites in a target nucleic acid. 1. A method of performing a digital amplification on a target nucleic acid in a sample comprising:partitioning a sample comprising a target nucleic acid into aliquots,conducting amplification reactions in the aliquots, wherein an amplified segment of the target nucleic acid is formed by extension of a pair of forward and reverse primers on the target nucleic acid if the target nucleic acid is present in the aliquot; whereinthe primers are underrepresented in one or more of the four standard nucleotide types, the underrepresented nucleotide type(s) being the same in the primers;and detecting an amplified segment, if present, in each aliquot.2. The method of claim 1 , wherein the copy number of the target nucleic acid is determined by the number of aliquots containing or lacking the amplified segment claim 1 , optionally following a Poisson distribution.3. The method of claim any preceding claim claim 1 , wherein the sample comprises a plurality of target nucleic acids claim 1 , and the amplification is performed with a plurality of forward and reverse primer pairs corresponding to the respective targets claim 1 , each of which is underrepresented in the same standard nucleotide type(s) claim 1 , optionally wherein the pluralities are each at least 2 claim 1 , 3 claim 1 , 4 claim 1 , 5 claim 1 , 6 claim 1 , 7 claim 1 , 8 claim 1 , 9 or 10.4. The method of claim 3 , wherein each of the primer pairs is underrepresented in the same one and only one standard nucleotide type.5. The method of claim 4 , wherein the target nucleic acids are from different chromosomes or the same ...

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Номер записи: 42
28-06-2018 дата публикации

Assays for Single Molecule Detection and Use Thereof

Номер: US20180179586A1
Автор: Collins Patrick James, Fehr Adrian Nielsen, Herschleb Jill Lyndon, Jones Hywel Bowden
Принадлежит: Singular Bio, Inc.

The invention relates to methods of detecting a genetic variation in a genetic sample from a subject using labeled probes and counting the number of labels in the probes. 183-. (canceled)85. The method according to claim 84 , wherein the first probe set further comprises a first gap probe claim 84 , and the second probe set further comprises a second gap probe.86. The method according to claim 85 , wherein each of the first and second gap probes comprises an artificial nucleotide sequence.87. The method according to claim 85 , whereinin the first probe set, the first gap probe, the first labeling probe, and the first tagging probe are adjacent to each other, andin the second probe set, the second gap probe, the second labeling probe, and the second tagging probe are adjacent to each other.88. The method according to claim 85 , further comprising claim 85 , after the hybridizing before the amplifying claim 85 , (i) ligating the first gap probe claim 85 , the first labeling probe claim 85 , and the first tagging probe in the first probe set claim 85 , and (ii) ligating the second gap probe claim 85 , the second labeling probe claim 85 , and the second tagging probe in the second probe set.89. The method according to claim 84 , further ligating probes in each of the first and second probe sets after the hybridizing before the amplifying.90. The method according to claim 84 , wherein the amplifying comprises linear amplification.91. The method according to claim 84 , further immobilizing probe products to a well.92. The method according to claim 84 , wherein each of the first and second labeling probes and the first and second tagging probes independently comprises a naturally-occurring nucleotide sequence.93. The method according to claim 84 , wherein the amplifying and labeling comprise amplifying and labeling the first and second probe products with first and second labels by using labelled first and second primers.94. The method according to claim 84 , wherein the ...

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Номер записи: 43
04-06-2020 дата публикации

METHODS AND PROBES FOR PERFORMING PCR WITH MELT ANALYSIS FOR INCREASED MULTIPLEXING

Номер: US20200172964A1
Автор: WHITMAN Doug
Принадлежит: Luminex Corporation

Methods and compositions for the detection and quantification of nucleic acids are provided. In certain embodiments, methods involve the use of cleavable probes capable of forming double-stranded structures, such as hairpin structures, which probes can be distinguished from one another on the basis of reporter signal, melt properties, or both. 1. A probe for detecting the presence of a target nucleic acid , the probe comprising , from 5′ to 3′:(a) a target-specific region comprising a first nucleotide sequence of from 5 to 36 contiguous nucleotides complementary to a target nucleotide sequence, a quencher, and a fluorophore, wherein the quencher is coupled to the target-specific region at a first location that is 5′ relative to the fluorophore, the fluorophore is coupled to the target-specific region at a second location that is 3′ relative to the quencher, and the first location and the second location are separated by at least 4 nucleotides of the first nucleotide sequence;(b) a polymerase extension-blocking moiety;(c) a melt-signature region comprising a second nucleotide sequence that is not complementary to the target nucleotide sequence and includes at least one non-naturally occurring nucleotide;(d) a loop region; and(e) a melt-signature complementary region comprising a third nucleotide sequence that is complementary to a portion of the second nucleotide sequence.2. The probe of claim 1 , wherein the first nucleotide sequence comprises from 20 to 36 nucleotides.3. The probe of claim 1 , wherein the quencher is coupled to the 5′-most nucleotide of the first nucleotide sequence.4. The probe of claim 1 , wherein the first location and the second location are separated by at least 10 nucleotides of the first nucleotide sequence.5. The probe of claim 1 , wherein the polymerase extension-blocking moiety comprises a carbon spacer or an inverted nucleotide sequence.6. The probe of claim 1 , wherein the at least one non-naturally occurring nucleotide is an isoC ...

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Номер записи: 44
18-06-2020 дата публикации

Processes and systems for preparation of nucleic acid sequencing libraries and libraries prepared using same

Номер: US20200190551A1
Автор: Benjamin Hindson, Indira Wu, Keith Bjornson, Paul Hardenbol, Paul William Wyatt, Pranav Patel, Zahra Kamila Belhocine
Принадлежит: 10X Genomics Inc

This disclosure provides methods for preparing a sequencing library including the steps of providing a template nucleic acid sequence, dNTPs, dUTP, a primer, a polymerase, a dUTP excising enzyme, and a plurality of beads including oligonucleotide adapter sequence segments; amplifying the template nucleic acid with the polymerase, dNTPs, dUTP and random hexamer to provide a complementary nucleic acid sequence including occasional dUTPs; and excising the incorporated dUTPs with the dUTP excising enzyme to provide nicks in the complementary nucleic acid sequence to provide a sequencing library.

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Номер записи: 45
19-07-2018 дата публикации

Massive parallel method for decoding dna and rna

Номер: US20180201642A1
Автор: Jingyue Ju, John Robert Edwards, Yasuhiro Itagaki, Zengmin Li
Принадлежит: Columbia University of New York

This invention provides methods for attaching a nucleic acid to a solid surface and for sequencing nucleic acid by detecting the identity of each nucleotide analogue after the nucleotide analogue is incorporated into a growing strand of DNA in a polymerase reaction. The invention also provides nucleotide analogues which comprise unique labels attached to the nucleotide analogue through a cleavable linker, and a cleavable chemical group to cap the —OH group at the 3′-position of the deoxyribose.

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Номер записи: 46
04-07-2019 дата публикации

METHODS FOR LABELLING NUCLEIC ACIDS

Номер: US20190203283A1
Автор: FORSHEW Tim, LENSING Stefanie Viola, WOODHOUSE Samuel
Принадлежит:

The invention relates to methods for labelling individual nucleic acid molecules present in a sample, comprising contacting the nucleic acid molecules with an adaptor or mixture of adaptors, wherein the adaptor or adaptors comprise one or more universal nucleotide bases and a ligation moiety at their 3′ end, and ligating an adaptor to the nucleic acid of interest, wherein the adaptor is ligated to the nucleic acid molecules at the 3′ end of the adaptor. A random tag is then generated in situ by conducting an extension reaction over the ligated adaptor. Methods of the invention may be used to detect genetic alterations or variants in any nucleic acid with high specificity and high sensitivity, including mutations in nucleic acids such as ctDNA, cfDNA, and in viral, microbiome and plant nucleic acids. Methods of the invention may also be used in detection and correction of errors introduced into nucleic acids during processing. 1. A double-stranded nucleic acid adaptor comprising two strands , wherein the first strand comprises one or more universal nucleotide bases and a ligation moiety at its 3′ end , and wherein the second strand comprises a ligation block at its 5′ end.2. The double-stranded nucleic acid adaptor of claim 1 , wherein the 3′ ligation moiety is selected from the group consisting of an overhang claim 1 , a blunt end claim 1 , and any other ligatable sequence.3. The double-stranded nucleic acid adaptor of claim 2 , wherein the 3′ ligation moiety is an overhang of one or more bases.4. The double-stranded nucleic acid adaptor of claim 3 , wherein the overhang is an overhang of one or more non-universal bases.5. The double-stranded nucleic acid adaptor of or claim 3 , wherein said non-universal bases are selected from the group consisting of A claim 3 , T claim 3 , C claim 3 , G and U (Uracil).6. The double-stranded nucleic acid adaptor of or claim 3 , wherein the overhang is a C or T overhang.7. The double-stranded nucleic acid adaptor of any preceding ...

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Номер записи: 47
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: 48
09-08-2018 дата публикации

Nucleic Acid Sample Preparation

Номер: US20180223332A1
Автор: BELL Neil Matthew, Ost Tobias William Barr
Принадлежит:

This invention relates to the preparation of nucleic acid samples for analysis. The invention may be particularly useful for single stranded samples. Embodiments of the invention involve the attachment of double stranded or hairpin oligonucleotides using template independent polymerase enzymes in the preparation of nucleic acid sequencing libraries. 124.-. (canceled)25. A method comprising: contacting a single stranded oligonucleotide with an enzyme , wherein said enzyme catalyzes an addition of a nucleotide to an end of said single stranded oligonucleotide in a template independent manner , wherein said nucleotide comprises a 5′-triphosphate.26. The method of claim 25 , wherein said enzyme comprises a terminal transferase (TdT).27. The method of claim 26 , wherein said terminal transferase comprises a terminal deoxynucleotidyl transferase (TdT) claim 26 , a polyadenylate polymerase (PAP) or a poly(U)polymerase (PUP).28. The method of claim 25 , wherein said nucleotide comprises a plurality of nucleotides.29. The method of claim 25 , wherein said single stranded oligonucleotide is produced by at least one of (i) a bisulfite treatment; (ii) a chemical or enzymatic cleavage; or (iii) use of an enzyme that is a restriction endonuclease to form said single stranded oligonucleotide.30. The method of claim 25 , wherein a second oligonucleotide strand comprises said nucleotide.31. The method of claim 30 , further comprising associating at least a portion of said single stranded oligonucleotide with at least a portion of said second oligonucleotide strand to form a double-stranded oligonucleotide.32. The method of claim 31 , wherein said second oligonucleotide strand comprises a hairpin.33. The method of claim 32 , wherein said hairpin comprises an extendable 3′-end.34. The method of claim 31 , wherein said second oligonucleotide strand is associated with a solid support.35. The method of claim 34 , wherein said second oligonucleotide strand comprises a moiety for ...

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Номер записи: 49
19-08-2021 дата публикации

ELIMINATION OF PRIMER-PRIMER INTERACTIONS DURING PRIMER EXTENSION

Номер: US20210254146A1
Автор: Godwin Brian
Принадлежит:

The invention comprises a method of amplifying nucleic acids by primer extension with reduced formation of primer-primer byproducts. 13-. (canceled)4. A method of amplifying a target nucleic acid in a sample with reduced primer-primer interaction comprising:(a) a primer extension step, wherein the sample is contacted with a nucleic acid polymerase and a first primer which is a single-stranded oligonucleotide consisting of two arms complementary to a target sequence separated by a linker sequence non-complementary to the target sequence and comprising at least one modified nucleotide that stalls nucleotide incorporation by the polymerase, to fill the gap between the ends of the primer; and(b) an exponential amplification step wherein the sample is contacted with a second primer complementary to the primer extension product and a polymerase tolerant of the modified nucleotide.5. (canceled)6. The method of claim 4 , wherein the first primer comprises binding sites for universal amplification primers and the second primer is a universal primer.79-. (canceled)10. The method of claim 4 , wherein the modified nucleotide is uracil.1113-. (canceled)14. A reaction mixture for synthesizing nucleic acid strands according to claim 4 , comprising a target nucleic acid claim 4 , a nucleic acid polymerase and a first primer which is a single-stranded oligonucleotide consisting of two arms complementary to a target sequence separated by a linker sequence non-complementary to the target sequence and comprising at least one modified nucleotide that stalls nucleotide incorporation by the polymerase.15. (canceled)16. The method of claim 4 , further comprising a step of joining the ends of the primer by ligation.17. The method of claim 16 , further comprising prior to step (b) claim 16 , contacting the sample with an exonuclease.18. The method of claim 4 , wherein the first primer comprises a unique molecular barcode.19. The method of claim 4 , wherein the second primer comprises a ...

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Номер записи: 50
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: 51
30-08-2018 дата публикации

PROBES FOR IMPROVED MELT DISCRIMINATION AND MULTIPLEXING IN NUCLEIC ACID ASSAYS

Номер: US20180245139A1
Автор: Arab Nicolas, Johnson Scott C., WHITMAN Doug
Принадлежит: Luminex Corporation

Methods and compositions for the detection and quantification of nucleic acids are provided. In certain embodiments, methods involve the use of cleavable probes that comprise a ribonucleotide position that is susceptible to endoribonuclease (e.g., RNase H) cleavage in the presence of target nucleic acid molecules. Probes of the embodiments may also comprise non-natural nucleotide linked to a reporter and/or quenching moiety. 1. A composition comprising a first cleavable probe , said probe comprising , from 5′ to 3′ , (i) a first sequence region comprising at least a one non-natural nucleotide labeled with a first member of a reporter-quencher pair; (ii) a second sequence region; (iii) a sequence that is the reverse complement of the second sequence region; and (iv) a sequence comprising one or more ribonucleotide base(s) that is complimentary to a first region on a first strand of a target nucleic acid.2. The composition of claim 1 , further comprising a second cleavable probe claim 1 , said second cleavable probe comprising claim 1 , from 5′ to 3′ claim 1 , (i) a first sequence region comprising at least a one non-natural nucleotide labeled with a first member of a reporter-quencher pair; (ii) a second sequence region; (iii) a sequence that is the reverse complement of the second sequence region; and (iv) a sequence comprising one or more ribonucleotide base(s) that is complimentary to a first region on a first strand of a second target nucleic acid.3. The composition of claim 2 , wherein the first and second probes comprise distinguishable reporters or form hairpin probes having distinguishable melt points.4. The composition of claim 2 , comprising at least four probes claim 2 , each specific for a different target nucleic acid and each capable of forming a hairpin probe with a distinguishable melt point.5. The composition of claim 1 , further comprising a reporter-labeled or quencher-labeled non-natural nucleotide.6. The composition of claims 1 , further ...

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Номер записи: 52
31-08-2017 дата публикации

Polymerase Chain Reaction Primers and Probes for Mycobacterium Tuberculosis

Номер: US20170247747A1
Автор: ALLAND David, Chakravorty Soumitesh
Принадлежит:

The present invention relates to novel primers and sloppy molecular beacon and molecular beacon probes for amplifying segments from different genes in for identifying the presence of DNA and/or resistance to anti-tuberculosis drugs. 1M. tuberculosis. An oligonucleotide set for amplifying a portion of a region selected from the group consisting of rpoB gene , gyrA gene , gyrB gene , inhA promoter , rrs gene , eis promoter , embB gene , katG gene , dosR gene , IS6110 gene , IS1081 gene , comprising , a pair of forward and reverse primers specific for said portion , wherein each primer has a sequence that is substantially identical to an oligonucleotide sequence selected from those listed in Tables 1A and 1B.2. The oligonucleotide set of claim 1 , wherein the sequence is identical to said oligonucleotide sequence selected from those listed in Tables 1A and 1B.3. An isolated nucleic acid comprising a sequence that is substantially identical to one selected from those listed in Table 2.4. The nucleic acid of claim 3 , comprising the sequence of one selected from those listed in Table 2.5. The nucleic acid of or claim 3 , wherein the nucleic acid is labeled.6. The nucleic acid of claim 5 , wherein the nucleic acid is labeled with a fluorophore and a quencher at its two ends respectively.7. The nucleic acid of claim 6 , wherein the fluorophore is fluorescein claim 6 , cyanine 5 claim 6 , or TexasRed or TAMRA.8. The nucleic acid of claim 6 , wherein the quencher is BHQ1 claim 6 , BHQ2 claim 6 , or DABCYL.9. A kit comprising an oligonucleotide set or nucleic acid of any one of -.10. The kit of further comprises a DNA polymerase claim 9 , extension nucleotides claim 9 , and a buffer.12. The method of claim 11 , wherein the detecting step is conducted by sequencing.14. The method of claim 13 , wherein the test Tm value claim 13 , if lower than the pre-determined reference Tm value claim 13 , indicates the presence of the mutation.15. The method of any one of - claim 13 , ...

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Номер записи: 53
08-08-2019 дата публикации

METHODS FOR MULTIPLEX PCR

Номер: US20190241935A1
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-based enrichment of a target substrate comprising the steps of:(i) introducing a plurality of polymerase chain reaction components into a sample comprising a nucleic acid substrate to provide a PCR reaction mixture, wherein the plurality of polymerase chain reaction components comprise a plurality of different target-specific primer pairs for amplifying a plurality of different loci of the nucleic acid substrate, a universal primer, a DNA polymerase, and dNTPs, wherein each of the plurality of different target-specific primer pairs comprise a forward primer and a reverse primer, wherein the forward primer and the reverse primer comprise a 3′ complementary sequence that is complementary to a first sequence of the nucleic acid substrate and a second sequence of the nucleic acid substrate, respectively, wherein the first sequence and second sequence is different for each of the plurality of different target-specific primer pairs, and wherein the forward primer and the reverse primer of each of the plurality of different target-specific primer pairs further comprise a 5′ terminal sequence that is not complementary to the nucleic acid substrate, wherein the 5′ terminal sequence comprises a universal adaptor sequence, wherein the universal primer comprises a modified universal adaptor sequence, wherein the modified universal adaptor sequence possesses a modified base, wherein the modified base targets cleavage of the universal primer by an endonuclease, wherein the modified universal adaptor sequence and the universal adaptor sequence are complementary to a common sequence, and wherein the universal primer is at a final concentration in the PCR reaction mixture that is in excess of the final concentration of each of the plurality of different target-specific primer pairs;(ii) ...

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Номер записи: 54
15-09-2016 дата публикации

Methods, compositions and kits for small rna capture, detection and quantification

Номер: US20160265031A1
Автор: Chunmei Liu, Linda Wong, Shoulian Dong
Принадлежит: Life Technologies Corp

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.

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Номер записи: 55
06-08-2020 дата публикации

Unbiased detection of nucleic acid modifications

Номер: US20200248229A1
Автор: David Arthur Scott, Feng Zhang, Winston Xia YAN
Принадлежит: Broad Institute Inc, Massachusetts Institute of Technology

Provided herein are methods of detecting a nucleic acid modification, methods for detecting off-target activity of a targeted nuclease specific for a selected target sequence, methods for determining cleavage efficiency of a targeted nuclease specific for a selected target sequence, methods for selecting a guide RNA from a plurality of guide RNAs specific for a selected target sequence, methods for enrichment of one or more nucleic acid molecules wherein a nucleic acid modification is made and kits of parts for use in such methods.

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Номер записи: 56
28-09-2017 дата публикации

METHODS AND SOLUTIONS FOR INHIBITING UNDESIRED CLEAVING OF LABELS

Номер: US20170275671A1
Автор: Guggenheim Evan, Meyyappan Visalakshi, Olejnik Jerzy
Принадлежит:

The invention provides methods and compositions, including, without limitation, algorithms, computer readable media, computer programs, apparatus, and systems for determining the identity of nucleic acids in nucleotide sequences using, for example, data obtained from sequencing by synthesis methods. The methods of the invention include correcting one or more phenomena that are encountered during nucleotide sequencing, such as using sequencing by synthesis methods. These phenomena include, without limitation, sequence lead, sequence lag, spectral crosstalk, and noise resulting from variations in illumination and/or filter responses. 115-. (canceled)16. A method of incorporating labeled nucleotides into nucleic acid , comprising: a) providing a plurality of nucleic acid template molecules , a polymerase , a cleaving agent , a cleaving agent scavenger , and a plurality of nucleotide analogues wherein each nucleotide analogue is labeled with a unique label attached through a cleavable disulfide linker and contains a removable chemical moiety capping the 3′-OH group , wherein said removable chemical moiety comprises a disulfide bond; b) incorporating a first nucleotide analogue with said polymerase; c) detecting the label of the incorporated nucleotide analogue; d) removing the unique label and the chemical moiety of the incorporated nucleotide analogue capping the 3′-OH group with said cleaving agent; and e) introducing said cleaving agent scavenger.17. The method of claim 16 , wherein said cleaving agent is a phosphine.18. The method of claim 17 , wherein said phosphine is Tris(2-carboxy-ethyl)phosphine.19. The method of claim 16 , wherein said cleaving agent scavenger does not contain a nucleic acid base.20. The method of claim 16 , further comprising claim 16 , prior to step b) hybridizing a primer to said plurality of nucleic acid template molecules claim 16 , such that said first nucleotide analogue is incorporated into said primer at step b).21. A method of ...

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Номер записи: 57
28-09-2017 дата публикации

METHODS FOR ANALYZING NUCLEIC ACID

Номер: US20170275676A1
Автор: Umbarger Mark
Принадлежит:

The invention generally relates to methods for analyzing nucleic acids. In certain aspects, methods of the invention involve obtaining a sample including a nucleic acid template. A plurality of molecular inversion probes are tiled across a portion of the template. The probes are designed such that immediately adjacent probes hybridize to opposite strands of the nucleic acid template and probes on the same strand hybridize to the template in an overlapping manner. A region between targeting arms of a plurality of the molecular inversion probes is filled-in with nucleotides, and the filled-in region of a plurality of the probes is analyzed to obtain sequence information about the nucleic acid template. 1. A method for analyzing a nucleic acid template , the method comprising:obtaining a sample comprising a nucleic acid template;tiling a plurality of molecular inversion probes across a portion of the template, wherein immediately adjacent probes hybridize to opposite strands of the nucleic acid template and probes on the same strand hybridize to the template in an overlapping manner;filling-in a region between targeting arms of a plurality of the molecular inversion probes with nucleotides; andanalyzing the filled-in region of a plurality of the probes to obtain sequence information about the nucleic acid template.2. The method according to claim 1 , wherein analyzing is by sequencing.3. The method according to claim 2 , wherein prior to sequencing claim 2 , the method further involves amplifying the filled-in region of the plurality of the probes.4. The method according to claim 2 , wherein sequencing is sequencing-by-synthesis.5. The method according to claim 4 , wherein sequencing-by-synthesis is single molecule sequencing-by-synthesis.6. The method according to claim 2 , wherein sequencing information comprises a mutation in the nucleic acid sequence.7. The method according to claim 6 , wherein the mutation is selected from the group consisting of: a single ...

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Номер записи: 58
27-08-2020 дата публикации

DETECTION OF TARGET NUCLEIC ACID SEQUENCES BY PTO CLEAVAGE AND EXTENSION-DEPENDENT EXTENSION ASSAY

Номер: US20200270679A1
Автор: KIM Dae Young, LEE Han Bit, Lee Young Jo
Принадлежит:

The present invention relates to the detection of a target nucleic acid sequence by a PTOCE-E (PTO Cleavage and Extension-dependent Extension) assay. The PTOCE-E assay of the present invention can reduce the non-target signal and increase the target signal as compared with the conventional PTOCE and PCE-SH methods, thereby enabling more accurate detection of the target nucleic acid sequence. 1. A method for detecting a target nucleic acid sequence from a DNA or a mixture of nucleic acids by a PTOCE-E (PTO Cleavage and Extension-dependent Extension) assay , comprising:(a) hybridizing the target nucleic acid sequence with an upstream oligonucleotide and a PTO (Probing and Tagging Oligonucleotide); wherein the upstream oligonucleotide comprises a hybridizing nucleotide sequence to the target nucleic acid sequence; wherein the PTO comprises (i) a 3′-targeting portion comprising a hybridizing nucleotide sequence to the target nucleic acid sequence and (ii) a 5′-tagging portion comprising a non-hybridizing nucleotide sequence to the target nucleic acid sequence; wherein the 3′-targeting portion of the PTO is hybridized with the target nucleic acid sequence and the 5′-tagging portion of the PTO is not hybridized with the target nucleic acid sequence; the upstream oligonucleotide is located upstream of the PTO;(b) contacting the resultant of the step (a) to an enzyme having a 5′ nuclease activity under conditions for cleavage of the PTO; wherein the upstream oligonucleotide or its extended strand induces cleavage of the PTO by the enzyme having the 5′ nuclease activity to release a PTO fragment comprising the 5′-tagging portion or a part of the 5′-tagging portion of the PTO;(c) hybridizing the PTO fragment with a first CTO (Capturing and Templating Oligonucleotide); wherein the first CTO comprises in a 3′ to 5′ direction (i) a capturing portion comprising a hybridizing nucleotide sequence to the PTO fragment and (ii) a templating portion comprising a non-hybridizing ...

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Номер записи: 59
12-09-2019 дата публикации

Reusable initiators for synthesizing nucleic acids

Номер: US20190275492A1
Автор: J. William Efcavitch, Matthew T. Holden
Принадлежит: Molecular Assemblies Inc

The invention provides improved methods for synthesizing polynucleotides, such as DNA and RNA, using renewable initiators coupled to a solid support. Using the methods of the invention, specific sequences of polynucleotides can be synthesized de novo, base by base, in an aqueous environment, without the use of a nucleic acid template.

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Номер записи: 60
03-09-2020 дата публикации

REVERSIBLE THERMODYNAMIC TRAP (THERMOTRAP) IN AMPLIFICATION OF NUCLEIC ACIDS

Номер: US20200277665A1
Автор: LIPINSKI Kamil Andrzej, MCKEOWN Brian James, PAILLIER Francois Joel
Принадлежит: DNAe Diagnostics Ltd

Described is a method and kit for efficiently amplifying and detecting certain nucleic acid sequences from a population. The invention is intended to provide increased assay specificity by minimizing unwanted interactions between priming oligonucleotides (primers). 1. A method for the amplification of nucleic acid sequences comprising: i. a nucleic acid sample;', 'ii. a first nucleic acid amplification primer having a 3′ region which is complementary to a first target region of the sample and a 5′ region which is not complementary to a region of the sample and has a sequence which does not occur in nature; wherein the 5′ region is either self-complementary such that the 5′ ends of a first strand of the first nucleic acid amplification primer are capable of hybridising to the 5′ ends of a second strand of the first nucleic acid amplification primer, or the 5′ region of the first nucleic acid amplification primer is complementary to the 5′ region of a second nucleic acid amplification primer, and wherein the 5′ non complementary region of the first nucleic acid amplification primer is attached to the primer via a spacer unit which can not be copied by the polymerase;', 'iii. a nucleic acid polymerase;', 'iv. nucleotide triphosphate monomers; and optionally', 'v. a second nucleic acid amplification primer having a 3′ region which is complementary to an extension product of the first primer and a 5′ region which is complementary to the 5′ region of the first primer and is not complementary to a region of the sample;, 'a. taking a reaction mixture comprisingb. hybridising the first primer to the sample,c. extending the first primer using the nucleic acid polymerase and nucleotide triphosphate monomers; andd. repeating steps b and c, thereby amplifying target sequences where the first nucleic acid amplification primer hybridises to the sample.2. The method according to wherein the amplification is carried out with only a first amplification primer.3. The method according ...

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Номер записи: 61
11-10-2018 дата публикации

QUANTITATIVE ASSESSMENT FOR CAP EFFICIENCY OF MESSENGER RNA

Номер: US20180291425A1
Автор: DeRosa Frank, Dias Anusha, Heartlein Michael
Принадлежит:

The present invention provides, among other things, methods of quantitating mRNA capping efficiency, particularly for mRNA synthesized in vitro. In some embodiments, the methods comprise chromatographic methods of quantifying capping efficiency and methylation status of the caps. 1. A method of quantifying mRNA capping efficiency , the method comprising:(1) providing an mRNA sample comprising capped mRNA and uncapped mRNA;(2) contacting the mRNA sample with a DNA oligonucleotide complimentary to a sequence in the 5′ untranslated region of the mRNA adjacent to the cap or uncapped penultimate base of mRNA under conditions that permit the DNA oligonucleotide anneal to the sequence;(3) providing one or more nucleases that selectively degrade DNA/RNA hybrid and/or unannealed mRNA, resulting in capped and uncapped fragments;(4) separating the capped and uncapped fragments by chromatography; and(5) determining relative amount of the capped and uncapped fragments, thereby quantifying mRNA capping efficiency.3. The method of claim 2 , wherein the nucleobase is guanine.5. The method of claim 1 , wherein step (4) further separates methylated and unmethylated capped RNA.6. (canceled)7. The method of claim 5 , wherein the method further comprises a step of quantitatively determining methylation percentage of the capped RNA.8. The method of claim 1 , wherein the DNA oligonucleotide is 10-80 nucleotides in length.9. The method of claim 1 , wherein the DNA oligonucleotide is flanked on both sides by one or more RNA nucleotides.10. The method of claim 1 , wherein the sequence in the 5′ untranslated region of the mRNA is within 1 or 2 bases from the cap or uncapped penultimate base of mRNA.11. (canceled)12. The method of claim 1 , wherein the one or more nucleases comprise a nuclease that generates blunt-ended capped and uncapped fragments.13. The method of claim 1 , wherein the nuclease is nuclease S1 claim 1 , RNAse H claim 1 , or another 5′ exonuclease.14. The method of claim 1 , ...

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Номер записи: 62
19-09-2019 дата публикации

THERAPEUTIC OLIGONUCLEOTIDES CAPTURE AND DETECTION

Номер: US20190284621A1
Автор: Jensen Mads Aaboe, JOENSON Lars, Kielpinski Lukasz, LINDOW Morten, VIKESAA Jonas
Принадлежит:

The present invention relates to the detection of therapeutic modified oligonucleotides in biological samples and an adaptor oligonucleotide (capture probe) which enables a quantitative PCR based detection method and the sequencing of modified oligonucleotides. The invention provides novel adaptor probes for use in detecting therapeutic oligonucleotides and for in vivo discovery of preferred therapeutic oligonucleotide sequences. 1. A capture probe oligonucleotide , for use in PCR or sequencing of a nucleoside modified oligonucleotide , comprising 5′-3′: a. at least 3 5′ contiguous nucleotides of predetermined sequence (region 1A), wherein the 5′ most nucleotide is a DNA nucleotide with a terminal 5′ phosphate group, and', 'b. optionally a region of degenerate or predetermined nucleotides, positioned 3′ of region 1A (region 1B), and', 'c. a 3′ region which comprises a universal primer binding site (region 1C); and, 'i) a first nucleotide segment comprising a. a contiguous sequence of nucleotides which are complementary to the predetermined sequence 1A of the first nucleotide segment (region 2A), and', 'b. a region of at least 2 nucleotides, wherein the 3′ most nucleotide is a terminal nucleotide with a blocked 3′ terminal group (region 2B); wherein the first and second regions are covalently linked via a non-hybridizing linker moiety., 'ii) a second nucleotide segment, comprising2. The capture probe oligonucleotide of claim 1 , wherein the non-hybridizing linker moiety is selected from the group consisting of an alkyl linker claim 1 , a polyethylene glycol linker claim 1 , a non nucleosidic carbohydrate linker claim 1 , a photocleavable linker (PC spacer) claim 1 , an alkyl disulfide linker claim 1 , a region of 1 claim 1 ,2-dideoxy ribose or abasic furan claim 1 , or a region of nucleosides which comprise non-hybridising base groups.3. The capture probe oligonucleotide of claim 1 , wherein region 1A comprises at least 2 or at least 3 contiguous DNA nucleotides.4. ...

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Номер записи: 63
25-10-2018 дата публикации

METHODS AND APPARATUS FOR SYNTHESIZING NUCLEIC ACIDS

Номер: US20180305746A1
Автор: Efcavitch J. William, Siddiqi Suhaib
Принадлежит:

The invention provides improved methods for synthesizing polynucleotides, such as DNA and RNA, using enzymes and specially designed nucleotide analogs. Using the methods of the invention, specific sequences of polynucleotides can be synthesized de novo, base by base, in an aqueous environment, without the use of a nucleic acid template. Because the nucleotide analogs have an unmodified 3′ OH, i.e., as found in “natural” deoxyribose and ribose molecules, the analogs result in natural polynucleotides suitable for incorporation into biological systems. 1. A method for non-template dependent oligonucleotide synthesis , the method comprising:exposing a nucleic acid strand to a terminal transferase enzyme capable of incorporating a single nucleotide and remaining bound to the strand and preventing further nucleotide incorporation until exposed to a releasing agent or releasing condition.2. The method of claim 1 , wherein said single nucleotide is a nucleotide analog.3. The method of claim 1 , wherein the terminal transferase enzyme is a modified terminal deoxynucleotidyl transferase (TdT) enzyme.4. The method of claim 3 , wherein the modification comprises a mutation allowing the covalent attachment of a nucleotide analog to the TdT enzyme.5. The method of claim 1 , wherein the releasing reagent comprises a salt buffer or a denaturant or a reducing agent or elevated pH.6. The method of claim 1 , wherein the releasing condition is a temperature increase or agitation.7. A nucleotidyl transferase enzyme modified to remain bound to a nucleic acid strand after incorporating a nucleotide into the nucleic acid strand and to prevent subsequent incorporation of nucleotide analogs until released by a releasing agent or releasing condition.8. The nucleotidyl transferase enzyme of claim 7 , wherein the nucleotidyl transferase enzyme incorporates the nucleotide analog into the nucleic acid strand in an absence of a nucleic acid template.9. The nucleotidyl transferase enzyme of claim 7 ...

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Номер записи: 64
10-11-2016 дата публикации

Compositions and methods for the construction of a random allelic series

Номер: US20160326517A1
Автор: Christine Gurnett, David Alvarado, Gabriel Haller, Matthew Dobbs
Принадлежит: Shriners Hospitals For Children

The present disclosure provides a method of making a systematic single point mutation in a target nucleic acid and a method of generating a mutational library comprising target nucleic acids with single point mutations. The mutational library comprises target nucleic acids with single point mutations distributed evenly throughout the target nucleic acid.

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Номер записи: 65
01-11-2018 дата публикации

Methods, Kits and Compositions Pertaining to the Suppression of the Detectable Probe Binding to Randomly Distributed Repeat Sequences Genomic Nucleic Acid

Номер: US20180312912A1
Автор: HYLDIG-NIELSEN Jens, NIELSEN Kirsten Vang, WILLIAMS Brett F.
Принадлежит:

This invention is directed to methods, kits, non-nucleotide probes as well as other compositions pertaining to the suppression of binding of detectable nucleic acid probes to undesired nucleotide sequences of genomic nucleic acid in assays designed to determine target genomic nucleic acid. 1. A non-nucleotide probe of at least sixteen nucleobase containing subunits in length having an aggregate nucleobase sequence that is at least eighty percent homologous to a sixteen nucleotide segment of randomly distributed repeat sequence of genomic nucleic acid.2. The non-nucleotide probe of claim 1 , wherein the segment of randomly distributed repeat sequence is a SINE or LINE claim 1 , selected from the group consisting of: Alu-repeat claim 1 , Kpn-repeat claim 1 , di-nucleotide repeat claim 1 , tri-nucleotide repeat claim 1 , tetra-nucleotide repeat claim 1 , penta-nucleotide repeat and hexa-nucleotide repeat.3. The non-nucleotide probe of claim 2 , wherein the segment of randomly distributed repeat sequence contains at least ten consecutive nucleobases that are at least eighty percent homologous to a unit repeat consensus Alu-repeat sequence selected from the group consisting of: Seq. Id. No. 1 and Seq. Id. No. 2.4. The non-nucleotide probe of claim 3 , wherein the ten consecutive nucleobases are at least ninety percent homologous to the identified consensus sequences.5. The non-nucleotide probe of claim 4 , wherein the ten consecutive nucleobases are exactly homologous to the identified consensus sequences.6. The non-nucleotide probe of claim 1 , wherein the non-nucleotide probe is a peptide nucleic acid probe.7. (canceled)8. The non-nucleotide probe of claim 6 , wherein the PNA subunits of the non-nucleotide probe consist of a naturally or non-naturally occurring nucleobase attached to an aza nitrogen of an N-[2-(aminoethyl)] glycine backbone through a methylene carbonyl linkage.917.-. (canceled)18. A mixture of two or more non-nucleotide probes wherein each probe ...

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Номер записи: 66
09-11-2017 дата публикации

METHODS AND COMPOSITIONS FOR NUCLEIC ACID AMPLIFICATION

Номер: US20170321273A1
Автор: Arnold Lyle J., BECKER Michael M., BRENTANO Steven T., CARLSON James D., LYAKHOV Dmitry, NELSON Norman C.
Принадлежит:

Compositions that are used in nucleic acid amplification in vitro are disclosed, which include a target specific universal (TSU) promoter primer or promoter provider oligonucleotide that includes a target specific (TS) sequence that hybridizes specifically to a target sequence that is amplified and a universal (U) sequence that is introduced into the sequence that is amplified, by using a primer for the universal sequence. Methods of nucleic acid amplification in vitro are disclosed that use one or more TSU oligonucleotides to attached a U sequence to a target nucleic acid in a target capture step and then use a primer for a U sequence in subsequent amplification steps performed in substantially isothermal conditions to make amplification products that contain a U sequence that indicates the presence of the target nucleic acid in a sample. 1. A target capture reaction mixture for separating a target nucleic acid from a sample , the reaction mixture comprising: [{'b': '1', '. a TSU promoter oligonucleotide comprising a 5′ promoter sequence, an internal first universal sequence (U1), and a 3′ first target specific sequence (TS1) that binds specifically to a target sequence contained in a target nucleic acid, wherein the TSU promoter oligonucleotide is a TSU promoter primer that has a 3′ terminus that is capable of being extended by a polymerase, or is a TSU promoter provider oligonucleotide that has a blocked 3′ terminus that is incapable of being extended by a polymerase,'}, 'ii. a TSU non-promoter primer oligonucleotide made up of a 5′ second universal sequence (U2) and a 3′ second target specific sequence (TS2) which is different from the TS1,', (A) a covalent linkage that is a polynucleotide linker sequence or a non-nucleotide a basic linker compound;', '(B) a hybridization complex between the 5′ promoter sequence and a sequence on the TSU non-promoter primer that is complementary to the 5′ promoter sequence; or', '(C) a hybridization complex that includes an S- ...

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Номер записи: 67
08-11-2018 дата публикации

MICROFLUIDIC DEVICE

Номер: US20180320216A1
Автор: ANDERSSON Dan I., BALTEKIN Özden, ELF Johan
Принадлежит:

A microfluidic device comprises a substrate transparent for imaging and having a plurality of spatially defined and separated cell channels having a dimension to accommodate cells in monolayer. A respective first end of the cell channels is in fluid connection with a flow input channel having a first end in fluid connection with a first fluid port and a second end in fluid connection with a second fluid port. A respective second end of the cell channels is in fluid connection with a first end of a respective wash channel having a second end in fluid connection with a flow output channel. The flow output channel is in fluid connection with a third fluid port. The wash channels have a dimension too small to accommodate the cells. 1. A microfluidic device comprising:a substrate having a first set of cell channels and a second set of cell channels;a respective first end of the cell channels of the first set is in fluid connection with a first flow input channel;a respective second end of the cell channels of the first set is in fluid connection with a first flow output channel;a respective first end of the cell channels of the second set is in fluid connection with a second flow input channel;a respective second end of the cell channels of the second set is in fluid connection with a second flow output channel;the cell channels of the first set and of the second set comprise a respective channel restriction in connection with the respective second end to prevent target cells entering the cell channels from reaching the first flow output channel or the second flow output channel.2. The microfluidic device according to claim 1 , whereina first end of the first flow input channel is in fluid connection with a first input port;a second end of the first flow input channel is in fluid connection with a common input port;a first end of the second flow input channel is in fluid connection with a second input port; anda second end of the second flow input channel is in fluid ...

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Номер записи: 68
24-11-2016 дата публикации

Methods for processing dna substrates

Номер: US20160340746A1
Автор: Julie LALIBERTE, Vladimir Makarov
Принадлежит: Swift Biosciences Inc

The present disclosure describes a method of adapter ligation to the ends of fragmented double-stranded DNA molecules.

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Номер записи: 69
31-10-2019 дата публикации

DNA MUTATION DETECTION EMPLOYING ENRICHMENT OF MUTANT POLYNUCLEOTIDE SEQUENCES AND MINIMALLY INVASIVE SAMPLING

Номер: US20190330692A1
Автор: Powell Michael J, Sha Michael Y, Zhan Ke, ZHANG AIGUO
Принадлежит:

The invention relates to a method for enriching a target polynucleotide sequence containing a genetic variation said method comprising: (a) providing two primers targeted to said target polynucleotide sequence; (b) providing a target specific xenonucleic acid clamp oligomer specific for a wildtype polynucleotide sequence; (c) generating multiple amplicons using PCR under specific temperature cycling conditions; and (d) detecting said amplicons. We introduce a novel molecule, Xenonucleic Acid (XNA) for the NGS library preparation. XNA is able to selectively suppress amplification of DNA with wild type alleles and amplify DNA containing mutant alleles. Mutants with low allelic frequency will be easily detectable without deep sequencing after enrichment by adding XNA in multiplex PCR. The 17 actionable mutants related to lung or colorectal cancer diseases at different variant allelic frequency (VAF)% were investigated. Clinical sensitivity is significantly improved with XNA in various types of samples.

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Номер записи: 70
22-10-2020 дата публикации

Methods Of Producing Nucleic Acids Using Oligonucleotides Modified By A Stimulus

Номер: US20200332341A1
Автор: TORI Kazuo
Принадлежит:

Provided are methods of producing product nucleic acids involving the use of oligonucleotides that are modified by the application of a stimulus. Aspects of such methods may include producing product nucleic acids using de-activatable oligonucleotides that are deactivated by a de-activating stimulus, as well as methods that may include producing product nucleic acids using activatable oligonucleotides that are activated by an activating stimulus and de-activatable oligonucleotides that are deactivated by a de-activating stimulus. Also provided are kits, compositions and devices that include de-activatable oligonucleotides or activatable and de-activatable oligonucleotides, e.g., for use in performing the methods as described herein. 1. A method comprising:elongating a de-activatable oligonucleotide via a template nucleic acid-mediated primer extension reaction to produce an extended nucleic acid; anddeactivating the de-activatable oligonucleotide.2. The method according to claim 1 , wherein deactivating the de-activatable oligonucleotide comprises applying a deactivating stimulus selected from the group consisting of: an enzyme claim 1 , a chemical agent and an electromagnetic stimulus.3. The method according to claim 2 , wherein the deactivating stimulus is an enzyme and the enzyme is ribonuclease H (RNase H).4. The method according claim 1 , comprising a template switching reaction comprising a template switching oligonucleotide (TSO) to produce an extended nucleic acid that comprises a sequence complementary to the TSO.5. The method according to claim 4 , wherein the TSO is de-activatable.6. The method according to claim 5 , wherein the de-activatable oligonucleotide and the de-activatable TSO are deactivated by the same stimulus.7. The method according claim 1 , wherein the de-activatable oligonucleotide claim 1 , the TSO claim 1 , or both comprise an adapter sequence claim 1 , a barcode sequence claim 1 , or a combination thereof.8. The method according claim 1 ...

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Номер записи: 71
13-12-2018 дата публикации

METHODS, COMPOSITIONS, AND KITS FOR DETECTING ALLELIC VARIANTS

Номер: US20180355420A1
Автор: Chen Caifu, Tan Ruoying
Принадлежит:

In some embodiments, the present inventions relates generally to compositions, methods and kits for use in discriminating sequence variation between different alleles. More specifically, in some embodiments, the present invention provides for compositions, methods and kits for quantitating rare (e.g., mutant) allelic variants, such as SNPs, or nucleotide (NT) insertions or deletions, in samples comprising abundant (e.g., wild type) allelic variants with high specificity and selectivity. In particular, in some embodiments, the invention relates to a highly selective method for mutation detection referred to as competitive allele-specific TaqMan PCR (“cast-PCR”). 1. A method for detecting a first allelic variant of a target sequence in a nucleic acid sample suspected of comprising at least a second allelic variant of the target sequence , comprising: i) the nucleic acid sample;', 'ii) a first allele-specific primer, wherein an allele-specific nucleotide portion of the first allele-specific primer is complementary to the first allelic variant of the target sequence;', 'iii) a first allele-specific blocker probe that is complementary to a region of the target sequence comprising the second allelic variant, wherein said region encompasses a position corresponding to the binding position of the allele-specific nucleotide portion of the first allele-specific primer, and wherein the first allele-specific blocker probe comprises a minor groove binder;', 'iv) a first locus-specific primer that is complementary to a region of the target sequence that is 3′ from the first allelic variant and on the opposite strand; and', 'v) a first detector probe;, 'a) forming a first reaction mixture by combiningb) carrying out an amplification reaction on the first reaction mixture using the first locus-specific primer and the first allele-specific primer to form a first amplicon; andc) detecting the first amplicon by detecting a change in a detectable property of the first detector probe, ...

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Номер записи: 72
13-12-2018 дата публикации

APPARATUS FOR SINGLE MOLECULAR SEQUENCING AND METHOD OF SEQUENCING NUCLEIC ACID MOLECULES

Номер: US20180355422A1
Автор: Chen Bor-Huah, Chiou Chung-Fan, CIOU Jian-Hao, Pan Chao-Chi, Tsai Ching-Wei
Принадлежит: Personal Genomics Taiwan, Inc.

An apparatus suitable for single molecule sequencing. The apparatus includes at least one nanowell, a plurality of nucleic acid immobilization moieties, and a plurality of types of nucleic acid fragments. The nanowell has an observation zone. The nucleic acid immobilization moieties are disposed in or proximate to the observation zone. The nucleic acid fragments are immobilized to the nucleic acid immobilization moieties, respectively. At least one polymerase is disposed in the observation zone. A method of sequencing nucleic acid molecules using the above-mentioned apparatus is provided. 1. An apparatus suitable for single molecule sequencing comprising:at least one nanowell, wherein the at least one nanowell has an observation zone;a plurality of nucleic acid immobilization moieties, disposed in or proximate to the observation zone;a plurality of types of nucleic acid fragments, immobilized to the plurality of nucleic acid immobilization moieties respectively; andat least one polymerase, disposed in the observation zone.2. The apparatus as claimed in claim 1 , wherein a diameter of the observation zone ranges from 10 nm to 500 nm claim 1 , and a height of the observation zone is less than 200 nm.3. The apparatus as claimed in claim 1 , wherein the plurality of types of nucleic acid fragments are linear.4. The apparatus as claimed in claim 1 , wherein the plurality of types of nucleic acid fragments comprise 50 nucleotides to 200 nucleotides in length.5. The apparatus as claimed in claim 4 , wherein the at least one polymerase is one polymerase.6. The apparatus as claimed in claim 1 , wherein the plurality of types of nucleic acid fragments comprise more than 200 nucleotides in length.7. The apparatus as claimed in claim 6 , wherein the at least one polymerase comprises a plurality of polymerases claim 6 , the apparatus further comprises a plurality of polymerase immobilization moieties in the observation zone claim 6 , and the plurality of polymerases are ...

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Номер записи: 73
20-12-2018 дата публикации

COMPOSITIONS AND METHODS FOR QUANTIFYING A NUCLEIC ACID SEQUENCE IN A SAMPLE

Номер: US20180363046A1
Автор: JUDICE STEPHEN A., Shaffer Daniel
Принадлежит: ENVIROLOGIX INC.

The present invention features compositions and methods for quantifying detection of a target oligonucleotide in a sample in real time. These methods are compatible with target oligonucleotides amplified using a NEAR reaction. 1i) a first region, andii) a second region,wherein the first region comprises a nicking enzyme recognition sequence; wherein the second region comprises at least 9 nucleotides that specifically bind a complementary sequence on a target nucleic acid molecule; and wherein the second region comprises one or more 2′ modified nucleotides.. An isolated oligonucleotide comprising from 5′ to 3′, This application is a continuation of U.S. patent application Ser. No. 15/808,442, allowed, filed Nov. 9, 2017; which is a continuation of U.S. patent application Ser. No. 15/438,330, filed Feb. 21, 2017, abandoned; which is a continuation of U.S. patent application Ser. No. 14/989,687, filed Jan. 6, 2016, now U.S. Pat. No. 9,631,231, which is a continuation of U.S. patent application Ser. No. 14/789,545, filed Jul. 1, 2015, now U.S. Pat. No. 9,322,053; which is a continuation of U.S. patent application Ser. No. 14/342,766, filed Mar. 4, 2014, now U.S. Pat. No. 9,096,897, which is the U.S. national phase application, pursuant to 35 U.S.C. § 371, of PCT International Application Ser. No. PCT/US2013/035750, filed Apr. 9, 2013, designating the United States and published in English, which claims priority to and the benefit of U.S. Provisional Application No. 61/621,975, filed Apr. 9, 2012; each of the aforementioned applications are incorporated herein by reference in their entirety.The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Aug. 28, 2018, is named 167665.010633_SL.txt and is 26,102 bytes in size.Nucleic acid amplification technologies have provided a means of understanding complex biological processes, detection, ...

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Номер записи: 74
20-12-2018 дата публикации

METHODS OF LOWERING THE ERROR RATE OF MASSIVELY PARALLEL DNA SEQUENCING USING DUPLEX CONSENSUS SEQUENCING

Номер: US20180363051A1
Автор: LOEB Lawrence A., SALK Jesse, SCHMITT Michael
Принадлежит:

Next Generation DNA sequencing promises to revolutionize clinical medicine and basic research. However, while this technology has the capacity to generate hundreds of billions of nucleotides of DNA sequence in a single experiment, the error rate of approximately 1% results in hundreds of millions of sequencing mistakes. These scattered errors can be tolerated in some applications but become extremely problematic when “deep sequencing” genetically heterogeneous mixtures, such as tumors or mixed microbial populations. To overcome limitations in sequencing accuracy, a method Duplex Consensus Sequencing (DCS) is provided. This approach greatly reduces errors by independently tagging and sequencing each of the two strands of a DNA duplex. As the two strands are complementary, true mutations are found at the same position in both strands. In contrast, PCR or sequencing errors will result in errors in only one strand. This method uniquely capitalizes on the redundant information stored in double-stranded DNA, thus overcoming technical limitations of prior methods utilizing data from only one of the two strands. 136-. (canceled)37. A method of analyzing a plurality of double-stranded nucleic acid fragments comprising: a double-stranded portion at the proximal end, and', 'a single-stranded portion at a distal end, wherein the single-stranded portion comprises a single-stranded tag substantially unique to the adapter;, 'a) attaching both ends of each of the plurality of double-stranded nucleic acid fragments to proximal ends of adapters selected from a pool of adapters, and wherein the pool of adapters comprises adapters havingb) amplifying both strands of the adapter-nucleic acid products to produce first amplicons and second amplicons, wherein the first amplicons are derived from a first strand of the double-stranded nucleic acid fragments and the second amplicons are derived from a second strand of the double-stranded nucleic acid fragments, and wherein each of the first ...

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Номер записи: 75
20-12-2018 дата публикации

METHODS OF LOWERING THE ERROR RATE OF MASSIVELY PARALLEL DNA SEQUENCING USING DUPLEX CONSENSUS SEQUENCING

Номер: US20180363052A1
Автор: LOEB Lawrence A., SALK Jesse, SCHMITT Michael
Принадлежит:

Next Generation DNA sequencing promises to revolutionize clinical medicine and basic research. However, while this technology has the capacity to generate hundreds of billions of nucleotides of DNA sequence in a single experiment, the error rate of approximately 1% results in hundreds of millions of sequencing mistakes. These scattered errors can be tolerated in some applications but become extremely problematic when “deep sequencing” genetically heterogeneous mixtures, such as tumors or mixed microbial populations. To overcome limitations in sequencing accuracy, a method Duplex Consensus Sequencing (DCS) is provided. This approach greatly reduces errors by independently tagging and sequencing each of the two strands of a DNA duplex. As the two strands are complementary, true mutations are found at the same position in both strands. In contrast, PCR or sequencing errors will result in errors in only one strand. This method uniquely capitalizes on the redundant information stored in double-stranded DNA, thus overcoming technical limitations of prior methods utilizing data from only one of the two strands. 136-. (canceled)37. A method for sequencing nucleic acid molecules from a sample using unique molecular identifiers (UMIs) , wherein each unique molecular identifier (UMI) is an oligonucleotide sequence that can be used to identify an individual molecule of a double-stranded DNA fragment in the sample , comprising:(a) applying adapters to both ends of double-stranded DNA fragments in the sample to obtain DNA-adapter products, wherein the adapters each comprise a double-stranded hybridized region, a single-stranded 5′ arm, a single-stranded 3′ arm, and an adapter-specific UMI on one strand or each strand of the adapter, the adapter-specific UMI being selected from a plurality of adapter-specific UMIs, and wherein each double-stranded DNA fragment in the sample comprises a fragment-specific UMI on one strand or each strand of the double-stranded DNA fragment;(b) ...

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Номер записи: 76
20-12-2018 дата публикации

METHODS OF LOWERING THE ERROR RATE OF MASSIVELY PARALLEL DNA SEQUENCING USING DUPLEX CONSENSUS SEQUENCING

Номер: US20180363053A1
Автор: LOEB Lawrence A., SALK Jesse, SCHMITT Michael
Принадлежит:

Next Generation DNA sequencing promises to revolutionize clinical medicine and basic research. However, while this technology has the capacity to generate hundreds of billions of nucleotides of DNA sequence in a single experiment, the error rate of approximately 1% results in hundreds of millions of sequencing mistakes. These scattered errors can be tolerated in some applications but become extremely problematic when “deep sequencing” genetically heterogeneous mixtures, such as tumors or mixed microbial populations. To overcome limitations in sequencing accuracy, a method Duplex Consensus Sequencing (DCS) is provided. This approach greatly reduces errors by independently tagging and sequencing each of the two strands of a DNA duplex. As the two strands are complementary, true mutations are found at the same position in both strands. In contrast, PCR or sequencing errors will result in errors in only one strand. This method uniquely capitalizes on the redundant information stored in double-stranded DNA, thus overcoming technical limitations of prior methods utilizing data from only one of the two strands. 136-. (canceled)37. A method for detecting double-stranded deoxyribonucleic acid (DNA) molecules in a biological sample , comprising:(a) tagging said double-stranded DNA molecules in said biological sample with a set of duplex tags, wherein said set of duplex tags comprises a plurality of different tag sequences, wherein each duplex tag of said set of duplex tags differently tags complementary strands of a double-stranded DNA molecule of said double-stranded DNA molecules in said biological sample to provide tagged strands, and wherein said tagging is performed with an excess of duplex tags as compared to said double-stranded DNA molecules;(b) for each genetic locus in a set of one or more genetic loci in a reference genome, selectively enriching said tagged strands for subset of said tagged strands that map to said genetic locus, to provide enriched tagged strands ...

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Номер записи: 77
05-12-2019 дата публикации

METHODS FOR HAPLOTYPE AND DIPLOTYPE DETERMINATION

Номер: US20190367971A1
Автор: Fan Tsz Wing, Hsing I Ming, Lee Yu Henson Lim
Принадлежит: THE HONG KONG UNIVERSITY OF SCIENCE AND TECHNOLOGY

The present invention provides methods for determining a haplotype or a diplotype in a genetic sample. The method comprises the steps of contacting a probe-complex with the genetic sample, wherein the probe complex comprises at least two probes, hybridizing at least two probes to a polynucleotide sequence, each of which is specific to one of two or more genetic variants, determining the presence or absence of at least one genetic variant by detecting a signal emitted from at least one probe, wherein detection of said signal is indicative of the the presence of a genetic variant, removing or displacing at least one of said probes from said sample, and detecting a change in the signal to determine the haplotype or a diplotype in the genetic sample. Kits for use in the method of the invention are also provided. 1. A method for determining a haplotype or a diplotype in a genetic sample comprising the steps of:a) contacting a probe-complex with the genetic sample, wherein the probe complex comprises at least two probes;b) hybridising the at least two probes to a polynucleotide sequence, wherein each of the at least two probes is specific to one of two or more genetic variants in said polynucleotide sequence;c) determining the presence or absence of at least one genetic variant by detecting a signal emitted from at least one probe, wherein detection of said signal is indicative of the the presence of a genetic variant;d) removing or displacing at least one of said probes from said sample; ande) detecting a change in the signal to determine the haplotype or a diplotype in the genetic sample.2. The method of claim 1 , wherein the probe-complex comprises a double-stranded DNA (dsDNA) molecule claim 1 , a three-stranded DNA molecule or a four-stranded DNA molecule.3. The method of claim 2 , wherein the probe-complex comprises a three stranded DNA molecule comprising a first and second probe hybridised to a connector strand claim 2 , wherein the first and second probes are ...

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Номер записи: 78
24-12-2020 дата публикации

MODIFIED NUCLEOTIDES

Номер: US20200399692A1
Автор: Barnes Colin Lloyd, Brennan Joseph, Liu Xiaohai, Milton John, Ruediger Silke, Smith Mark Edward Brennan, Wu Xiaolin
Принадлежит:

The invention provides modified nucleotide or nucleoside molecule comprising a purine or pyrimidine base and a ribose or deoxyribose sugar moiety having a removable 3′-OH blocking group covalently attached thereto, such that the 3′ carbon atom has attached a group of the structure —O—Z wherein Z is any of —C(R′)2-O—R″, —C(R′)2-N(R″)2, —C(R′)2-N(H)R″, —C(R′)2-S—R″ and —C(R′)2-F, wherein each R″ is or is part of a removable protecting group; each R′ is independently a hydrogen atom, an alkyl, substituted alkyl, arylalkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclic, acyl, cyano, alkoxy, aryloxy, heteroaryloxy or amido group, or a detectable label attached through a linking group; or (R′)2 represents an alkylidene group of formula ═C(R′″)2 wherein each R′″ may be the same or different and is selected from the group comprising hydrogen and halogen atoms and alkyl groups; and wherein said molecule may be reacted to yield an intermediate in which each R″ is exchanged for H or, where Z is —C(R′)2-F, the F is exchanged for OH, SH or NH2, preferably OH, which intermediate dissociates under aqueous conditions to afford a molecule with a free 3′OH; with the proviso that where Z is —C(R′)2-S—R″, both R′ groups are not H. 1. (canceled)2. (canceled)3. A composition comprising:a planar solid support;a plurality of different target polynucleotides immobilized at distinct regions on the solid support, wherein the distinct regions comprise multiple copies of one of the plurality of different target polynucleotides;a plurality of different complementary oligonucleotides hybridized to the different target polynucleotides; andan aqueous solution contacting the solid support, wherein the aqueous solution comprises:at least 75% by volume water as a continuous phase of the aqueous solution;at least one fluorescent label; and{'sub': '1-6', 'a water-soluble tri-C-alkyl phosphine substituted with a plurality of hydroxyl, carboxyl, carboxylate, amino, or sulfonate groups.'}4. The ...

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Номер записи: 79
03-11-2022 дата публикации

METHODS FOR LABELLING NUCLEIC ACIDS

Номер: US20220348998A1
Автор: FORSHEW Tim, Lensing Stefanie, WOODHOUSE Samuel
Принадлежит:

The invention relates to methods for labelling individual nucleic acid molecules present in a sample, comprising contacting the nucleic acid molecules with an adaptor or mixture of adaptors, wherein the adaptor or adaptors comprise one or more universal nucleotide bases and a ligation moiety at their 3′ end, and ligating an adaptor to the nucleic acid of interest, wherein the adaptor is ligated to the nucleic acid molecules at the 3′ end of the adaptor. A random tag is then generated in situ by conducting an extension reaction over the ligated adaptor. Methods of the invention may be used to detect genetic alterations or variants in any nucleic acid with high specificity and high sensitivity, including mutations in nucleic acids such as ctDNA, cfDNA, and in viral, microbiome and plant nucleic acids. Methods of the invention may also be used in detection and correction of errors introduced into nucleic acids during processing. 1189-. (canceled)190. A double-stranded nucleic acid adaptor comprising a first strand and a second strand , wherein the first strand comprises one or more universal nucleotide bases and a ligation moiety at its 3′ end , and the second strand comprises a ligation block at its 5′ end.191. The double-stranded nucleic acid adaptor of claim 190 , wherein the ligation moiety comprises an overhang of one or more non-universal bases at its 3′ end.192. The double-stranded nucleic acid adaptor of claim 191 , wherein the one or more universal nucleotide bases are in the double-stranded region of the adaptor.193. The double-stranded nucleic acid of claim 190 , wherein the one or more non-universal bases are selected from the group consisting of A claim 190 , T claim 190 , C claim 190 , G claim 190 , and U (Uracil).194. The double-stranded nucleic acid adaptor of claim 191 , wherein the overhanging 3′ end of the first strand is a C or T overhang.195. The double-stranded nucleic acid adaptor of claim 190 , wherein the 5′ ligation block on the second strand ...

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Номер записи: 80
10-10-2013 дата публикации

Reagent and method of preventing at least one false start in polymerase chain reaction amplification, sets of reagents and oligonucleotides for amplification by polymerase chain reaction

Номер: RU2495046C2
Автор: АКИЛЕС Дж. САНЧЕС, Артур РЕЙС, Джесс СОЛК, Джон РАЙС, Кеннет ПИРС, Лоренс Дж. УОНГ
Принадлежит: Брандейс Юнивесити

FIELD: chemistry. SUBSTANCE: invention relates to biotechnology, specifically to a reagent for preventing at least one false start in polymerase chain reaction (PCR) amplification, a method of preventing at least one false start in PCR amplification and sets which include said reagent. The reagent is used in PCR which is carried out in the presence of 1.25 units of thermally stable DNA polymerase per 25 mcl of the reaction mixture with concentration of not more than 650 nM. The reagent is a non-extending DNA polymerase oligonucleotide, having a barrel-loop structure and is not a hybridisation probe for said amplified DNA product. The barrel has a length greater than six nucleotides, is stabilised at the end far from the loop by non-fluorescent substances selected from: fluorescence extinguisher and a nucleotide analogue which facilitates a tighter connection between oligonucleotides in the barrel than a DNA-DNA hybrid. The melting point of the barrel (Tm) is below 94°C. The loop is an oligonucleotide and/or carbon linker. EFFECT: invention enables to prevent false start (unintentional hybridisation) in amplifications and analyses using PCR. 20 cl, 25 dwg, 3 tbl, 14 ex РОССИЙСКАЯ ФЕДЕРАЦИЯ (19) RU (11) 2 495 046 (13) C2 (51) МПК C07H 21/00 (2006.01) C12Q 1/68 (2006.01) ФЕДЕРАЛЬНАЯ СЛУЖБА ПО ИНТЕЛЛЕКТУАЛЬНОЙ СОБСТВЕННОСТИ (12) ОПИСАНИЕ ИЗОБРЕТЕНИЯ К ПАТЕНТУ (21)(22) Заявка: 2007118546/10, 17.10.2005 (24) Дата начала отсчета срока действия патента: 17.10.2005 C 2 C 2 (56) Список документов, цитированных в отчете о поиске: WO 0102559 A1, 11.01.2001. US 6183967 B1, 06.02.2001. SANCHEZ J.А. ET AL: "LinearAfter-The-Exponential (LATE)-PCR: An advanced method of asymmetric PCR and its uses in quantitative real-time analysis." 2004, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, v.101, no.7, pp.19331938. US 5866336, 02.02.1999. US (см. прод.) R U 2 4 9 5 0 4 6 (85) Дата начала рассмотрения заявки PCT на национальной фазе: 18.05.2007 (86) ...

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Номер записи: 81
11-08-2015 дата публикации

Manipulation of microparticles in microfluidic systems

Номер: US9101928B2
Автор: Andrea W. Chow, Anne R. Kopf-Sill, J. Wallace Parce, Luc J. Bousse, Michael R. Knapp, Steve Gallagher, Tammy Burd Mehta, Theo T. Nikiforov
Принадлежит: Caliper Life Sciences Inc

An array of transportable particle sets is used in a microfluidic device for performing chemical reactions in the microfluidic device. The microfluidic device comprises a main channel and intersecting side channels, the main channel and side channels forming a plurality of intersections. The array of particle sets is disposed in the main channel, and the side channels are coupled to reagents. As the particle sets are transported through the intersections of the main channel and the side channels, reagents are flowed through the side channels into contact with each array member (or selected array members), thereby providing a plurality of chemical reactions in the microfluidic system.

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Номер записи: 82
21-02-2017 дата публикации

Секвенирование днк с повторным использованием реагентов на проволочной сетке

Номер: RU2611207C2
Автор: ДЕР ЗАГ Питер Ян ВАН, Йохан ЛУБ, Райнхольд ВИМБЕРГЕР-ФРИДЛЬ
Принадлежит: КОНИНКЛЕЙКЕ ФИЛИПС Н.В.

Изобретения относятся к области определения последовательности нуклеиновой кислоты. Предложена группа изобретений, включающая устройство и способ для оптического контроля секвенирования нуклеиновой кислоты, машиночитаемый носитель с компьютерной программой и программный элемент, используемые в вышеуказанном способе, а также применение 5-метил-(2-(2-нитрофенил)пропил)карбонат-dUTP, 5-метил-(2-оксо-1,2-дифенилэтил)карбонат-dUTP в качестве блокатора в секвенировании ДНК в вышеуказанном способе. Устройство включает подложку в виде проволочной сетки для связывания молекулы на поверхности; оптическую схему, выполненную с возможностью направления возбуждающего излучения на подложку, приема, детектирования излучения и направления расщепляющего излучения на подложку; раствор с нуклеотидами и ферментом, где нуклеотиды содержат блокатор. Способ включает обеспечение подложки с молекулой, облучение подложки возбуждающим излучением, ограничение возбуждающего излучения с помощью подложки, прием и детектирование флуоресценции возбужденной флуоресцентной метки нуклеотида, выполнение облучения подложки расщепляющим излучением, ограничение расщепляющего излучения и обеспечение раствора с многочисленными нуклеотидами и ферментом. Изобретения обеспечивают усовершенствование способа определения последовательности нуклеиновой кислоты. 5 н. и 9 з.п. ф-лы, 11 ил. РОССИЙСКАЯ ФЕДЕРАЦИЯ (19) RU (11) (13) 2 611 207 C2 (51) МПК C12Q 1/68 (2006.01) G01N 21/64 (2006.01) B01J 19/00 (2006.01) ФЕДЕРАЛЬНАЯ СЛУЖБА ПО ИНТЕЛЛЕКТУАЛЬНОЙ СОБСТВЕННОСТИ (12) ФОРМУЛА (21)(22) Заявка: ИЗОБРЕТЕНИЯ К ПАТЕНТУ РОССИЙСКОЙ ФЕДЕРАЦИИ 2014133184, 09.01.2013 (24) Дата начала отсчета срока действия патента: 09.01.2013 Дата регистрации: (72) Автор(ы): ВИМБЕРГЕР-ФРИДЛЬ Райнхольд (NL), ЛУБ Йохан (NL), ВАН ДЕР ЗАГ Питер Ян (NL) Приоритет(ы): (30) Конвенционный приоритет: (56) Список документов, цитированных в отчете о поиске: WO 00/50642 A1, 31.08.2000. WO 13.01.2012 US 61/586,260 2006/007207 A2, 19.01.2006. WO 2006/044078 ...

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Номер записи: 83
20-01-2012 дата публикации

A DNA sequencing method using novel nucleoside triphosphates with a fluorescent 3'-O-blocking group as reversible terminators

Номер: KR101107315B1
Автор: 신동윤, 안대로, 안희철
Принадлежит: 한국과학기술연구원

본 발명은 3'-하이드록실기에 형광을 띄는 장애그룹이 부착된 뉴클레오시드 삼인산을 가역적 종결자로서 이용한 DNA 염기서열 분석 방법에 관한 것으로, 보다 상세하게는 3'-하이드록실기 부분에 형광신호를 내면서 화학적으로 제거가 가능한 가역적 장애그룹(reversible blocking group)을 부착한 새로운 뉴클레오티드 단량체인 단일 부분이 변형된 가역적 종결자(mono-modified reversible terminator, MRT)를 이용한 합성에 의한 염기서열 분석 방법(sequencing-by-synthesis)에 관한 것이다. 본 발명의 염기서열 분석 방법은 상기 새로운 뉴클레오티드 단량체가 염기서열 사슬의 연장을 중단시킴과 동시에 3'-하이드록실기 부분에 부착된 형광 신호를 감지함으로써 삽입된 염기의 종류를 분석할 수 있으며, 형광 신호 분석 이후 3'-하이드록실기에 부착된 장애그룹이 효과적으로 제거되어서 3'-OH 작용기로 복구되기 때문에 다음 단량체의 삽입을 가능하게 하여 연이어 염기서열 분석을 가능하게 한다. The present invention relates to a DNA sequencing method using a nucleoside triphosphate having a fluorescence group of 3'-hydroxyl groups attached as a reversible terminator, and more specifically, to a 3'-hydroxyl group. A method for sequencing by synthesis using a mono-modified reversible terminator (MRT), a single nucleotide monomer that attaches a reversible blocking group that can be chemically removed while signaling. sequencing-by-synthesis). The sequencing method of the present invention can analyze the type of the inserted base by detecting the fluorescent signal attached to the 3'-hydroxyl group portion while the new nucleotide monomer stops the extension of the nucleotide sequence. After the signal analysis, the obstacle group attached to the 3'-hydroxyl group is effectively removed and restored to the 3'-OH functional group, thereby enabling the insertion of the next monomer, thereby enabling subsequent sequencing. DNA 염기서열 분석(DNA sequencing), 중합효소에 의한 염기서열 분 석(sequencing-by-synthesis), 3'-하이드록실기가 변형된 뉴클레오티드(3'-O-modified nucleotide), 형광(fluorescence), 가역적 종결자(reversible terminator). DNA sequencing, sequencing-by-synthesis by polymerases, 3'-O-modified nucleotides, fluorescence, Reversible terminator.

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Номер записи: 84
26-11-2013 дата публикации

Methods and compositions for long fragment read sequencing

Номер: US8592150B2
Автор: Andrei Alexeev, Brock A. Peters, Peter Hong, Radoje Drmanac
Принадлежит: Complete Genomics Inc

The present invention is directed to methods and compositions for long fragment read sequencing. The present invention encompasses methods and compositions for preparing long fragments of genomic DNA, for processing genomic DNA for long fragment read sequencing methods, as well as software and algorithms for processing and analyzing sequence data.

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Номер записи: 85
15-10-2019 дата публикации

Helper oligonucleotide for improved efficiency of amplification and detection/quantitation of nucleic acids

Номер: US10443095B2
Автор: Rochak Mehta
Принадлежит: Roche Molecular Systems Inc

Improved methods for the detection and quantitation of a target nucleic acid in a sample using a non-extending helper oligonucleotide are described. The methods include contacting nucleic acids in a sample with amplification reagents including one or more primers, one or more non-extending helper oligonucleotides, and one or more probes. The non-extending helper oligonucleotide facilitates and increases the target nucleic acid accessibility of one or more of the primers, result in greater accumulation of amplicon production, thereby increasing the efficiency and sensitivity of the amplification assay, including amplification assays for Hepatitis C Virus (HCV), for example, HCV Genotype 5. Kits, articles of manufacture, and reaction mixtures are also provided.

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Номер записи: 86
11-12-2008 дата публикации

Methods and compositions for nucleic acid amplification

Номер: US20080305482A1
Автор: Dmitry LYAKHOV, James D. Carlson, Norman C. Nelson, Steven T. Brentano
Принадлежит: Gen Probe Inc

Compositions that are used in nucleic acid amplification in vitro are disclosed, which include a target specific universal (TSU) promoter primer or promoter provider oligonucleotide that includes a target specific (TS) sequence that hybridizes specifically to a target sequence that is amplified and a universal (U) sequence that is introduced into the sequence that is amplified, by using a primer for the universal sequence. Methods of nucleic acid amplification in vitro are disclosed that use one or more TSU oligonucleotides to attached a U sequence to a target nucleic acid in a target capture step and then use a primer for a U sequence in subsequent amplification steps performed in substantially isothermal conditions to make amplification products that contain a U sequence that indicates the presence of the target nucleic acid in a sample.

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Номер записи: 87
17-04-2018 дата публикации

Multiplex quantitative PCR

Номер: US9944978B2
Автор: Calvin Harley, Jue Lin, Karl Guegler
Принадлежит: TELOMERE DIAGNOSTICS Inc

Disclosed are methods and compositions for determining the average length or abundance of a first target nucleic by calculating the abundance of a first target nucleic acid (T) relative to the average abundance (S) of a second and a third target nucleic acid, in a single well using a separate detection label for each target nucleic acid. In various aspects, the first target nucleic acid is a telomere. In exemplary aspects, the disclosed methods and compositions can be used to determine the average telomere length in a biological sample. The average telomere length determined using the disclosed methods and compositions can be correlated to a variety of clinically important conditions and indices. This abstract is intended as a scanning tool for purposes of searching in the particular art and is not intended to be limiting of the present invention.

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Номер записи: 88
12-06-2012 дата публикации

Methods and compositions for nucleic acid amplification

Номер: US8198027B2
Автор: Dmitry LYAKHOV, James D. Carlson, Lyle J. Arnold, Jr., Norman C. Nelson, Steven T. Brentano
Принадлежит: Gen Probe Inc

Compositions that are used in nucleic acid amplification in vitro are disclosed, which include a target specific universal (TSU) promoter primer or promoter provider oligonucleotide that includes a target specific (TS) sequence that hybridizes specifically to a target sequence that is amplified and a universal (U) sequence that is introduced into the sequence that is amplified, by using a primer for the universal sequence. Methods of nucleic acid amplification in vitro are disclosed that use one or more TSU oligonucleotides to attached a U sequence to a target nucleic acid in a target capture step and then use a primer for a U sequence in subsequent amplification steps performed in substantially isothermal conditions to make amplification products that contain a U sequence that indicates the presence of the target nucleic acid in a sample.

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Номер записи: 89
10-09-2019 дата публикации

Methods and compositions for nucleic acid amplification

Номер: US10407723B2
Автор: Dmitry LYAKHOV, James D. Carlson, Lyle J. Arnold, Michael M. Becker, Norman C. Nelson, Steven T. Brentano
Принадлежит: Gen Probe Inc

Compositions that are used in nucleic acid amplification in vitro are disclosed, which include a target specific universal (TSU) promoter primer or promoter provider oligonucleotide that includes a target specific (TS) sequence that hybridizes specifically to a target sequence that is amplified and a universal (U) sequence that is introduced into the sequence that is amplified, by using a primer for the universal sequence. Methods of nucleic acid amplification in vitro are disclosed that use one or more TSU oligonucleotides to attached a U sequence to a target nucleic acid in a target capture step and then use a primer for a U sequence in subsequent amplification steps performed in substantially isothermal conditions to make amplification products that contain a U sequence that indicates the presence of the target nucleic acid in a sample.

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Номер записи: 90
17-09-2019 дата публикации

Methods and compositions for nucleic acid amplification

Номер: US10415092B2
Автор: Dmitry LYAKHOV, James D. Carlson, Lyle J. Arnold, Michael M. Becker, Norman C. Nelson, Steven T. Brentano
Принадлежит: Gen Probe Inc

Compositions that are used in nucleic acid amplification in vitro are disclosed, which include a target specific universal (TSU) promoter primer or promoter provider oligonucleotide that includes a target specific (TS) sequence that hybridizes specifically to a target sequence that is amplified and a universal (U) sequence that is introduced into the sequence that is amplified, by using a primer for the universal sequence. Methods of nucleic acid amplification in vitro are disclosed that use one or more TSU oligonucleotides to attached a U sequence to a target nucleic acid in a target capture step and then use a primer for a U sequence in subsequent amplification steps performed in substantially isothermal conditions to make amplification products that contain a U sequence that indicates the presence of the target nucleic acid in a sample.

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Номер записи: 91
13-06-2017 дата публикации

Methods and compositions for nucleic acid amplification

Номер: US9677135B2
Автор: Dmitry LYAKHOV, James D. Carlson, Lyle J. Arnold, Jr., Michael M. Becker, Norman C. Nelson, Steven T. Brentano
Принадлежит: Gen Probe Inc

Compositions that are used in nucleic acid amplification in vitro are disclosed, which include a target specific universal (TSU) promoter primer or promoter provider oligonucleotide that includes a target specific (TS) sequence that hybridizes specifically to a target sequence that is amplified and a universal (U) sequence that is introduced into the sequence that is amplified, by using a primer for the universal sequence. Methods of nucleic acid amplification in vitro are disclosed that use one or more TSU oligonucleotides to attached a U sequence to a target nucleic acid in a target capture step and then use a primer for a U sequence in subsequent amplification steps performed in substantially isothermal conditions to make amplification products that contain a U sequence that indicates the presence of the target nucleic acid in a sample.

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Номер записи: 92
04-02-2014 дата публикации

Methods and compositions for nucleic acid amplification

Номер: US8642268B2
Автор: Dmitry LYAKHOV, James D. Carlson, Lyle J. Arnold, Jr., Michael M. Becker, Norman C. Nelson, Steven T. Brentano
Принадлежит: Gen Probe Inc

Compositions that are used in nucleic acid amplification in vitro are disclosed, which include a target specific universal (TSU) promoter primer or promoter provider oligonucleotide that includes a target specific (TS) sequence that hybridizes specifically to a target sequence that is amplified and a universal (U) sequence that is introduced into the sequence that is amplified, by using a primer for the universal sequence. Methods of nucleic acid amplification in vitro are disclosed that use one or more TSU oligonucleotides to attached a U sequence to a target nucleic acid in a target capture step and then use a primer for a U sequence in subsequent amplification steps performed in substantially isothermal conditions to make amplification products that contain a U sequence that indicates the presence of the target nucleic acid in a sample.

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Номер записи: 93
23-06-2020 дата публикации

Methods, compositions, and kits for detecting allelic variants

Номер: US10689694B2
Автор: Caifu Chen, Ruoying Tan
Принадлежит: Life Technologies Corp

In some embodiments, the present inventions relates generally to compositions, methods and kits for use in discriminating sequence variation between different alleles. More specifically, in some embodiments, the present invention provides for compositions, methods and kits for quantitating rare (e.g., mutant) allelic variants, such as SNPs, or nucleotide (NT) insertions or deletions, in samples comprising abundant (e.g., wild type) allelic variants with high specificity and selectivity. In particular, in some embodiments, the invention relates to a highly selective method for mutation detection referred to as competitive allele-specific TaqMan PCR (“cast-PCR”).

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Номер записи: 94
05-03-2019 дата публикации

Processes and systems for preparation of nucleic acid sequencing libraries and libraries prepared using same

Номер: US10221436B2
Автор: Benjamin Hindson, Indira Wu, Kamila Belhocine, Keith Bjornson, Paul Hardenbol, Paul William Wyatt, Pranav Patel
Принадлежит: 10X Genomics Inc

This disclosure provides methods for preparing a sequencing library including the steps of providing a template nucleic acid sequence, dNTPs, dUTP, a primer, a polymerase, a dUTP excising enzyme, and a plurality of beads including oligonucleotide adapter sequence segments; amplifying the template nucleic acid with the polymerase, dNTPs, dUTP and random hexamer to provide a complementary nucleic acid sequence including occasional dUTPs; and excising the incorporated dUTPs with the dUTP excising enzyme to provide nicks in the complementary nucleic acid sequence to provide a sequencing library.

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Номер записи: 95
11-02-2020 дата публикации

Processes and systems for preparation of nucleic acid sequencing libraries and libraries prepared using same

Номер: US10557158B2
Автор: Benjamin Hindson, Indira Wu, Kamila Belhocine, Keith Bjornson, Paul Hardenbol, Paul William Wyatt, Pranav Patel
Принадлежит: 10X Genomics Inc

This disclosure provides methods for preparing a sequencing library including the steps of providing a template nucleic acid sequence, dNTPs, dUTP, a primer, a polymerase, a dUTP excising enzyme, and a plurality of beads including oligonucleotide adapter sequence segments; amplifying the template nucleic acid with the polymerase, dNTPs, dUTP and random hexamer to provide a complementary nucleic acid sequence including occasional dUTPs; and excising the incorporated dUTPs with the dUTP excising enzyme to provide nicks in the complementary nucleic acid sequence to provide a sequencing library.

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Номер записи: 96
16-08-2022 дата публикации

Processes and systems for preparation of nucleic acid sequencing libraries and libraries prepared using same

Номер: US11414688B2
Автор: Benjamin Hindson, Indira Wu, Keith Bjornson, Paul Hardenbol, Paul William Wyatt, Pranav Patel, Zahra Kamila Belhocine
Принадлежит: 10X Genomics Inc

This disclosure provides methods for preparing a sequencing library including the steps of providing a template nucleic acid sequence, dNTPs, dUTP, a primer, a polymerase, a dUTP excising enzyme, and a plurality of beads including oligonucleotide adapter sequence segments; amplifying the template nucleic acid with the polymerase, dNTPs, dUTP and random hexamer to provide a complementary nucleic acid sequence including occasional dUTPs; and excising the incorporated dUTPs with the dUTP excising enzyme to provide nicks in the complementary nucleic acid sequence to provide a sequencing library.

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Номер записи: 97
06-06-2006 дата публикации

Isothermal chimeric primer nucleic acid amplification methods using blocking oglionucleotide

Номер: US7056671B2
Автор: Hiroaki Sagawa, Ikunoshin Kato, Tatsuji Enoki
Принадлежит: Takara Bio Inc

Methods of amplifying a target nucleic acid whereby the target nucleic acid is amplified in the presence of a deoxyribonucleotide triphosphate, a DNA polymerase having strand displacement activity, at least one chimeric oligonucleotide primer, at least one upstream block oligonucleotide and a RNase H; wherein the chimeric oligonucleotide primer contains a ribonucleotide positioned at the 3′-terminus; wherein the upstream block oligonucleotide is capable of annealing to a region 3′ to a portion in the nucleic acid as the template to which the chimeric oligonucleotide primer anneals; and compositions and kits thereof.

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Номер записи: 98
23-11-2016 дата публикации

Methods and compositions for nucleic acid amplification

Номер: EP3095873A1
Автор: Dmitry LYAKHOV, James D Carlson, Lyle J ARNOLD Jr, Michael M Becker, Norman C NELSON, Steven T BRENTANO
Принадлежит: Gen Probe Inc

There is described a target capture reaction mixture for separating a target nucleic acid from a sample, the reaction mixture comprising: a. a target specific universal (TSU) primer complex made up of i. a TSU promoter oligonucleotide comprising a 5' promoter sequence, an internal first universal sequence (U1), and a 3' first target specific sequence (TS1) that binds specifically to a target sequence contained in a target nucleic acid, wherein the TSU promoter oligonucleotide is a TSU promoter primer that has a 3' terminus that is capable of being extended by a polymerase, or is a TSU promoter provider oligonucleotide that has a blocked 3' terminus that is incapable of being extended by a polymerase, directly or indirectly joined to, ii. a TSU non-promoter primer oligonucleotide made up of a 5' second universal sequence (U2) and a 3' second target specific sequence (TS2) which is different from the TS1, wherein the TSU promoter oligonucleotide is joined to the TSU non-promoter primer via: (A) a covalent linkage that is a polynucleotide linker sequence or a non-nucleotide abasic linker compound; (B) a hybridization complex between a first sequence on the TSU promoter oligonucleotide and a second sequence on the TSU non-promoter primer that is complementary to the first sequence on the TSU promoter oligonucleotide; or (C) a hybridization complex that includes an S-oligonucleotide that contains a first sequence complementary to a sequence in the TSU promoter oligonucleotide and a second sequence complementary to a sequence in the TSU non-promoter primer oligonucleotide; and b. a target specific capture oligonucleotide that contains a target specific sequence (TS3) that hybridizes specifically to a sequence in the target nucleic acid that is different from the sequence in the target nucleic acid that hybridizes to the TS sequence of the TSU promoter oligonucleotide or the TS sequence of the TSU non-promoter primer, and contains a means for ...

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Номер записи: 99
12-02-2019 дата публикации

DNA sequencing

Номер: US10202642B2
Автор: Mark W. Eshoo
Принадлежит: Ibis Biosciences Inc

Provided herein is technology relating to sequencing nucleic acids and particularly, but not exclusively, to methods, compositions, systems, and kits for sequencing a nucleic acid using a degenerate two-base code. Particular embodiments provide: 1) that the two-base degenerate code relates a first element to a base comprising adenine (A) or guanine (G) and a second element to a base comprising cytosine (C) or thymine (T); 2) that the two-base degenerate code relates a first element to a base comprising A or C and a second element to a base comprising G or T; and 3) that the two-base degenerate code relates a first element to a base comprising G or C and a second element to a base comprising A or T.

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Номер записи: 100