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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 955. Отображено 100.
01-03-2012 дата публикации

Method and Device for Microparticle Assay Fluorescence Intensity Reference Intraplex

Номер: US20120053085A1
Автор: Brian P. Hanley
Принадлежит: Individual

A method for making suspended microarray readings from a single sample more reliably accurate. It can be applied to any assay system that uses discrete particles coupled with an assay. Most of these are fluidic systems that read the assay result using flow cytometry. However, other methods such as the distribution of tiny assay devices coupled with miniature transponders, where the sampling is of the environment, can also make use of this method. This invention combines a reference set of signal levels on particles with separately identified assays of one sample. By elimination of outliers, averaging, taking ratios of averages, and then taking ratios of assay signal levels against the reference set this method makes possible highly reliable diagnostics. When used standalone, the method uses different signal intensities to better calibrate an instrument. This method compensates for multiple sources of errors that can occur in this type of assay system.

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Номер записи: 1
01-03-2012 дата публикации

Methods of creating and screening dna-encoded libraries

Номер: US20120053091A1
Автор: Richard W. Wagner
Принадлежит: X Chem Inc

The present invention features a number of methods for identifying one or more compounds that bind to a biological target. The methods include synthesizing a library of compounds, wherein the compounds contain a functional moiety having one or more diversity positions. The functional moiety of the compounds is operatively linked to an initiator oligonucleotide that identifies the structure of the functional moiety.

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Номер записи: 2
17-05-2012 дата публикации

Rna labeling method

Номер: US20120122702A1
Автор: Emily Marine Leproust, Gusti Zeiner, Petula N. D'Andrade
Принадлежит: AGILENT TECHNOLOGIES INC

A method of sample analysis is provided. In certain embodiments, the method involves: a) obtaining a fragmented RNA sample comprising fragments of long RNA molecules and short RNA molecules; b) ligating an adaptor to an end of the RNA of the fragmented RNA sample to produce an adaptor-ligated sample; c) hybridizing said adaptor-ligated sample to an array of nucleic acid probes; and d) reading said array to obtain an estimate of the abundance of a long RNA in the RNA sample and an estimate of the abundance a small RNA in the RNA sample.

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Номер записи: 3
24-05-2012 дата публикации

Method for screening new drug candidate inhibiting target protein-protein interaction for development of first-in-class drug

Номер: US20120129722A1
Автор: So Youn Kim
Принадлежит: Industry Academic Cooperation Foundation of Dongguk University

The present invention relates to a method for screening a substance inhibiting protein-protein interactions, and more particularly to a method for screening a substance inhibiting protein-protein interactions, the method comprising using a protein chip having immobilized thereon spots comprising a mixture of a sol-gel material and a protein. According to the invention, a protein chip can be easily manufactured in a 96-well plate using a sol-gel material, whereby an inhibitor that inhibits protein-protein interactions can be easily screened from a library of natural substances.

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Номер записи: 4
18-10-2012 дата публикации

Donor specific antibody libraries

Номер: US20120264647A1
Автор: Arun K. Kashyap, Lawrence Horowitz, Ramesh Bhatt
Принадлежит: Kashyap Arun K, Lawrence Horowitz, Ramesh Bhatt

The present invention concerns donor-specific antibody libraries derived from a patient donor who has suffered from, or is suffering from one or more diseases discussed herein. The present invention also concerns the method of making and using the donor-specific antibodies. The present invention further concerns the neutralizing antibodies obtained from the donor-specific antibody libraries and the methods of using these antibodies for the prevention/treatment of human disease.

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Номер записи: 5
21-02-2013 дата публикации

Library characterization by digital assay

Номер: US20130045875A1
Автор: Benjamin J. Hindson, Michael Y. Lucero, Ryan T. Koehler, Serge Saxonov, Svilen S. Tzonev
Принадлежит: Bio Rad Laboratories Inc

Methods of characterizing a nucleic acid library by digital assay.

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Номер записи: 6
18-07-2013 дата публикации

METHOD FOR IDENTIFYING COMPOUNDS

Номер: US20130184163A1
Автор: Ottmann Udo, Ross Guenther
Принадлежит: PRIAXON AG

The present invention relates to a method for identifying compounds comprising the steps of: (a) providing a set of compounds; (b) optionally selecting a sub-set from the set of compounds based on one or more specific compound properties; (c) generating a 3D structure of each of the compounds provided and/or selected in step (a) or (b); (d) encoding each 3D structure; (e) providing at least one known compound having at least one desired property and/or providing a target molecule; (f) encoding the 3D structure of (each of) the known compound(s) provided in step (e) and/or the active site of the target molecule provided in step (e); (g) comparing said encoded 3D structure(s) of step (d) with the encoded 3D structure(s) of step (f); and (h) selecting all compounds falling within a specified similarity range. 19-. (canceled)10. A method for identifying compounds comprising the steps of:(a) providing a set of compounds;(b) optionally selecting a sub-set from the set of compounds based on one or more specific compound properties;(c) generating a 3D structure of each of the compounds provided in step (a) or optionally selected in step (b);(d) encoding each 3D structure;(e) providing at least one known compound having at least one desired property or providing a target molecule;(f) encoding a 3D structure of each known compound provided in step (e) or an active site of the target molecule provided in step (e);(g) comparing each encoded 3D structure of step (d) with each encoded 3D structure of step (f); and(h) selecting all compounds falling within a specified similarity range.11. The method of claim 10 , further comprising the steps of:(i) optionally selecting a further sub-set of the compounds provided in step (h) based on one or more specific compound properties;(j) preparing the selected compounds of step (h) or optionally selected compounds of step (i) and testing the prepared compounds for activity;(k) optionally repeating steps (g) to (j) or (h) to (j).12. The ...

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Номер записи: 7
10-04-2014 дата публикации

Microvessels, microparticles, and methods of manufacturing and using the same

Номер: US20140100123A1
Автор: John A. Moon, M. Shane Bowen, Michal Lebl, Michel Perbost, Ryan C. Smith, Steven H. Modiano
Принадлежит: Illumina Inc

A plurality of isolated microvessels including a plurality of encoded microvessels each having a microbody and a reservoir core. The microbody is configured to separate a biological or chemical substance in the reservoir core from an ambient environment surrounding the microbody. The microbody includes a transparent material that at least partially surrounds the reservoir core and facilitates detection of an optical characteristic of the substance within the reservoir core. The microbody of each microvessel includes an identifiable code that distinguishes individual microvessels of the plurality of encoded microvessels from each other. The plurality of isolated microvessels also includes a plurality of compartments each configured to separate individual microvessels of the plurality of encoded microvessels from each other.

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Номер записи: 8
16-01-2020 дата публикации

COMPOSITIONS AND METHODS FOR STUDYING THE TAT GENE

Номер: US20200017883A1
Автор: Benjamin Ronald, Schiller Martin R.
Принадлежит: The Board Of Regents Of The Nevada System Of Higher Education On Behalf Of The University of Nevada

Disclosed are compositions and methods for studying a Tat gene. Specifically, the disclosure provides a vector comprising a double-stranded nucleic acid construct which comprises a Tat gene and a green fluorescent protein (GFP) reporter element, and further wherein the double-stranded nucleic acid construct comprising AAVS1 (adeno-associated virus integration site, a safe harbor locus) arms that flank on both sides of the Tat gene and the reporter element for integration at the human AAVS1 site by homologus recombination. Further provided are methods of using a cell comprising the vector for studying the effects of exogenous conditions on expression of the Tat gene. 1. (canceled)2. A vector comprising a double-stranded nucleic acid construct , wherein the double-stranded nucleic acid construct comprises a first strand and a second strand ,wherein the first strand comprises from 5′ to 3′ a 5′ ARM sequence, a GADE sequence and a 3′ ARM sequence;wherein the second strand comprises from 5′ to 3′ a 3′ ARM sequence, a GARE sequence and a 5′ ARM sequence; andwherein the first strand is complementary to the second strand.3. The vector of claim 2 , wherein the GADE sequence comprises from 5′ to 3′ a transcriptional control element operably linked to one or more driver elements and a 3′UTR claim 2 , wherein the 3′UTR comprises a barcode sequence claim 2 , wherein the barcode sequence of the GADE sequence is complementary to a barcode sequence in the 3′ UTR of the GARE sequence.4. The vector of claim 2 , wherein the GARE sequence comprises from 5′ to 3′ a transcriptional control element operably linked to one or more reporter elements and a 3′UTR claim 2 , wherein the 3′UTR comprises a barcode sequence claim 2 , wherein the barcode sequence of the GARE sequence is complementary to a barcode sequence in the 3′ UTR of the GADE sequence.5. The vector of claim 2 , wherein the GADE and GARE sequences both comprise a barcode sequence claim 2 , wherein the barcode sequence of the ...

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Номер записи: 9
24-01-2019 дата публикации

MICROFLUIDIC ANALYSIS OF LIGAND INDUCED CELL EXPRESSION

Номер: US20190022645A1
Автор: HU Hongxing, MERTEN Christoph A., SEAH Samantha
Принадлежит:

The present invention relates to the field of microfluidics and in particular to analysing the gene expression of a cell in response to a ligand expressed in the same microfluidic compartment. By barcoding the transcriptome of the cell and of the expression system generating the ligand, the effect of the ligand on the cell expression can be discerned. The invention provides microfluidic compartments and methods for this purpose. 1. A plurality of microfluidic compartments , wherein at least 1% of said compartments form a subset in which each compartment comprises(i) a first cell,(ii) one second cell or one cell-free expression system expressing a polypeptide ligand intended to specifically bind to a molecule or part thereof accessible on the surface of the first cell, and(iii) a set of barcode oligonucleotides each comprising a barcode sequence unique to the set and a sequence capable of binding specifically to mRNA and/or cDNA.2. The plurality of microfluidic compartments of claim 1 , wherein the size of said subset of compartments is at least 5 or 10%.3. The plurality of microfluidic compartments of claim 1 , wherein the first cell and the second cell or polypeptide ligand expressed by the cell-free expression system are derived from different species.4. The plurality of microfluidic compartments of claim 1 , wherein the target molecule accessible on the surface of the first cell is a cell signaling receptor.5. The plurality of microfluidic compartments of claim 1 , wherein the first cell is a diseased cell claim 1 , a stem or pluripotent cell or a cell in which pluripotency is inducible by a polypeptide ligand.6. The plurality of microfluidic compartments of claim 1 , wherein the second cell is a non-human plasma cell of the B-cell lineage and the polypeptide ligand is an antibody produced by the plasma cell.7. The plurality of microfluidic compartments of claim 1 , wherein the polypeptide ligand is selected from the group consisting of an antibody claim 1 , an ...

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Номер записи: 10
25-01-2018 дата публикации

Methods And Kits For Theranostic Applications

Номер: US20180023123A1
Автор: Dunn Ian, Lawler Matthew
Принадлежит:

The present disclosure is directed to methods and kits for identifying, enriching, and evaluating templated assembly reactants. Some embodiments disclose methods for identifying templated assembly targets by synthesizing templated assembly reactants, hybridizing the templated assembly reactants to target nucleic acids, performing a templated assembly reaction, and identifying the target nucleic acids that hybridized to the templated assembly reactants. Libraries of templated assembly reactants, a kit for identifying templated assembly targets, and a pair of templated assembly targets enriched from a library of chemically-ligated oligonucleotides spatially elicited (CLOSE) products are also disclosed. 1. A method for identifying templated assembly targets comprising:synthesizing a first population of templated assembly reactants and a second population of corresponding templated assembly reactants, wherein the first and second populations of templated assembly reactants comprise oligonucleotide sequences;hybridizing both populations of templated assembly reactants to target nucleic acids;performing a templated assembly reaction, wherein the hybridized first population of templated assembly reactants and the hybridized second population of corresponding templated assembly reactants undergo templated assembly; andidentifying the target nucleic acids that hybridized to either the first or second population of templated assembly reactants that underwent templated assembly, wherein the hybridized target nucleic acids are the templated assembly targets.2. The method of claim 1 , wherein the steps of synthesizing the first and second population of templated assembly reactants further comprises synthesizing random oligonucleotides sequences of about 7 to about 30 nucleotides long claim 1 , or synthesizing gene specific sequences of about 7 to about 30 nucleotides long.3. (canceled)4. The method of claim 1 , wherein the templated assembly reactants comprise a 5′ or a 3′ ...

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Номер записи: 11
24-01-2019 дата публикации

NUCLEAR RECEPTOR SET DOMAIN CONTAINING PROTEIN 2 TRANSITION STATE AND USES THEREOF

Номер: US20190026440A1
Автор: Poulin Myles B., Schramm Vern L.
Принадлежит: ALBERT EINSTEIN COLLEGE OF MEDICINE, INC.

Methods and systems for obtaining inhibitors of Nuclear receptor SET Domain containing protein 2 (NSD2) are disclosed where the methods involve designing compounds that resemble the NSD2 transition state. 1. A system comprising a non-transitory computer-readable medium coupled to one or more data processing apparatus having instructions stored thereon which , when executed by the one or more data processing apparatus , cause the one or more data processing apparatus to perform a method comprising:(i) obtaining kinetic isotope effects on human Nuclear receptor SET Domain containing protein 2 (NSD2)-catalyzed methylation of histone H3 lysine 36 to obtain the NSD2 transition state structure,wherein the NSD2 transition state comprises the structure{'img': {'@id': 'CUSTOM-CHARACTER-00001', '@he': '7.53mm', '@wi': '135.93mm', '@file': 'US20190026440A1-20190124-P00999.TIF', '@alt': 'text missing or illegible when filed', '@img-content': 'character', '@img-format': 'tif'}}(ii) obtaining the molecular electrostatic potential at the van der Waals surface computed from the wave function of the NSD2 transition state and the geometric atomic volume of the NSD2 transition state; and(iiia) identifying from a library of compounds a chemically stable compound that resembles the molecular electrostatic potential at the van der Waals surface computed from the wave function of the NSD2 transition state and the geometric atomic volume of the NSD2 transition state, or(iiib) designing a chemically stable compound that resembles the molecular electrostatic potential at the van der Waals surface computed from the wave function of the NSD2 transition state and the geometric atomic volume of the NSD2 transition state;wherein the chemically stable compound that resembles the molecular electrostatic potential at the van der Waals surface computed from the wave function of the NSD2 transition state and the geometric atomic volume of the NSD2 transition state is a putative inhibitor of NSD2.2. ( ...

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Номер записи: 12
30-01-2020 дата публикации

Directed evolution of enzymes by plasmid tagging in droplets

Номер: US20200032247A1
Автор: Marc Prindle, Mark Stamatios KOKORISKokoris, Michael KOVARIK, Miranda LAHAM, Robert BUSAM, Salka KELLER
Принадлежит: Stratos Genomics Inc

Methods and compositions for the selection of nucleic acid processing and other enzymes, and more specifically for the selection of DNA polymerases and other enzymes with desired properties employing the directed evolution of enzymes by plasmid tagging in droplets.

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Номер записи: 13
30-01-2020 дата публикации

Methods, systems, computer readable media, and kits for sample identification

Номер: US20200032334A1
Автор: Earl Hubbell
Принадлежит: Life Technologies Corp

A method for sequencing a polynucleotide sample having a barcode sequence includes: introducing a series of nucleotides to the polynucleotide sample according to a predetermined order of nucleotide flows; obtaining a series of signals resulting from the introducing of nucleotides to the polynucleotide sample; and resolving the series of signals over the barcode sequence to render a flowspace string, wherein the flowspace string is a codeword of an error-correcting code that is (i) designed based on and adapted for use with the predetermined order of nucleotide flows, and (ii) capable of distinguishing any codeword in the error-correcting code from the other codewords in the error-correcting code in the presence of zero, one, and two errors.

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Номер записи: 14
18-02-2021 дата публикации

HIGHLY MULTIPLEXED PHYLOGENETIC IMAGING OF MICROBIAL COMMUNITIES

Номер: US20210047634A1
Автор: De Vlaminck Iwijn, Shi Hao
Принадлежит: CORNELL UNIVERSITY

Micron scale biogeography is a major driver of physiology and ecology of complex microbial biofilm communities, which remains elusive largely due to the lack of tools for spatially resolved phylogenetic mapping. This disclosure provides methods, computer-readable storage devices and kits that allow highly multiplexed and spatially resolved imaging of microbial community spatial organization. The disclosure provides a highly-multiplexed approach to resolve the spatial structure of complex microbial community at high taxonomic resolution. 1. A computer-readable storage device storing computer readable instructions for:assigning each taxon in a list of taxa of microorganisms a unique n-bit binary code selected from a plurality of unique n-bit binary codes, wherein n is an integer greater than 1;designing a set of n number of decoding probes, wherein each decoding probe corresponds to a digit in the n-bit binary code, and wherein each decoding probe is substantially complementary to a readout sequence selected from a set of n number of readout sequences; and each encoding probe comprises a targeting sequence and one or more readout sequences,', 'the encoding probes within each subset comprise a targeting sequence that is specific to a taxon in the list of taxa of microorganisms and is different from a targeting sequence of the encoding probes of another subset, and', 'the readout sequences in the encoding probes within a subset are selected from the set of n number of readout sequences based on the unique n-bit binary code assigned to the taxon which the targeting sequence of the subset is specific to., 'designing a set of encoding probes, wherein the set of encoding probes includes a plurality of subsets of encoding probes, wherein'}2. The computer-readable storage device of claim 1 , wherein the targeting sequence in the encoding probes of a subset is substantially complementary to a consensus 16S ribosomal sequence specific to a taxon.3. The computer-readable storage ...

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Номер записи: 15
14-02-2019 дата публикации

IMPROVED AMPLIFICATION AND SEQUENCING METHODS

Номер: US20190048335A1
Автор: Chen Shiaw-Min, JOUN David, LI Chieh-Yuan
Принадлежит:

In some embodiments, the disclosure relates generally to methods, as well as related systems, compositions, kits, and apparatuses for any one or any combination of: conducting a library preparation method which generates a mixture of desirable template polynucleotides and non-desirable polynucleotide byproducts, amplifying the resulting library, enriching the desirable template polynucleotides, and sequencing the enriched template polynucleotides. The methods, as well as related systems, compositions, kits, and apparatuses, of the present teachings can be used to improve sequencing data. 1108.-. (canceled)109. A method of producing at least one particle comprising a template sequence , comprising:a) forming a reaction mixture by contacting:(i) a template polynucleotide, wherein the template polynucleotide comprises, 5′ to 3′, a second adaptor sequence, a template sequence, and a first adaptor sequence,(ii) at least one particle having a plurality of a capture primer attached thereon, wherein the capture primer is capable of hybridizing to the first adaptor sequence, and(iii) a plurality of a solution-phase primer, wherein the solution phase primer comprises, 5′ to 3′, a fourth adaptor sequence and a third adaptor sequence, wherein the third adaptor sequence is capable of hybridizing to the complement of the second adaptor sequence;b) subjecting the reaction mixture to a nucleic acid amplification condition, thereby generating at least one particle attached to a polynucleotide population containing at least one polynucleotide that comprises, 5′ to 3′, the capture primer sequence, the complement of the template sequence, the complement of the third adaptor sequence, and the complement of the fourth adaptor sequence.110. A method of producing at least a set of particles , wherein a first set of particles comprises a first template sequence and an optional second set of particles comprises a second template sequence , wherein the first and second template sequences may ...

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Номер записи: 16
25-02-2021 дата публикации

OPTIMIZING HIGH-THROUGHPUT SEQUENCING CAPACITY

Номер: US20210054451A1
Автор: JUNEAU Kara
Принадлежит:

Provided are methods and compositions for preparing nucleic acid fragments for sequencing by synthesis on a flow cell. The methods and compositions described herein introduce nucleotide diversity into a sample preparation that would otherwise lack nucleotide diversity due to homogeneity of the sequencing target. 1. A set of oligonucleotides configured to introduce nucleotide diversity into sample DNA to be sequenced comprising:a first primer comprising from 5′ to 3′, a first SP2 binding site, a first variable-length phase-shift sequence n nucleotides in length, and a region homologous to a 5′ region of the sample DNA;a second primer comprising from 5′ to 3′, the first SP2 binding site, a second variable-length phase-shift sequence n+1 nucleotides in length, and the region homologous to a 5′ region of the sample DNA;a third primer comprising from 5′ to 3′, the first SP2 binding site, a third variable-length phase-shift sequence n+2 nucleotides in length, and the region homologous to a 5′ region of the sample DNA;a fourth primer comprising from 5′ to 3′, the first SP2 binding site, a fourth variable-length phase-shift sequence n+3 nucleotides in length, and the region homologous to a 5′ region the sample DNA;a fifth primer comprising from 5′ to 3′, a second SP2 binding site, a fifth variable-length phase-shift sequence n nucleotides in length, and a region homologous to a 3′ region of the sample DNA;a sixth primer comprising from 5′ to 3′, the second SP2 binding site, a sixth variable-length phase-shift sequence n+1 nucleotides in length, and the region homologous to a 3′ region of the sample DNA;a seventh primer comprising from 5′ to 3′ the second SP2 binding site, a seventh variable-length phase-shift sequence n+2 nucleotides in length, and the region homologous to a 3′ region of the sample DNA; andan eighth primer comprising from 5′ to 3′, the second SP2 binding site, an eighth variable-length phase-shift sequence n+3 nucleotides in length, and the region ...

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Номер записи: 17
25-02-2021 дата публикации

ENCAPSULATION OF EUKARYOTIC CELLS FOR CELLULAR SCREENING OF EXPRESSED SEQUENCES

Номер: US20210054530A1
Автор: Bathgate Ross, Scott Daniel James
Принадлежит:

Provided herein are methods for selecting polypeptides or proteins having one or more desired properties from a library of sequences expressed in eukaryotic cells, comprising: encapsulating said cells by photopolymerization; solubilizing said encapsulated cells to produce semipermeable microcapsules; optionally contacting said cells and/or said microcapsules with one or more agents to facilitate detection of activity or function of polypeptides or proteins of interest; and selecting polypeptides or proteins of interest having one or more desired properties. Also provided are methods for encapsulating eukaryotic cells for use in the selection of polypeptides and proteins as described above. 1. A method for selecting polypeptides or proteins having one or more desired properties from a library of sequences expressed in eukaryotic cells , comprising:(i) encapsulating said cells by photopolymerization;(ii) solubilizing said encapsulated cells to produce semipermeable microcapsules;(iii) optionally contacting said cells and/or said microcapsules with one or more agents to facilitate detection of activity or function of polypeptides or proteins of interest; and(iv) selecting polypeptides or proteins of interest having one or more desired properties.2. The method according to claim 1 , wherein step (iii) occurs prior to step (i) or step (ii) claim 1 , concurrently with step (i) or step (ii) claim 1 , or subsequent to step (ii).3. The method according to claim 1 , wherein said contacting comprises two or more contacting steps claim 1 , with the same or different agents claim 1 , and wherein at least one contacting step occurs prior to said encapsulating or solubilizing and at least one contacting step occurs subsequent to said encapsulating or solubilizing.4. The method according to claim 1 , wherein the eukaryotic cells are insect cells or mammalian cells.5. The method according to claim 1 , wherein the photopolymerization comprises photopolymerization of a poly(ethylene ...

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Номер записи: 18
21-02-2019 дата публикации

IDENTIFICATION AND ISOLATION OF ANTIBODIES FROM WHITE BLOOD CELLS

Номер: US20190056398A1
Автор: Ao Zheng, Collins Nathan, Johanning Gary L., Liu Xiaohe, Sambucetti Lidia, WANG-JOHANNING Feng
Принадлежит:

Embodiments in accordance with the present disclosure include apparatuses, devices, and methods. For example, a method is directed to method exposing immobilized white blood cells from a blood sample to an antigen. And, scanning the immobilized white blood cells and, therefrom, identify and isolate white blood cells from among the immobilized white blood cells that produce an antibody in response to the exposure to the antigen. 1. A method comprising:exposing immobilized white blood cells from a blood sample to an antigen; andscanning the immobilized white blood cells and, therefrom, identify and isolate white blood cells from among the immobilized white blood cells that produce an antibody (Ab) in response to the exposure to the antigen.2. The method of claim 1 , further including scanning a whole white blood cell complement of the blood sample at a rate of 1 million to 25 million cells per minute.3. The method of claim 1 , further including immobilizing the white blood cells on a substrate while maintaining cell viability and exposing the immobilized white blood cells to the antigen by treating the white blood cells with the antigen which is labeled.4. The method of claim 1 , further including isolating the white blood cells individually using cell picking circuitry by identifying antigens bound to an antibody on a surface of or near white blood cells.5. The method of claim 1 , further including assessing efficacy of the produced Abs.6. The method of claim 1 , further including assessing an ability of the produced Abs to neutralize target cells associated with the antigen.7. The method of claim 1 , further including cloning at least one of the produced Abs and using the cloned Ab as at least one selected from the group consisting of: a diagnostic agent claim 1 , a sensor claim 1 , a therapeutic agent claim 1 , and a combination thereof.8. A method comprising:causing a substrate coated with an antigen to contact a multiple-well array and thereby exposing a ...

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Номер записи: 19
08-03-2018 дата публикации

OLIGONUCLEOTIDE PROBES AND USES THEREOF

Номер: US20180066262A1
Автор: DOMENYUK Valeriy, MIGLARESE Mark, Spetzler David, STARK Adam, XIAO Nianqing, ZHONG Zhenyu
Принадлежит:

Methods and compositions are provided for oligonucleotide probes and oligonucleotide probe libraries that recognize targets of interest. The targets include circulating biomarkers such as microvesicles, including those derived from various diseases. 1. An oligonucleotide comprising a sequence according to any one of SEQ ID NOs 137-969 and 1072-4150.2. An oligonucleotide comprising a sequence according to any one of the SEQ ID NOs in Table 40.3. An oligonucleotide comprising a sequence according to any one of the SEQ ID NOs in the row “2000v1” in Table 43.4. An oligonucleotide comprising a sequence according to any one of the SEQ ID NOs in the row “2000v2” in Table 43.5. An oligonucleotide comprising a sequence according to any one of the SEQ ID NOs in the row “Common” in Table 43.6. An oligonucleotide comprising a sequence according to any one of the SEQ ID NOs in any preceding claim and further having a 5′ region with sequence 5′-CTAGCATGACTGCAGTACGT (SEQ ID NO. 131) and/or a 3′ region with sequence 5′-CTGTCTCTTATACACATCTGACGCTGCCGACGA (SEQ ID NO. 132).7. An oligonucleotide comprising a nucleic acid sequence or a portion thereof that is at least 50 , 55 , 60 , 65 , 70 , 75 , 80 , 85 , 90 , 95 , 96 , 97 , 98 , 99 or 100 percent homologous to an oligonucleotide sequence according to any preceding claim.8. A plurality of oligonucleotides comprising at least 1 claim 7 , 2 claim 7 , 3 claim 7 , 4 claim 7 , 5 claim 7 , 6 claim 7 , 7 claim 7 , 8 claim 7 , 9 claim 7 , 10 claim 7 , 11 claim 7 , 12 claim 7 , 13 claim 7 , 14 claim 7 , 15 claim 7 , 16 claim 7 , 17 claim 7 , 18 claim 7 , 19 claim 7 , 20 claim 7 , 25 claim 7 , 30 claim 7 , 40 claim 7 , 45 claim 7 , 50 claim 7 , 55 claim 7 , 60 claim 7 , 65 claim 7 , 70 claim 7 , 75 claim 7 , 80 claim 7 , 85 claim 7 , 90 claim 7 , 95 claim 7 , 100 claim 7 , 125 claim 7 , 150 claim 7 , 175 claim 7 , 200 claim 7 , 300 claim 7 , 400 claim 7 , 500 claim 7 , 600 claim 7 , 700 claim 7 , 800 claim 7 , 900 claim 7 , 1000 claim 7 , 2000 ...

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Номер записи: 20
27-02-2020 дата публикации

FUNCTIONALIZED GEL BEADS

Номер: US20200063191A1
Автор: Bent Zachary, Gagnon Alex, Meer Elliott, RIORDAN Daniel, Ryvkin Paul, Srinivas Niranjan, Terry Jessica
Принадлежит:

The present disclosure provides methods of generating supports (e.g., beads) comprising barcode molecules coupled thereto. A barcode molecule coupled to a support may comprise a barcode sequence and a functional sequence. A barcode molecule may be generated using two or more ligation reactions in a combinatorial fashion. A support comprising two or more different barcode molecules may be useful for analyzing or processing one or more analytes such as nucleic acid molecules, proteins, and/or perturbation agents. 130-. (canceled)31. A composition comprising a nucleic acid molecule comprising a sequence selected from the group consisting of SEQ ID NO: 1-163.32. The composition of claim 31 , wherein said nucleic acid molecule further comprises a nucleic acid barcode sequence.33. The composition of claim 31 , wherein said sequence is SEQ ID NO: 155.34. The composition of claim 31 , wherein said sequence is SEQ ID NO: 156.35. The composition of claim 31 , further comprising a support having said nucleic acid molecule coupled thereto.36. The composition of claim 35 , wherein said support is a bead.37. The composition of claim 36 , wherein said bead is a gel bead.38. The composition of claim 35 , wherein said nucleic acid molecule is coupled to said support via a labile moiety.39. The composition of claim 38 , wherein said labile moiety is a disulfide bond.40. The composition of claim 35 , wherein said support comprises a labile moiety.41. The composition of claim 40 , wherein said labile moiety is a disulfide bond.42. The composition of claim 35 , wherein said support is coupled to an additional nucleic acid molecule.43. The composition of claim 42 , wherein said additional nucleic acid molecule comprises an additional sequence selected from the group consisting of SEQ ID NO: 1-163.44. The composition of claim 43 , wherein said sequence is different than said additional sequence.45. The composition of claim 44 , wherein said sequence is SEQ ID NO: 155 and said additional ...

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Номер записи: 21
27-02-2020 дата публикации

AUTOMATED INSTRUMENTATION FOR PRODUCTION OF T-CELL RECEPTOR PEPTIDE LIBRARIES

Номер: US20200063288A1
Автор: Church Deanna, Federowicz Stephen, Graige Michael
Принадлежит:

The present disclosure provides instrumentation and automated methods for creating cell surface display libraries, where the cells of the library display engineered peptides on their cell surfaces for identification of antigens that bind to T-cell receptors. The engineered peptides may be putative antigens or binding regions of the T-cell receptors. 1. An automated method of creating a cell library co-expressing engineered peptides and MHC molecules using instrumentation , the method comprising:providing a population of cells;processing the population of cells using an instrument for multiplexed nuclease-directed genome editing using introduced nucleic acids and a nucleic acid-directed nuclease to create cells, wherein the introduced nucleic acids comprise nucleic acids that encode engineered peptides and MHC molecules configured to be displayed on a surface of the cells;incubating the processed cells to facilitate nucleic acid editing in the cells, wherein the edited cells co-express the engineered peptide antigens and MHC molecules in the cells; andallowing the cells to display the engineered peptides and MHC molecules on the surface of the cells.2. The method of claim 1 , wherein the engineered peptides are putative TCR binding antigens.3. The method of claim 1 , wherein the engineered peptides comprise predicted TCR binding regions.4. The method of claim 1 , wherein the population of cells are yeast cells.5. The method of claim 1 , wherein the nuclease is an RNA-directed nuclease.6. A cell library produced using the method of .7. An automated method of creating a cell library co-expressing engineered putative T-cell receptor (TCR) antigens and MHC molecules on a surface of cells claim 1 , the method comprising:providing a population of cells;processing the population of cells using an instrument for multiplexed nuclease-directed genome editing using introduced nucleic acids and a nuclease to create cells, wherein the introduced nucleic acids comprise nucleic ...

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Номер записи: 22
12-03-2020 дата публикации

Expressed Barcode Libraries and Uses Thereof

Номер: US20200080076A1
Автор: Yaara Oren
Принадлежит: Broad Institute Inc

Disclosed herein are nucleic acid molecules comprising molecular barcodes having a variable sequence operably linked to a promoter and compositions comprising such nucleic acid molecules. Further disclosed herein are methods for simultaneously mapping the lineage and the transcriptional state of a cell.

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Номер записи: 23
25-03-2021 дата публикации

METHODS FOR ANALYZING T CELL RECEPTORS AND B CELL RECEPTORS

Номер: US20210087610A9
Автор: Gierahn Todd Michael, Love J. Christopher, Tu Ang
Принадлежит: Massachusetts Institute of Technology

The invention includes methods for enriching for T cell receptor- or B cell receptor-encoding transcripts from a single cell RNA sequencing library, (ii) sequencing T cell receptor- or B cell receptor-encoding transcripts from a single cell RNA sequencing library, and (iii) targeted tagmentation of T cell receptor- or B cell receptor-encoding transcripts. 1. A method for enriching for T cell receptor- or B cell receptor-encoding transcripts in a library of transcripts comprising:providing a library of transcripts from a plurality of cells, with transcripts from each cell identified by a unique barcode;contacting the library of transcripts with a labeled oligonucleotide that is complementary to a constant region of the T cell receptor- or B cell receptor-encoding transcripts; andseparating the labeled oligonucleotide hybridized to the T cell receptor- or B cell receptor-encoding transcripts from the library of transcripts,thereby obtaining a population of transcripts enriched for T cell receptor- or B cell receptor-encoding transcripts.2. A method for identifying a T cell receptor- or B cell receptor in a cell comprising:providing a library of transcripts from a plurality of cells, with each transcript having a 5′ universal primer site followed by a barcode that is unique to each cell;contacting the library of transcripts with a first sequencing primer that is complementary to the universal primer site and a second sequencing primer that is complementary to a constant region of T cell receptor- or B cell receptor-encoding transcripts;sequencing from both primers in the same read, such that barcode sequence data for a T cell receptor- or B cell receptor-encoding transcript is generated in conjunction with CDR3 sequence data.3. A method for producing a library of T cell receptor- or B cell receptor-encoding transcripts , each comprising a same primer sequence at a uniform position in the variable region comprising:providing:(i) a library of T cell receptor- or B cell ...

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Номер записи: 24
25-03-2021 дата публикации

Methods And Kits For Theranostic Applications

Номер: US20210087612A1
Автор: Dunn Ian, Lawler Matthew
Принадлежит:

The present disclosure is directed to methods and kits for identifying, enriching, and evaluating templated assembly reactants. Some embodiments disclose methods for identifying templated assembly targets by synthesizing templated assembly reactants, hybridizing the templated assembly reactants to target nucleic acids, performing a templated assembly reaction, and identifying the target nucleic acids that hybridized to the templated assembly reactants. Libraries of templated assembly reactants, a kit for identifying templated assembly targets, and a pair of templated assembly targets enriched from a library of chemically-ligated oligonucleotides spatially elicited (CLOSE) products are also disclosed. 1. A method for identifying templated assembly targets comprising:synthesizing a first population of templated assembly reactants and a second population of corresponding templated assembly reactants, wherein the first and second populations of templated assembly reactants comprise oligonucleotide sequences;hybridizing both populations of templated assembly reactants to target nucleic acids;performing a templated assembly reaction, wherein the hybridized first population of templated assembly reactants and the hybridized second population of corresponding templated assembly reactants undergo templated assembly; andidentifying the target nucleic acids that hybridized to either the first or second population of templated assembly reactants that underwent templated assembly, wherein the hybridized target nucleic acids are the templated assembly targets.214-. (canceled)15. A library of templated assembly reactants for identifying templated assembly targets comprising at least a first and second population of templated assembly reactants , wherein the templated assembly reactants comprise an oligonucleotide sequence and a modification for reaction in a templated assembly reaction.16. The library of claim 15 , wherein the templated assembly reactants further comprise either a ...

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Номер записи: 25
19-03-2020 дата публикации

SYSTEM AND METHOD FOR ISOLATION AND QUALIFICATION OF NUCLEIC ACIDS

Номер: US20200087708A1
Автор: Beckert Sophie, Constandse Victoria, Hinzmann Bernd, Klass Daniel, Lovejoy Alexander, Loyzer Melissa, Miller Bronwen
Принадлежит:

Present disclosure provides a method including isolating DNA from a source, thereby providing a composition including the isolated DNA. The isolated DNA has at least first and second target regions, where the length of the second target region is greater than the length of the first target region. The method further includes quantifying a total mass of the isolated DNA, quantifying a first quantification cycle (C) of the first target region and a second Cof the second target region, and calculating a Q-ratio for the isolated DNA by dividing the second Cby the first C. The method further includes determining a value for a quality-mass constant (k), estimating a required input mass by dividing kby the Q-ratio, and preparing the isolated DNA for sequencing if the total mass of the isolated DNA in the composition is equal or greater than the required input mass. 1. A method , comprising:(a) isolating DNA from a source, thereby providing a composition comprising the isolated DNA, the isolated DNA having at least a first target region having a first length in nucleotides and a second target region having a second length in nucleotides, the second length being greater than the first length;(b) quantifying a total mass of the isolated DNA in the composition;{'sub': q', 'q, '(c) quantifying a first quantification cycle (C) of the first target region and a second Cof the second target region in the composition;'}{'sub': q', 'q, '(d) calculating a Q-ratio for the isolated DNA by dividing the second Cby the first C;'}{'sub': 'Qm', '(e) determining a value for a quality-mass constant (k);'}{'sub': 'Qm', '(f) estimating a required input mass for the isolated DNA by dividing the quality-mass constant (k) by the Q-ratio; and'}(g) preparing the isolated DNA for sequencing if the total mass of the isolated DNA in the composition is equal or greater than the required input mass.2. The method of claim 1 , wherein the length of the second target region is about 2 to about 10 times ...

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Номер записи: 26
01-04-2021 дата публикации

NUCLEIC ACID-GUIDED EDITING OF EXOGENOUS POLYNUCLEOTIDES IN HETEROLOGOUS CELLS

Номер: US20210095394A1
Автор: Fox Richard, Held Daniel
Принадлежит:

The present disclosure provides shuttle vectors for editing exogenous polynucleotides in heterologous live cells, as well as automated methods, modules, and multi-module cell editing instruments and systems for performing the editing methods. 1. A method of nucleic acid-guided nuclease editing of five hundred or more exogenous polynucleotides from source cells within heterologous editing cells in a single multiplexed automated operation , comprising:inserting five hundred or more different target polynucleotides from the source cells into shuttle vector backbones to form a library of shuttle vectors;transferring the library of shuttle vectors into a first receptacle;providing heterologous editing cells in a second receptacle;growing the heterologous editing cells in a growth module;transferring the heterologous editing cells from the growth module to a transformation module;transferring the library of shuttle vectors from the first receptacle to the transformation module;introducing the library of shuttle vectors into the heterologous editing cells in the transformation module;providing one or more editing vectors wherein the editing vectors comprise a coding sequence for a nuclease, a guide nucleic acid and a DNA donor sequence in a third receptacle;transferring the editing vectors from the third receptacle to the transformation module;introducing the one or more editing vectors into the heterologous editing cells in the transformation module;transferring the heterologous editing cells from the transformation module to an editing module;allowing editing to take place in the editing module under conditions that allow the editing vectors to edit the one or more target polynucleotides in the shuttle vectors thereby forming edited shuttle vectors; andisolating the living edited cells comprising the edited shuttle vectors; andisolating the edited shuttle vectors; wherein the first receptacle, second receptacle, third receptacle, growth module, cell concentration module, ...

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Номер записи: 27
28-03-2019 дата публикации

Enrichment of short nucleic acid fragments in sequencing library preparation

Номер: US20190093102A1
Автор: Alex Aravanis, Byoungsok Jung
Принадлежит: Grail LLC

Methods for preparing enriched sequencing libraries from test samples that contain double-stranded deoxyribonucleic acid (dsDNA) are provided.

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Номер записи: 28
28-03-2019 дата публикации

DNA BARCODING OF DESIGNER MONONUCLEOSOME AND CHROMATIN ARRAY LIBRARIES FOR THE PROFILING OF CHROMATIN READERS, WRITERS, ERASERS, AND MODULATORS THEREOF

Номер: US20190093159A1
Автор: MUELLER Manuel M., Muir Tom W., NGUYEN Uyen T. T.
Принадлежит: THE TRUSTEES OF PRINCETON UNIVERSITY

Compositions and methods are provided for DNA barcoding of designer mononucleosome and polynucleosome (chromatin array) libraries for use, for example, for the profiling of chromatin readers, writers, erasers, and modulators thereof. 1. A library of synthetic mononucleosomes claim 15 , comprising two or more synthetic mononucleosomes of claim 15 ,wherein each synthetic mononucleosome member of the library hasone or more unique DNA barcode(s) indicative of the pattern of nucleosomal modifications in that member of the library.2. A synthetic polynucleosome claim 15 , comprising two or more synthetic mononucleosomes bonded together by a defined DNA molecule claim 15 , the mononucleosomes having a defined connectivity claim 15 ,wherein at least one of the mononucleosomes comprises a complex of(a) a protein octamer, containing 2 copies each of histones H2A, H2B, H3, and H4, and optionally, a linker histone, wherein at least one of the histones is unmodified, and/or at least one of the histones is modified to form a pattern of mononucleosomal histone modifications, and (i) a nucleosome positioning sequence (NPS), wherein the NPS is a nucleotide sequence that stably complexes with one or more of the histones, and', '(ii) DNA extension(s), on the 5′- and/or 3′-end of the NPS and/or within the NPS,', 'wherein the DNA extension is unmodified and/or wherein at least one nucleotide in the DNA extension is modified, to form a pattern of mononucleosomal DNA modifications,', 'and optionally, '(b) a mononucleosomal DNA molecule, comprising'}(c) one or more non-histone chromatin-associated protein(s),wherein the pattern of mononucleosomal histone modifications and/or the pattern of mononucleosomal DNA modifications in the polynucleosome may be uniform or may be different, resulting in a pattern of polynucleosomal modifications,wherein the polynucleosome comprises one or more unique DNA barcode(s) located at defined position(s) in the DNA, and whose sequence and position in the DNA ...

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Номер записи: 29
12-04-2018 дата публикации

METHOD FOR NUCLEOTIDE DETECTION

Номер: US20180100193A1
Автор: Hall Kevin, Klausing Kay, MOORE John, Shen Min-Jui Richard, Smith Vincent Peter
Принадлежит:

A method of inhibiting light-induced degradation of nucleic acids includes irradiating a portion of the nucleic acids in the presence of a detection solution comprising a polyphenolic compound. A method of detecting a nucleic acid having a fluorescent tag includes irradiating at least a portion of the nucleic acid with light of a suitable wavelength to induce a fluorescence emission and detecting the fluorescence emission. Optionally, the polyphenolic compound is gallic acid, a lower alkyl ester thereof, or mixtures thereof. A kit includes one or more nucleotides, an enzyme capable of catalyzing incorporation of the nucleotides into a nucleic acid strand and a polyphenolic compound suitable for preparing a detection solution. 2. The method of claim 1 , wherein said nucleic acid is in an array of nucleic acids attached to a support.3. The method of claim 1 , further comprising adding a fluorescently tagged nucleotide to said nucleic acid.4. The method of claim 1 , further comprising replacing a solution with said detection solution prior to the irradiation step.5. The method of claim 2 , wherein inhibiting light-induced degradation to said nucleic acid comprises reducing the cleavage of a nucleic acid member from said array.6. The method of claim 1 , wherein said gallic acid claim 1 , said lower alkyl ester thereof claim 1 , or said mixtures thereof is present in a concentration ranging from between about 10 mM to about 200 mM.7. The method of claim 1 , further comprising one or more compound(s) claim 1 , wherein said one or more compound(s) further enhances preservation of the integrity of said nucleic acid.8. The method of claim 7 , wherein said one or more compound(s) is selected from urea claim 7 , ascorbic acid or salt thereof claim 7 , and isoascorbic acid or salt thereof.9. The method of claim 1 , comprising at least 50 cycles repeating step c.10. The method of claim 1 , comprising repeating said adding and detection steps for a number of cycles in a range ...

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Номер записи: 30
26-03-2020 дата публикации

Methods for analyzing t cell receptors and b cell receptors

Номер: US20200095625A1
Автор: Ang TU, J. Christopher Love, Todd Michael GIERAHN
Принадлежит: Massachusetts Institute of Technology

The invention includes methods for enriching for T cell receptor- or B cell receptor-encoding transcripts from a single cell RNA sequencing library, (ii) sequencing T cell receptor- or B cell receptor-encoding transcripts from a single cell RNA sequencing library, and (iii) targeted tagmentation of T cell receptor- or B cell receptor-encoding transcripts.

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Номер записи: 31
02-06-2022 дата публикации

OLIGONUCLEOTIDE PROBES AND USES THEREOF

Номер: US20220170021A1
Автор: DOMENYUK Valeriy, MIGLARESE Mark, Spetzler David, STARK Adam, XIAO Nianqing, ZHONG Zhenyu
Принадлежит:

Methods and compositions are provided for oligonucleotide probes and oligonucleotide probe libraries that recognize targets of interest. The targets include circulating biomarkers such as microvesicles, including those derived from various diseases. 169-. (canceled)70. A method of generating a single-stranded DNA (ssDNA) molecule comprising: [ (1) a 5′ leader region comprising a lengthener region, a terminator region, and a forward primer region;', '(2) a variable region positioned 3′ of the leader region; and', '(3) a tail region positioned 3′ of the variable region and comprising a reverse primer region; and, '(i) a nucleic acid molecule comprising, '(ii) forward and reverse primers configured to amplify the nucleic acid molecule from the forward primer region and reverse primer region, respectively; and, '(a) providing a mixture comprising(b) performing asymmetric polymerase chain reaction (PCR) on the mixture in a) to favorably amplify the reverse strand of the nucleic acid molecule, wherein the forward and reverse primers in the mixture are at a ratio of about 1:37.5 (F/R) in favor of the reverse primers; thereby generating the ssDNA molecule.71. (canceled)72. (canceled)73. The method of claim 70 , further comprising isolating the amplified reverse strand of the nucleic acid molecule on a native gel.74. The method of claim 70 , further comprising:(c) denaturing the amplified nucleic acid molecules from b); and(d) isolating the denatured reverse strand of the nucleic acid molecules from c).75. The method of claim 74 , wherein the denatured reverse strand of the nucleic acid molecules is isolated on a denaturing gel.76. The method of claim 70 , wherein the mixture in a) further comprises at least one of an enrichment buffer claim 70 , non-target molecules claim 70 , proteins claim 70 , microvesicles claim 70 , and polyethylene glycol.7779-. (canceled)80. The method of claim 70 , wherein the lengthener region comprises at least 10 claim 70 , 11 claim 70 , 12 claim ...

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Номер записи: 32
02-04-2020 дата публикации

METHODS AND COMPOSITIONS FOR IDENTIFYING EPITOPES

Номер: US20200102553A1
Автор: Dezfulian Mohammad Haj, Elledge Stephen, Kula Tomasz
Принадлежит:

Described herein, in one aspect, are antigen presenting cells (APCs) comprising an exogenous nucleic acid encoding one or more candidate antigens, wherein the one or more candidate antigens are expressed and presented with MHC class I or MC class II molecules; a molecular reporter of Granzyme B (GzB) activity; and c) an exogenous inhibitor of caspase-activated deoxyribonuclease (CAD)-mediated DNA degradation, a CAD knockout, or a caspase knockout (e.g., caspase 3 knockout). Described herein, in another aspect, is a system for detection of recognized antigen presentation by an antigen presenting cell to a cytotoxic lymphocyte or NK cell. 1. An antigen presenting cell (APC) comprising:a) an exogenous nucleic acid encoding one or more candidate antigens, wherein the one or more candidate antigens are expressed and presented with MHC class I or MHC class II molecules;b) a molecular reporter of Granzyme B (GzB) activity; andc) an exogenous inhibitor of caspase-activated deoxyribonuclease (CAD)-mediated DNA degradation, a CAD knockout, or a caspase knockout.2. The APC of claim 1 , wherein the exogenous nucleic acid is stably introduced into the genome of the APC claim 1 , optionally via a lentiviral vector claim 1 , a retroviral vector claim 1 , or a transposon.3. The APC of any one of - claim 1 , wherein the exogenous nucleic acid is flanked on each side by predetermined primer recognition sequences.4. The APC of any one of - claim 1 , wherein the molecular reporter of GzB activity comprises a fusion polypeptide comprising a GzB cleavage site (VGPD claim 1 , SEQ ID NO:1) linked to a detection molecule.5. The APC of claim 4 , wherein the molecular reporter comprises a modified infrared fluorescent protein claim 4 , a membrane tethered CRE recombinase claim 4 , an antibody-based reporter of GzB activity claim 4 , an ER retention-based reporter of GzB activity claim 4 , a cell surface detectable-based reporter of GzB activity claim 4 , or combinations thereof.6. The APC of ...

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Номер записи: 33
02-04-2020 дата публикации

EFFICIENT COMBINATORIAL BEAD BARCODING

Номер: US20200102556A1
Автор: da Veiga Beltrame Eduardo, Dilanyan Taleen, Gehring Jase, Pachter Lior S., Tambe Akshay
Принадлежит:

Disclosed herein include methods, compositions, and systems for extending particle-associated oligonucleotides. In some embodiments, the methods of extending particle-associated oligonucleotides enable efficient methods of preparing a library of barcoded beads. It is also provided, in some embodiments, hydrogel beads degradable upon application of a chemical stimulus that comprise releasably attached oligonucleotides. 1. A method of extending particle-associated oligonucleotides , comprising:(a) providing a particle comprising a plurality of oligonucleotides each comprising an input primer; (i) an unpaired first 3′ toehold domain complementary to the input primer,', '(ii) a first paired stem domain formed by intramolecular nucleotide base pairing between a 3′ subdomain of the first hairpin molecule and a 5′ subdomain of the first hairpin molecule, wherein the first paired stem domain comprises a first coupling segment, a first payload segment, and complements thereof, and', '(iii) a first hairpin loop domain; and, '(b) contacting the particle with a first hairpin molecule, wherein the first hairpin molecule comprises(c) extending the input primer hybridized to the first 3′ toehold domain through the first paired stem domain of the first hairpin molecule, thereby displacing the 5′ subdomain of the first hairpin molecule and forming a first extended oligonucleotide comprising the input primer, the first payload segment and the first coupling segment.2. The method of claim 1 , further comprising: (i) an unpaired second 3′ toehold domain complementary to the first coupling segment,', '(ii) a second paired stem domain formed by intramolecular nucleotide base pairing between a 3′ subdomain of the second hairpin molecule and a 5′ subdomain of the second hairpin molecule, wherein the second paired stem domain comprises a second coupling segment, a second payload segment, and complements thereof, and', '(iii) a second hairpin loop domain; and, '(d) contacting the particle ...

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Номер записи: 34
09-06-2022 дата публикации

METHODS AND SYSTEMS FOR MICROFLUIDIC SCREENING

Номер: US20220176374A1
Автор: CAYER Devon, CHUBUKOV Pavel, MACCONNELL Andrew, RAMJI Ramesh, STROMBERG Sean
Принадлежит:

Provided are methods and systems useful for screening large libraries of effector molecules. Such methods and systems are particularly useful in microfluidic systems and devices. The methods and systems provided herein utilize encoded effectors to screen large libraries of effectors. 1. A method for screening an encoded effector , the method comprising:(a) providing at least one cell and a scaffold in an encapsulation, wherein the scaffold comprises an encoded effector bound to the scaffold by a photocleavable linker and a nucleic acid encoding the effector;(b) cleaving the photocleavable linker to release the encoded effector from the scaffold; and(c) detecting a signal from the droplet, wherein the signal results from an interaction between the encoded effector and the at least one cell.2. The method of claim 1 , wherein cleaving the photocleavable linker releases a pre-determined amount of the encoded effector into the droplet.3. The method of claim 1 , wherein the photocleavable linker is cleaved using electromagnetic radiation.4. The method of claim 1 , wherein cleaving the photocleavable linker comprises exposing the encapsulation to a light from a light source.5. The method of claim 4 , wherein the light intensity of the light is from about 0.01 J/cmto about 200 J/cm.6. The method claim 1 , further comprising the step of lysing the one or more cells.7. The method of claim 1 , further comprising providing an activating reagent to activate the photocleavable linker claim 1 , so as to enable the photocleavable linker to be cleaved from the encoded effector.8. A system for screening an encoded effector claim 1 , the system comprising:(a) one or more cells;(b) a scaffold, wherein an encoded effector is bound to the scaffold by a cleavable linker, wherein a nucleic acid encoding the effector is bound to the scaffold; and (i) receive the one or more cells and scaffold;', '(ii) encapsulate the one or more cells and scaffold within an encapsulation;', '(iii) cleave ...

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Номер записи: 35
09-06-2022 дата публикации

METHODS AND SYSTEMS FOR MICROFLUIDIC SCREENING

Номер: US20220176375A1
Автор: CAYER Devon, CHUBUKOV Pavel, MACCONNELL Andrew, RAMJI Ramesh, STROMBERG Sean
Принадлежит:

Provided are methods and systems useful for screening large libraries of effector molecules. Such methods and systems are particularly useful in microfluidic systems and devices. The methods and systems provided herein utilize encoded effectors to screen large libraries of effectors. 1. A method for amplifying a primer to maximize cellular nucleic acid capture , the method comprising:(a) providing an encapsulation encapsulating a scaffold, one or more cells, an amplification mix, and a nicking enzyme, wherein a nucleic acid encoding is bound to the scaffold;(b) lysing the one or more cells to release one or more cellular nucleic acids;(c) nicking the nucleic acid encoding with the nicking enzyme, thereby creating a first encoded nucleic acid primer;(d) amplifying the first encoded nucleic acid primer via the nicking site and amplification mix; and(e) labeling the one or more cellular nucleic acids with the first encoded nucleic acid primer.2. The method of claim 1 , wherein the nicking enzyme targets a specific site in the nucleic acid encoding claim 1 , wherein the specific site comprises a specific nucleotide sequence.3. The method of claim 1 , wherein the amplifying of d) comprises i) creating a copy of the nucleic acid encoding that extends from the nicking site; and ii) nicking the copy of the nucleic acid encoding to create a second encoded nucleic acid primer.4. The method of claim 1 , wherein the amplifying of d) comprises simultaneously i) creating a copy of the nucleic acid encoding that extends from the nicking site; and ii) displacing the copy of the nucleic acid encoding to create a second encoded nucleic acid primer.5. The method of claim 4 , wherein the amplification mix comprises an amplification enzyme claim 4 , such that the amplification enzyme enables the copy of the nucleic acid encoding to be simultaneously created and displaced.6. The method of claim 5 , wherein the amplification enzyme comprises a polymerase.7. The method of claim 1 , wherein ...

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Номер записи: 36
18-04-2019 дата публикации

DROPLET-BASED LINKED-FRAGMENT SEQUENCING

Номер: US20190112654A1
Автор: Marziali Andrea, Pel Joel
Принадлежит:

The invention generally relates to sequencing library preparation methods. In certain embodiments, two or more template nucleic acids are joined together by a linking molecule, such as a PEG derivative. Identical copies of a nucleic acid fragment or both strands of a duplex fragment may be linked together. The linked nucleic acids are amplified, creating linked amplicons. Emulsion PCR with linked primers creates linked template nucleic acids for seeding sequencing clusters and errors can be readily identified by their presence on only one of the linked fragments. 1. A method of sequencing a nucleic acid , the method comprising:joining a plurality of amplification primers with a linker;forming a droplet comprising a nucleic acid fragment, the linked plurality of amplification primers, and multiplexed amplification primers;amplifying the nucleic acid fragment in the droplet, thereby creating a complex comprising joined copies of the nucleic acid fragment;annealing the complex to a solid support comprising binding sites for the complex;amplifying the complex to create a cluster of amplification products; andsequencing the amplification products.2. The method of claim 1 , wherein the linked plurality of amplification primers are universal amplification primers.3. The method of claim 1 , further comprising ligating an adapter to the nucleic acid fragment claim 1 , said adapter comprising a universal primer site corresponding to the universal amplification primers.4. The method of claim 1 , wherein the multiplexed amplification primers are gene specific to a region of interest to be sequenced.5. The method of claim 1 , wherein the linker is selected from the group consisting of a polyethylene glycol derivative claim 1 , an oligosaccharide claim 1 , a lipid claim 1 , a hydrocarbon claim 1 , a polymer claim 1 , an inverted base claim 1 , and a protein.6. The method of claim 1 , wherein the linker comprises streptavidin and the plurality of amplification primers comprise ...

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Номер записи: 37
18-04-2019 дата публикации

PLATFORM FOR DISCOVERY AND ANALYSIS OF THERAPEUTIC AGENTS

Номер: US20190112730A1
Автор: Boutell Jonathan Mark, Golynskiy Misha, He Molly, KELLINGER Matthew William, Peisajovich Sergio, Previte Michael
Принадлежит: Illumina, Inc.

A method of characterizing candidate agents including steps of (a) providing a library of candidate agents attached to nucleic acid tags; (b) contacting the library with a solid support to attach the candidate agents to the solid support, whereby an array of candidate agents is formed; (c) contacting the array with a screening agent, wherein one or more candidate agents in the array react with the screening agent; (d) detecting the array to determine that at least one candidate agent in the array reacts with the screening agent; (e) sequencing the nucleic acid tag to determine the tag sequences attached to candidate agents in the array; and (f) identifying the at least one candidate agent in the array that reacts with the screening agent based on the tag sequence that is attached to the at least one candidate agent. 1. A method of characterizing candidate agents , comprising(a) providing a library of candidate agents, wherein each candidate agent is attached to a nucleic acid tag haying a tag sequence;(b) conflicting the library of candidate agents with a solid support to attach the candidate agents to the solid support, whereby an array of candidate agents is formed comprising individual features on the solid support that each attach to an individual candidate agent from the library;(c) contacting the array of candidate agents with a screening agent, wherein one or more candidate agents in the array react with the screening agent;(d) detecting the array during or after the contacting of the array with the screening agent, thereby determining that at least one candidate agent in the array reacts with the screening agent;(e) sequencing the nucleic acid tags on the array to determine the tag sequence that is attached to each of the candidate agents; and(f) identifying the at least one candidate agent in the array that reacts with the screening agent based on the tag sequence that is attached to the at least one candidate agent.2. The method of claim 1 , wherein the ...

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Номер записи: 38
03-05-2018 дата публикации

METHODS FOR SINGLE-STRANDED NUCLEIC ACID LIBRARY PREPARATION

Номер: US20180119216A1
Автор: Amini Hamed, Jamshidi Arash
Принадлежит:

Aspects of the invention relate to methods and compositions for preparing and analyzing a single-stranded sequencing library from a double-stranded DNA (e.g., double-stranded cfDNA) sample. In some embodiments, the sample includes double-stranded DNA (dsDNA) molecules, and damaged dsDNA (e.g., nicked dsDNA) molecules. In some embodiments, the sample includes single-stranded DNA (ssDNA) molecules. The subject methods facilitate the collection of information, including strand-pairing and connectivity information, from dsDNA, ssDNA and damaged DNA (e.g., nicked DNA) molecules in a sample, thereby providing enhanced diagnostic information as compared to sequencing libraries that are prepared using conventional methods. 1. A method for preparing a single-stranded DNA library for sequencing , the method comprising the following steps:a. obtaining a test sample comprising double stranded DNA (dsDNA) and isolating dsDNA from the test sample;b. partitioning the dsDNA sample into a plurality of individual reaction compartments;c. adding a reaction mixture to each of said individual reaction compartments, said reaction mixture including a plurality of oligonucleotide comprising a unique sequence tag;d. denaturing dsDNA to produce single-strand DNA (ssDNA) fragments; ande. ligating unique sequence tags to the ssDNA fragments.2. A method for preparing a cell-free DNA library for sequencing , the method comprising the following steps:a. obtaining a test sample comprising cell-free double stranded DNA (dsDNA) and isolating dsDNA from the test sample;b. partitioning the dsDNA sample into a plurality of individual reaction droplets;c. adding a reaction mixture to each of said individual droplets, said reaction mixture including a plurality of DNA capture beads, wherein each of said DNA capture beads includes a plurality of attached oligonucleotides comprising unique sequence tag;d. heating the droplets to denature the dsDNA or chemically denaturing the dsDNA to produce single-strand ...

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Номер записи: 39
25-08-2022 дата публикации

METHODS AND SYSTEMS FOR MICROFLUIDIC SCREENING

Номер: US20220266250A1
Автор: CAYER Devon, CHUBUKOV Pavel, MACCONNELL Andrew, RAMJI Ramesh, STROMBERG Sean
Принадлежит:

Provided are methods and systems useful for screening large libraries of effector molecules. Such methods and systems are particularly useful in microfluidic systems and devices. The methods and systems provided herein utilize encoded effectors to screen large libraries of effectors. 120.-. (canceled)21. A method comprising:(a) providing or obtaining a microfluidic device comprising a droplet formation region connected to a flow path;(b) providing or obtaining an effector library comprising a plurality of different effectors, wherein an effector of the plurality of different effectors is bound to a scaffold of a plurality of scaffolds via a cleavable linker and is releasable from the scaffold upon cleavage of the cleavable linker, and wherein the scaffold further comprises an encoding corresponding to and for identifying a structure of the effector;(c) generating a plurality of droplets in the droplet formation region and encapsulating the effector library in the plurality of droplets, wherein each droplet of a subset of the plurality of droplets comprises at least one scaffold of the plurality of scaffolds;(d) incubating the plurality of droplets in the flow path for an incubation time, wherein the incubation time is at least 14 minutes and a dispersion ratio of incubation times of the plurality of droplets is less than 4%;(e) measuring a signal indicative of an activity of the effector from the plurality of droplets; and (i) sorting a droplet, contents of a droplet, a scaffold, an encoding, or any combination thereof, based on the signal;', '(ii) measuring a property of an encoding to identify a structure of the effector in a droplet, based on the signal; or', '(iii) adding a barcode to tag an encoding in the droplet, based on the signal., '(f) performing at least one of22. The method of claim 21 , wherein the dispersion ratio is at most 2.5%.23. The method of claim 21 , wherein the dispersion ratio is at most 3%.24. The method of claim 21 , wherein the ...

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Номер записи: 40
02-05-2019 дата публикации

Attribute Sieving and Profiling By Pooled Sanger Sequencing

Номер: US20190127777A1
Автор: Ghazala Hashmi, Michael Seul
Принадлежит: Bioinventors and Entrepreneurs Network LLC

Disclosed is a novel method of allele profiling, or nucleic acid sieving, with pooled Sanger sequencing as a first (aka “screening”) stage; where the first step is: amplifying a single sequence, delineated by forward and reverse primers which may represent a single exon, or a segment thereof, or a contiguous stretch of multiple exons and introns. The amplicons produced from a pool of samples include the amplified sequence, and these are next converted into fragments in the standard Sanger labeling reaction. Ambiguities will appear as superposed peaks at any heterozygous position of interest, as the origin of the variant signal cannot be uniquely attributable to a specific sample, or samples, in the pool. These ambiguities may be resolved by the allele profiling process; or, resolution can be done with source-tagged primers generating source-tagged amplicons, which generate position shifts in labels, which can be decoded to resolve the ambiguities.

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Номер записи: 41
23-04-2020 дата публикации

PRODUCTION OF ENCODED CHEMICAL LIBRARIES

Номер: US20200123534A1
Автор: Decurtins Willy, Franzini Raphael, Neri Dario, SCHEUERMANN Jörg, Wichert Moreno
Принадлежит:

This invention relates to the synthesis of nucleic acid-encoded chemical libraries using common adaptor sequences. Nucleic acid strands coupled to chemical moieties may be contacted with identifier oligonucleotides comprising coding sequences encoding the chemical moieties and an adaptor oligonucleotides, such that the adaptor oligonucleotide hybridizes to both the nucleic acid strands and the identifier oligonucleotides to allow ligation of the identifier oligonucleotides to the nucleic acid strands. The adaptor oligonucleotide is then removed. Nucleic acid-encoded chemical libraries, and methods of producing or screening such libraries are provided. 140.-. (canceled)41. A method of producing a nucleic acid encoded chemical library comprising; (a) providing a population of first nucleic acid strands, each nucleic acid strand being coupled or couplable to a member of a first diverse population of chemical moieties and comprising a non-hybridisable spacer,', such that the one or more adaptor oligonucleotides hybridize to the nucleic acid strands and the identifier oligonucleotides to form a partially double-stranded complex,', 'wherein each first nucleic acid strand is contacted with an identifier oligonucleotide comprising a first coding sequence that encodes a chemical moiety that is coupled or couplable to the first nucleic acid strand, and;', 'wherein each of said one or more adaptor oligonucleotides hybridizes to more than one first nucleic acid strand in the population and more than one different identifier oligonucleotide,, '(b) contacting the first nucleic acid strands with identifier oligonucleotides comprising a first coding sequence and one or more adaptor oligonucleotides,'}, '(c) ligating the first nucleic acid strands to the identifier oligonucleotides in the partially double-stranded complexes, such that the identifier oligonucleotides are incorporated into the first nucleic acid strands,', 'thereby producing a sub-library comprising first nucleic acid ...

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Номер записи: 42
08-09-2022 дата публикации

METHODS FOR STANDARDIZED SEQUENCING OF NUCLEIC ACIDS AND USES THEREOF

Номер: US20220282303A1
Автор: Blomquist Thomas, Crawford Erin, Willey James C.
Принадлежит: The University of Toledo

Methods for controlling for non-systematic error in an amplification-based next generation sequencing (NGS) library preparation are described, which method includes using an internal amplification control (IAC) sharing identical priming sites to a native nucleic acid target template of interest in a NGS library preparation. 2. The method of claim 1 , wherein preparation of the sequencing library comprises one or more of the following steps:1) nucleic acid fragmentation;2) in vivo cloning to attach flanking nucleic acid adaptor sequences;3) in vitro adaptor ligation;4) PCR based adaptor addition; and,5) unimolecular inversion probe type technology with, or without, polymerase fill-in, and ligation of probe to capture the sequence by circularization, with adaptor contained within the probe sequence.3. The method of claim 2 , wherein the nucleic acid adaptor acts as one or more of:a) sequencing primer recognition site,b) barcode sequence of nucleotides to deconvolute the sample that was prepared for sequencing during analysis, andc) universal nucleic acid site which allows for multi-template amplification, or further addition of fusion-tail sequences through amplification.4. The method of claim 2 , wherein the prepared sequencing library is analyzed on a sequencing instrument claim 2 , and a representative sampling of the library is sequenced.5. The method of claim 2 , wherein controlling for the majority of non-systematic errors introduced during NGS library preparation enables inter-laboratory comparison of quantitative NGS data.6. The method of claim 5 , further comprising conducting an inter-laboratory comparison of clinical molecular diagnostic results.7. The method of claim 1 , wherein obtaining samples one or more of: use of microfluidic device claim 1 , capillary electrophoresis claim 1 , an oligonucleotide array claim 1 , mass spectrometry claim 1 , and chromatography.8. The method of claim 1 , wherein the nucleic acid acts as a template for synthesis of a ...

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Номер записи: 43
10-06-2021 дата публикации

COMPOSITIONS AND METHODS FOR IMPROVING SAMPLE IDENTIFICATION IN INDEXED NUCLEIC ACID LIBRARIES

Номер: US20210172014A1
Автор: Bevis-Mott Claire, Khosroheidari Mahdieh, Vermaas Eric Hans
Принадлежит:

The present invention is concerned with compositions and methods for improving the rate of correct sample identification in indexed nucleic acid library preparations for multiplex next generation sequencing by modifying or blocking 5′ and 3′ ends of pooled indexed polynucleotides from multiple samples, with an optional exonuclease treatment, prior to amplification and sequencing. 121-. (canceled)22. A polynucleotide prepared for sequencing , comprisingan adapter-target-adapter sequence,wherein the adapter sequence comprises a library specific sequence,wherein the polynucleotide is double stranded in a region comprising the target and at least a portion of the adapter on both ends of the target, andwherein 5′ and 3′ ends of the polynucleotide in a region of the adapter sequence are single stranded,wherein the 5′ ends are modified to prevent digestion by an enzyme having 5′ exonuclease activity, andwherein the 3′ ends are modified to inhibit digestion by an enzyme having 3′ exonuclease activity, to inhibit addition of nucleotides to the 3′ end by an enzyme having polymerase activity, or to inhibit digestion by an enzyme having 3′ exonuclease activity and to inhibit addition of nucleotides to the 3′ end by an enzyme having polymerase activity.23. The polynucleotide according to claim 22 , wherein the 3′ ends are modified to inhibit digestion by an enzyme having 3′ exonuclease activity.24. The polynucleotide according to claim 23 , wherein the 3′ ends are modified to inhibit addition of nucleotides to the 3′ end by an enzyme having polymerase activity.25. The polynucleotide according to claim 22 , wherein the 3′ ends are modified to inhibit addition of nucleotides to the 3′ end by an enzyme having polymerase activity.26. The polynucleotide according to claim 22 , wherein the 3′ ends comprise dideoxynucleotide.27. The polynucleotide according to claim 22 , wherein the 5′ ends comprise a phosphorothioate bond.28. The polynucleotide according to claim 22 , wherein the 5′ ...

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Номер записи: 44
24-05-2018 дата публикации

PLATFORM FOR DISCOVERY AND ANALYSIS OF THERAPEUTIC AGENTS

Номер: US20180142378A1
Автор: Boutell Jonathan Mark, Golynskiy Misha, He Molly, KELLINGER Matthew William, Peisajovich Sergio, Previte Michael
Принадлежит:

A method of characterizing candidate agents including steps of (a) providing a library of candidate agents attached to nucleic acid tags; (b) contacting the library with a solid support to attach the candidate agents to the solid support, whereby an array of candidate agents is formed; (c) contacting the array with a screening agent, wherein one or more candidate agents in the array react with the screening agent; (d) detecting the array to determine that at least one candidate agent in the array reacts with the screening agent; (e) sequencing the nucleic acid tag to determine the tag sequences attached to candidate agents in the array; and (f) identifying the at least one candidate agent in the array that reacts with the screening agent based on the tag sequence that is attached to the at least one candidate agent. 1. A method of characterizing candidate agents , comprising(a) providing a library of candidate agents, wherein each candidate agent is attached to a nucleic acid tag having a tag sequence;(b) contacting the library of candidate agents with a solid support to attach the candidate agents to the solid support, whereby an array of candidate agents is formed comprising individual features on the solid support that each attach to an individual candidate agent from the library;(c) contacting the array of candidate agents with a screening agent, wherein one or more candidate agents in the array react with the screening agent;(d) detecting the array during or after the contacting of the array with the screening agent, thereby determining that at least one candidate agent in the array reacts with the screening agent;(e) sequencing the nucleic acid tags on the array to determine the tag sequence that is attached to each of the candidate agents; and(f) identifying the at least one candidate agent in the array that reacts with the screening agent based on the tag sequence that is attached to the at least one candidate agent.2. The method of claim 1 , wherein the ...

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Номер записи: 45
15-09-2022 дата публикации

OLIGONUCLEOTIDE PROBES AND USES THEREOF

Номер: US20220290124A1
Автор: DOMENYUK Valeriy, Hornung Tassilo, MIGLARESE Mark, Spetzler David
Принадлежит:

Methods and compositions are provided to identify oligonucleotides that bind targets of interest. The targets include tissues, cells, circulating biomarkers such as microvesicles, including those derived from various diseases. The oligonucleotides can be used in diagnostic and therapeutic applications. 1102-. (canceled)103. A method of enriching an oligonucleotide probe library comprising a plurality of oligonucleotides , the method comprising:(a) providing a planar support arrayed with a plurality of samples, wherein a portion of the plurality of samples differs from another portion of the plurality of samples according to a phenotype of interest;(b) contacting the plurality of samples arrayed on the planar support with the plurality of oligonucleotides; and(c) recovering members of the oligonucleotide probe library that bound to members of the portion of samples of one phenotype of interest, thereby enriching the oligonucleotide probe library.104. The method of claim 103 , further comprising repeating steps (a)-(c) at least 5 times.105. The method of claim 103 , wherein the unenriched oligonucleotide probe library comprises at least 10different oligonucleotide sequences.106. The method of claim 103 , wherein the plurality of samples comprises a cell culture claim 103 , a tissue sample claim 103 , a tissue lysate claim 103 , a bodily fluid or a fraction claim 103 , derivative or combination thereof.107. The method of claim 106 , wherein the bodily fluid comprises at least one of blood or a derivative thereof claim 106 , sera claim 106 , plasma claim 106 , ascites claim 106 , urine claim 106 , cerebrospinal fluid (CSF) claim 106 , sputum claim 106 , saliva claim 106 , bone marrow claim 106 , synovial fluid claim 106 , aqueous humor claim 106 , amniotic fluid claim 106 , cerumen claim 106 , breast milk claim 106 , broncheoalveolar lavage fluid claim 106 , semen claim 106 , prostatic fluid claim 106 , Cowper's fluid claim 106 , pre-ejaculatory fluid claim 106 , female ...

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Номер записи: 46
15-09-2022 дата публикации

METHOD FOR IN SITU DETERMINATION OF NUCLEIC ACID PROXIMITY

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

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

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Номер записи: 47
16-05-2019 дата публикации

Targeted in situ protein diversification by site directed dna cleavage and repair

Номер: US20190144853A1
Автор: David Ng, Mutlu Erdogan, Oliver Griesbeck
Принадлежит: Max Planck Gesellschaft zur Foerderung der Wissenschaften eV

The present invention relates to a method for producing a panel of cells (i.e. a cell library) expressing various different mutant variants of a protein of interest, wherein only one of said mutant variants is expressed per cell from a single gene copy. The present invention also relates to a method or cell library for identifying a mutant variant of a protein of interest having a different or modified biological activity as compared to the corresponding wild-type protein of interest. According to the present invention the identified mutant variant of a protein of interest may be applied for white biotechnology.

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Номер записи: 48
16-05-2019 дата публикации

METHODS FOR IDENTIFICATION OF OLIGONUCLEOTIDES

Номер: US20190144918A1
Автор: Moeller Thorleif
Принадлежит:

The present invention discloses methods for identification of oligonucleotides by manipulation of the information content a plurality of oligonucleotides. A main object of the methods is the identification of new molecular activity such as new ligands of interest for the development of therapeutics or in the field of nanotechnology. 1) A method of specifically amplifying the sequence of a hybridised oligonucleotide specie comprising the steps:a. Incubating a plurality of oligonucleotides under conditions of hybridisation.b. Extending the 3′ end of one or more hybridised oligonucleotide species, such that the extended region generates a new primer binding site.c. Amplifying extended sequences by PCR using said new primer binding site.d. Optionally identifying the identity of amplified sequences thereby revealing the identity of hybridised oligonucleotide species in step a.2) A method according to claim 1 , wherein the amplified extended sequences of step c claim 1 , or oligonucleotide species derived thereof claim 1 , is comprised in a plurality of oligonucleotides used in a next round of steps a-c claim 1 , such that claim 1 , an iterative process is initiated wherein each repetition of steps a claim 1 , b and c is optionally followed by step d.3) A method according to claim 2 , wherein the iterative process comprise a number of repetitions of steps a claim 2 , b and c and optionally step d selected from the group of 2 claim 2 , 3 claim 2 , 4 claim 2 , 5 claim 2 , 6 claim 2 , 7 claim 2 , 8 claim 2 , 9 claim 2 , 10 claim 2 , 11 claim 2 , 12 claim 2 , 13 claim 2 , 14 claim 2 , 15 claim 2 , 16 claim 2 , 17 claim 2 , 18 claim 2 , 19 claim 2 , 20 claim 2 , 25 and 30 repetitions.4) A method according to any of the preceding claims claim 2 , wherein a qPCR is performed on at least a subset of the extended sequences of step C to estimate the complexity of the plurality of oligonucleotide species.5) A method according to any of the preceding claims claim 2 , wherein said ...

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Номер записи: 49
07-05-2020 дата публикации

METHODS FOR ISOLATION AND QUANTIFICATION OF SHORT NUCLEIC ACID MOLECULES

Номер: US20200140850A1
Автор: FISHMAN Alla, LAMM Ayelet
Принадлежит:

Methods, kits and compositions for separation, identification, and isolation of short nucleic acids (i.e., less than 100 nucleotides) of different length are provided. The invention further provides methods for preparation of small RNA libraries. 148-. (canceled)49. A method for separating a nucleic acid molecule of a desired length below 100 nucleotides from a solution comprising a nucleic acid molecule of the desired length and a nucleic acid molecule of a length at least 15 nucleotides shorter , the method comprising:(a) obtaining a solution comprising nucleic acid molecules of multiple lengths;(b) adding to said solution particles comprising a carboxyl-group coated surface, salt, polyalkylene glycol, and alcohol; and(c) isolating said particles;thereby separating a nucleic acid molecule of a desired length below 100 nucleotides from a solution comprising a nucleic acid molecule of the desired length and a nucleic acid molecule of a length at least 15 nucleotides shorter.50. The method of claim 49 , wherein said salt is sodium chloride claim 49 , said polyalkylene glycol is polyethylene glycol (PEG) claim 49 , said alcohol is isopropanol and wherein said adding produces a binding solution having concentrations of PEG and isopropanol suitable for selective binding of said nucleic acid molecule of a desired length to said particles; wherein(i) said desired length is at least 60 bases and said concentration of PEG and isopropanol is 7%-8.5% and 32%-41%, respectively;(ii) said desired length is at least 50 bases and said concentration of PEG and isopropanol is 7%-8.5% and 38%-45%, respectively;(iii) said desired length is at least 40 bases and said concentration of PEG and isopropanol is 7%-8.5% and 41%-50%, respectively; and(iv) said desired length is at least 30 bases and said concentration of PEG and isopropanol is 7%-8.5% and 45%-58%, respectively; and(v) said desired length is at least 20 bases and said concentration of PEG and isopropanol is 7%-8.5% and 49%-60 ...

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Номер записи: 50
22-09-2022 дата публикации

METHODS AND SYSTEMS FOR MICROFLUIDIC SCREENING

Номер: US20220297125A1
Автор: CAYER Devon, CHUBUKOV Pavel, MACCONNELL Andrew, RAMJI Ramesh, STROMBERG Sean
Принадлежит:

Provided are methods and systems useful for screening large libraries of effector molecules. Such methods and systems are particularly useful in microfluidic systems and devices. The methods and systems provided herein utilize encoded effectors to screen large libraries of effectors. 120.-. (canceled)21. A screening system comprising:(a) a microfluidic device comprising a droplet formation region connected to a flow path, wherein the droplet formation region is configured to generate a plurality of droplets, and wherein the flow path is configured to incubate the plurality of droplets for at least 14 minutes with a dispersion ratio of less than 4%; (i) an effector bound to the unique scaffold via a cleavable linker and configured to be released from the scaffold upon cleavage of the cleavable linker to generate a released effector in a droplet of the subset of the plurality of droplets; and,', '(ii) a barcode corresponding to and identifying the effector; and, '(b) a plurality of unique scaffolds encapsulated in the plurality of droplets wherein a subset of the plurality of droplets each comprises at least one unique scaffold of the plurality of unique scaffolds, and wherein at least a subset of the plurality of the unique scaffolds each comprises(c) at least one of: a sorting device for sorting a droplet of the plurality of droplets or an injection device for injecting a reagent into a droplet of the plurality of droplets, based on the signal.22. The system of claim 21 , wherein the droplet formation region is configured to generate droplets at a frequency of at least 70 Hz.23. The system of claim 21 , wherein the plurality of unique scaffolds comprise at least 80 claim 21 ,000 unique scaffolds claim 21 , wherein each unique scaffold comprises a unique effector.24. The system of claim 21 , wherein the effector comprises at least one effector subunit claim 21 , the barcode comprises at least one barcode subunit claim 21 , and the barcode subunit corresponds to and ...

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Номер записи: 51
22-09-2022 дата публикации

Selective degradation of proteins

Номер: US20220298503A1
Автор: Charly CHAHWAN, Maria SOLOVEYCHIK
Принадлежит: Synthex Inc

The present disclosure provides methods to identify peptides and small molecule moieties that are able to functionally bridge an interaction between a target protein and an E3 ubiquitin ligase to mediate the degradation of the target protein. Some moieties can degrade specific target variants, but not others. The moieties create a neosubstrate for an E3 ligase of interest. The methods described enable generation of compounds able to selectively degrade specific targets within cells with implications for drug development for pathological conditions. The disclosure also describes the generation of modified peptides using post-translational modification enzymes, such as N-methyltransferases, prolyloligopeptidases, lactamases, hydroxylases, and dehydratases, along with methods of using the same.

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Номер записи: 52
29-09-2022 дата публикации

METHODS AND SYSTEMS FOR MICROFLUIDIC SCREENING

Номер: US20220305492A1
Автор: CAYER Devon, CHUBUKOV Pavel, MACCONNELL Andrew, RAMJI Ramesh, STROMBERG Sean
Принадлежит:

Provided are methods and systems useful for screening large libraries of effector molecules. Such methods and systems are particularly useful in microfluidic systems and devices. The methods and systems provided herein utilize encoded effectors to screen large libraries of effectors. 120.-. (canceled)21. A screening method comprising: (i) an effector bound to the scaffold by a cleavable linker; and', '(ii) a barcode corresponding to and for identifying the effector;, '(a) providing at least one cell and a scaffold in a compartment, wherein the scaffold comprises(b) cleaving the cleavable linker to release the effector from the scaffold, thereby generating a released effector in the compartment;(c) detecting a signal from the compartment, wherein the signal results from an interaction between the released effector and the at least one cell;(d) identifying the compartment as a positive compartment or a negative compartment based on the signal; and, (i) sorting the positive compartment, sorting contents of the positive compartment, sorting the scaffold of the positive compartment, sorting the barcode of the positive compartment, physically separating the positive compartment from the negative compartment in space, or any combination thereof, based on the identifying of (d);', '(ii) measuring a property of the barcode in the positive compartment to elucidate the structure of the effector in the positive compartment, based on the identifying of (d); or', '(iii) adding a secondary barcode to tag the barcode in the positive compartment, based on the identifying of (d)., '(e) performing at least one of22. The method of claim 21 , wherein the barcode is a nucleic acid barcode.23. The method of claim 21 , wherein the barcode comprises a property or subunit unique to the effector.24. The method of claim 21 , wherein the barcode comprises a property or subunit unique to the scaffold.25. The method of claim 21 , wherein the barcode comprises at least one barcode subunit claim 21 ...

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Номер записи: 53
30-05-2019 дата публикации

High-Throughput Single Cell Barcoding

Номер: US20190161750A1
Автор: Francois Vigneault, George M. Church
Принадлежит: Harvard College

Methods and compositions for high-throughput, single cell analyses are provided. The methods and compositions can be used for analysis of genomes and transcriptomes, as well as antibody discovery, HLA typing, haplotyping and drug discovery.

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Номер записи: 54
30-05-2019 дата публикации

METHODS FOR SELECTING ENZYMES HAVING PROTEASE ACTIVITY

Номер: US20190161786A1
Автор: Blazej Robert, Emrich Charles, Toriello Nicholas
Принадлежит: NOVOZYMES A/S

Provided herein are systems and components thereof for improving protease activity. The systems make use of an emulsion for in vitro compartmentalization of a library of synthetic compounds, each compound having a gene linked to a protease substrate and selectable marker. Expressed enzymes with greater protease activity will preferentially hydrolyze the protease substrate, whereas enzymes with less protease activity will leave the substrate intact. Removal of the non-hydrolyzed compounds provides an enriched gene library encoding for more active protease variants. Also described are synthetic compounds and emulsions which can be used in the methods. 1. A method of selecting for a polypeptide having protease activity , the method comprising: (a) a polynucleotide encoding for a polypeptide;', '(b) a protease substrate linked to said polynucleotide; and', '(c) a selectable marker linked to said polynucleotide;, 'suspending a plurality of synthetic compounds in an aqueous phase, wherein the synthetic compounds individually comprisewherein the aqueous phase comprises components for expression of the polypeptide;(ii) forming a water-in-oil emulsion with the aqueous phase, wherein the synthetic compounds are compartmentalized in aqueous droplets of the emulsion;(iii) expressing the polypeptides within the aqueous droplets of the emulsion, wherein a polypeptide with protease activity in an aqueous droplet hydrolyzes the protease substrate in that droplet; and(iv) separating the synthetic compounds to recover synthetic compounds comprising the protease substrate and/or synthetic compounds wherein the protease substrate has been hydrolyzed.2. The method of claim 1 , wherein the polypeptide comprises a propeptide.3. The method of claim 1 , wherein the plurality of synthetic compounds comprises at least about 10different synthetic compounds.4. The method of claim 1 , wherein the emulsion comprises at least about 10aqueous droplets/mL of emulsion.5. The method of claim 1 , ...

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Номер записи: 55
21-05-2020 дата публикации

THERAPEUTIC DRUG FOR LIPID-PEROXIDATION-INDUCED DISEASES AND SCREENING METHOD FOR THERAPEUTIC DRUGS FOR LIPID-PEROXIDATION-INDUCED DISEASES

Номер: US20200158709A1
Автор: IDE Tomomi, SHINTO Saki, YAMADA Ken-ichi, Yamamoto Keiichi
Принадлежит:

The present invention provides: an assay method that uses a compound represented by formula (I) as a fluorescent probe molecule and that is for detecting the lipid peroxidation suppression activity of a test compound; an assay kit that uses the assay method; a screening method that uses the assay method; and a pharmaceutical composition that is for the treatment, etc. of diseases (such as age-related macular degeneration) that are induced by lipid peroxidation reactions. 2. The assay kit according to claim 1 , wherein the divalent iron ion source material is iron(II) sulfate.3. The assay kit according to or claim 1 , comprising a package insert showing an activity value of a compound having lipid peroxidation inhibitory activity.5. The assay kit according to claim 4 , wherein the cultured cell is a human hepatoma-derived HepG2 cell.6. The assay kit according to or claim 4 , comprising a package insert showing an activity value of a compound having lipid peroxidation inhibitory activity.7. An assay kit comprising any two or more of the assay kits according to to .8. An assay method for measuring lipid peroxidation inhibitory activity claim 4 , comprising:i) preparing a buffer containing a compound represented by formula (I) and a liposome;ii) adding at least one compound selected from the group consisting of 2,2′-azobis(2-aminopropane) dihydrochloride and a divalent iron ion source material;iii) adding a test compound;iv) measuring fluorescence; andv) determining an activity value of the test compound from the result of measuring the fluorescence.9. An assay method for measuring lipid peroxidation inhibitory activity claim 4 , comprising:i) preparing a buffer containing a compound represented by formula (I) and a cultured cell;ii) adding at least one compound selected from the group consisting of arachidonic acid and tert-butyl hydroperoxide,iii) adding a test compound;iv) measuring fluorescence; andv) determining an activity value of the test compound from the ...

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Номер записи: 56
23-06-2016 дата публикации

Genomic-scaled nucleic acid synthesis, and other combinatorial syntheses

Номер: US20160175801A1
Автор: Efrain "Frank" Rodriguez, Wlodek Mandecki, Ziye "Jay" Qian
Принадлежит: Pharmaseq Inc

Provided is a method of synthesis comprising: (I) providing separate reaction sequences to TABs; (II) utilizing reaction vessels configured to react a separate combinatorial building block with a moiety on a surface of a TAB; and (III) operating one or more TAB sorters comprising a TAB reader, a sorting tree comprising valves or switches and sorting nodes, and a monitor configured to detect TAB location, wherein the operating comprises serially conducting: (a) reacting distinct combinatorial building blocks in the reaction chambers with surfaces of TABs distributed in the reaction chambers; (b) operating a controller to operate the TAB sorters to segregate the TABs to allocations appropriate for the next assigned reaction, the operating including recycling TABs with ambiguous identity back through the sorter; and (c) repeating steps (a) and (b) as needed to complete 30% or more of the assigned sequences.

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Номер записи: 57
01-07-2021 дата публикации

Methods For Making and Using Polynucleotide Sequences in the Synthesis of Alkaloid Compounds

Номер: US20210198706A1
Автор: Peter James Facchini
Принадлежит: Antheia Inc, Willow Biosciences Inc

Novel methods that may be used for the manufacture of plant alkaloid compounds and novel polynucleotide compounds are provided. The plant alkaloid compounds are useful as medicinal compounds.

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Номер записи: 58
21-06-2018 дата публикации

COPY NUMBER PRESERVING RNA ANALYSIS METHOD

Номер: US20180171384A1
Автор: MOLL Pamela
Принадлежит:

The present invention provides a method for generating an amplified nucleic acid portion of a template RNA molecule, comprising after having obtained a template RNA, annealing a first oligonucleotide primer at a preselected 3′ terminal nucleic acid region of the template RNA, elongating the first oligonucleotide primer in a template specific manner thereby obtaining a first elongated strand, removing the RNA template, annealing one or more further oligonucleotide primers to the first elongated strand, elongating the one or more further oligonucleotide primers in a template specific manner without strand displacement of polynucleotides annealed to the first elongated strand or using a polymerase that destroys a displaced strand, thereby generating further elongation products, isolating and/or amplifying an elongation product of said further elongation product comprising a nucleic acid portion that is elongated complementary to the first oligonucleotide primer; as well as kits for performing the method. 1. A method for generating a nucleic acid product from a template RNA molecule , comprising after having obtained a template RNAa) annealing a first oligonucleotide primer at a preselected nucleic acid region of the template RNA,b) elongating the first oligonucleotide primer in a template specific manner thereby obtaining a first elongated strand,c) removing the RNA template,d) annealing one or more further oligonucleotide primers to the first elongated strand,e) elongating the one or more further oligonucleotide primers in a template specific manner without displacement of primers annealed to the first elongated strand or with a polymerase that destroys a displaced strand, thereby generating further elongation products,f) isolating and/or amplifying an elongation product of said further elongation product comprising a nucleic acid portion that is elongated complementary to the first oligonucleotide primer.2. The method of claim 1 , wherein the preselected nucleic acid ...

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Номер записи: 59
06-06-2019 дата публикации

Processes and Systems for Preparation of Nucleic Acid Sequencing Libraries and Libraries Prepared Using Same

Номер: US20190169666A1
Автор: Belhocine Kamila, Bjornson Keith, Hardenbol Paul, HINDSON Benjamin, Patel Pranav, Wu Indira, Wyatt Paul William
Принадлежит:

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. 130.-. (canceled)31. A method for nucleic acid processing , comprising:(a) synthesizing a double-stranded nucleic acid molecule comprising at least one uracil;(b) at one or more sites corresponding to said at least one uracil, generating a plurality of single-stranded nucleic acid molecules; and(c) using a nucleic acid barcode molecule comprising a barcode sequence to generate a barcoded nucleic acid molecule comprising (i) a sequence of a single-stranded nucleic acid molecule of said plurality of single-stranded nucleic acid molecules; and (ii) a sequence corresponding to said barcode sequence.32. The method of claim 31 , further comprising claim 31 , subsequent to (b) claim 31 , using a single-stranded nucleic acid molecule of said plurality of single-stranded nucleic acid molecules to generate an additional double-stranded nucleic acid molecule claim 31 , and using a ligating enzyme to ligate said nucleic acid barcode molecule to said additional double-stranded nucleic acid molecule.33. The method of claim 32 , further comprising claim 32 , subsequent to (b) claim 32 , hybridizing a primer to said single-stranded nucleic acid molecule and preforming a nucleic acid extension reaction to generate said additional double-stranded nucleic acid molecule.34. The method of claim 33 , wherein said primer comprises a random N-mer sequence of 5 to 25 nucleotides in length.35. The method ...

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Номер записи: 60
08-07-2021 дата публикации

METHODS FOR NEXT GENERATION GENOME WALKING AND RELATED COMPOSITIONS AND KITS

Номер: US20210207207A1
Автор: Ward Brian
Принадлежит:

Methods are provided herein for identifying rare and/or unknown DNA sequences by next-generation sequencing approaches. Isolated double-stranded (ds), single-stranded (ss), or ds/ss DNA is fragmented and the fragments are polished, phosphorylated, and tailed, as necessary. Fragmentation can be enzymatic or mechanical. A universal adapter sequence is ligated to each fragment, wherein the adapter can have a top strand without a 5′ phosphate, a 3′ with an —H in place of the —OH, and/or a 3′ extra base complementary to any base added to the polished fragments. The ligatamers may then serve as templates for amplification using a forward primer complementary to the adapter sequence and a reverse primer targeted to the fragment sequence. Compositions produced by these methods and kits adapted for performing these methods are also described herein. 1. A method for copying a sequence of interest , the method comprising amplifying a plurality of template polynucleotides , each comprising a native sequence and a universal adapter sequence on at least one end , the native sequence comprising the sequence of interest , and the universal adapter sequence comprising 5′ to 3′ an adapter priming domain and , optionally , a barcode domain consisting of 1 to 20 nucleotides; wherein the universal adapter sequence is located a fixed distance from the 5′ end of the sequence of interest , such that the nucleotide sequence between the universal adapter sequence and the sequence of interest defines an identification sequence that is unique to a given template and its progeny amplicons; and wherein the amplification is primed with a pair of primers comprising a universal primer that is identical to at least 10 bp of the adapter priming domain of the universal adapter sequence and a first reverse primer that is complementary to a region of the native sequence downstream of the sequence of interest.2. The method of claim 1 , wherein the sequence of interest is a mutation claim 1 , a SNP claim ...

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Номер записи: 61
08-07-2021 дата публикации

Methods for stable complex formation and related kits

Номер: US20210208150A1
Автор: Kevin L. Gunderson, Norihito MURANAKA
Принадлежит: Encodia Inc

The present disclosure relates to methods and kits for forming a stable complex comprising a binding agent and a target (e.g., a macromolecule). In some embodiments, the target comprises a peptide, a polypeptide, or a protein to be analyzed. In some embodiments, the present disclosure relates to formation of a stable complex comprising a binding agent and a target (e.g., a macromolecule) to be analyzed in a method which employs barcoding and nucleic acid encoding of molecular recognition events, and/or detectable labels. Provided herein is also a programmable system for information transfer comprising one or more adaptor molecules.

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Номер записи: 62
05-07-2018 дата публикации

METHOD FOR PROFILING PROTEIN METHYLATION

Номер: US20180188262A1
Автор: FIGEYS Daniel, MIERZWA Anna Sylvia, NING Zhibin
Принадлежит:

There is provided a method for identifying protein methylation on arginine and lysine residues. The method comprises obtaining a set of peptides; blocking un-methylated arginine and lysine residues and the free N-terminal amine of peptides in the set of peptides, so that un-methylated peptides are neutralized and only methylated peptides are positively charged at neutral or basic pH; isolating the methylated peptides based on charge; and performing mass spectrometry (MS) analysis on the isolated methylated peptides to detect methylated lysine and arginine residues. Methods provided herein can be used for large scale, high throughput profiling of protein methylation in a cell or tissue. 1. A method for identifying methylation on arginine and lysine residues in a set of peptides , the method comprising:i) obtaining the set of peptides;ii) blocking un-methylated arginine and lysine residues and the free N-terminal amine of peptides in the set of peptides, so that un-methylated peptides are neutralized and only methylated peptides are positively charged at neutral or basic pH;iii) isolating methylated peptides based on charge; andiv) performing mass spectrometry (MS) analysis on the isolated methylated peptides to detect methylated lysine and arginine residues.2. The method of claim 1 , wherein step (ii) comprises conversion of the guanidine group on un-methylated arginine residues to a 2-pyrimidine residue.3. The method of claim 2 , wherein said conversion of the guanidine group comprises reaction with malondialdehyde (MDA) or an acetal or derivative thereof claim 2 , or reaction with 1 claim 2 ,1 claim 2 ,3 claim 2 ,3-tetraisopropoxypropane (TiPP).4. The method of claim 1 , wherein step (ii) comprises blocking the epsilon primary amine on un-methylated lysine residues and blocking the free primary amine on the peptide N-terminals claim 1 , or wherein un-methylated arginine and lysine residues are blocked in step (ii) regardless of peptide sequence.5. (canceled)6. The ...

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Номер записи: 63
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: 64
26-07-2018 дата публикации

Color-Encoding and In-Situ Interrogation of Matrix-Coupled Chemical Compounds

Номер: US20180209067A1
Автор: Richard H. Ebright
Принадлежит: Rutgers State University of New Jersey

A method and apparatus for the physico-chemical encoding of a collection of beaded resin (“beads”) to determine the chemical identity of bead-anchored compounds by in-situ interrogation of individual beads. The present invention provides method and apparatus to implement color-coding strategies in applications and including the ultrahigh-throughput screening of bead-based combinatorial compounds libraries as well as multiplexed diagnostic and environmental testing and other biochemical assays.

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Номер записи: 65
25-06-2020 дата публикации

Nucleic acid-guided editing of exogenous polynucleotides in heterologous cells

Номер: US20200199781A1
Автор: Daniel Held, Richard Fox
Принадлежит: Inscripta Inc

The present disclosure provides shuttle vectors for editing exogenous polynucleotides in heterologous live cells, as well as automated methods, modules, and multi-module cell editing instruments and systems for performing the editing methods.

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Номер записи: 66
02-08-2018 дата публикации

METHODS FOR HIGH EFFICIENCY LIBRARY PREPARATION USING DOUBLE-STRANDED ADAPTERS

Номер: US20180216252A1
Автор: Jung Byoungsok
Принадлежит:

Methods for preparing a sequencing library from a DNA-containing test sample are provided, including methods for rescuing one or more partially ligated DNA fragments to enhance library preparation conversion efficiencies. The subject methods can further be used to improve recovery of duplex sequence information from double-stranded DNA. 1. (canceled)2. A method for preparing a sequencing library from a test sample comprising a plurality of double-strand DNA fragments:(a) obtaining a test sample comprising a plurality of double-stranded DNA (dsDNA) fragments, the dsDNA fragments comprising a forward strand and a reverse strand;(b) adding double-stranded adapters to the dsDNA sample and ligating the double-strand adapters to both ends of the dsDNA fragments;(c) extending unligated 3′-ends of the dsDNA fragments with a DNA polymerase to create dsDNA fragment-adapter templates, wherein the polymerase further comprises strand displacement activity;(d) adding a poly-adenine tail to the 3′-ends of the dsDNA fragment-adapter templates;(e) adding a set of ssDNA oligonucleotides and hybridizing the ssDNA adapters to the dsDNA fragment-adapter templates; and(f) extending the set of ssDNA oligonucleotides to create a dsDNA sequencing library.3. The method of claim 2 , wherein the dsDNA sequencing library is sequenced to obtain sequence reads.4. The method of claim 3 , wherein the sequence reads are obtained from next-generation sequencing (NGS).5. The method of claim 3 , wherein the sequence reads are obtained from massively parallel sequencing using sequencing-by-synthesis.6. The method of claim 2 , wherein the test sample comprises a plurality of cell-free DNA molecules.76. The method of claim 2 , wherein the test sample is from a whole blood claim 2 , a blood fraction claim 2 , plasma claim 2 , serum claim 2 , urine claim 2 , fecal claim 2 , saliva claim 2 , a tissue biopsy claim 2 , pleural fluid claim 2 , pericardial fluid claim 2 , cerebral spinal fluid claim 2 , or ...

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Номер записи: 67
12-08-2021 дата публикации

NUCLEIC ACID-GUIDED EDITING OF EXOGENOUS POLYNUCLEOTIDES IN HETEROLOGOUS CELLS

Номер: US20210246574A1
Автор: Fox Richard, Held Daniel
Принадлежит:

The present disclosure provides shuttle vectors for editing exogenous polynucleotides in heterologous live cells, as well as automated methods, modules, and multi-module cell editing instruments and systems for performing the editing methods. 1. A method of nucleic acid-guided nuclease editing of five hundred or more exogenous polynucleotides from source cells within heterologous editing cells in a single multiplexed automated operation , comprising:inserting five hundred or more different target polynucleotides from the source cells into shuttle vector backbones to form a library of shuttle vectors;transferring the library of shuttle vectors into a first receptacle;providing heterologous editing cells in a second receptacle;growing the heterologous editing cells in a growth module;transferring the heterologous editing cells from the growth module to a transformation module;transferring the library of shuttle vectors from the first receptacle to the transformation module;providing one or more editing vectors wherein the editing vectors comprise a coding sequence for a nuclease, a guide nucleic acid and a DNA donor sequence in a third receptacle;transferring the editing vectors from the third receptacle to the transformation module;introducing the library of shuttle vectors and the one or more editing vectors into the heterologous editing cells in the transformation module;transferring the heterologous editing cells from the transformation module to an editing module;allowing editing to take place in the editing module under conditions that allow the editing vectors to edit the one or more target polynucleotides in the shuttle vectors thereby forming edited shuttle vectors; andisolating the living edited cells comprising the edited shuttle vectors; andisolating the edited shuttle vectors; wherein the first receptacle, second receptacle, third receptacle, growth module, cell concentration module, transformation module and editing module are all part of a stand-alone ...

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Номер записи: 68
11-08-2016 дата публикации

SYSTEM AND METHOD FOR THE DECONVOLUTION OF MIXED DNA PROFILES USING A PROPORTIONATELY SHARED ALLELE APPROACH

Номер: US20160232282A1
Автор: Overson Thomas L.
Принадлежит: United States Government as represented by the Secretary of the Army

A total forensic DNA casework management system and method for the deconvolution of mixed DNA samples using a novel, 3-rule algorithm to determine the proportional allele sharing of the sample's contributors. The process is fully document, can assess and process DNA anomalies and artifacts, and transforms raw STR data to produce final DNA profile types, peak height ratios, proportions, fitting criteria and associated graphs. 1. A method of resolving a mixture comprising DNA of more than one individual into genotype profiles for individuals in the mixture comprising:(a) obtaining quantitative allele peak data for alleles present at a first locus in a DNA mixture comprising DNA of more than one individual;(b) defining a minimum contributor proportion;(c) defining a minimum peak height;(d) defining a minimum peak height ratio;(e) selecting at least one reference sample;(f) calculating the total sum of all relative fluorescent units at the at the first locus; 1) assuming, whenever possible that allele peak ratios at the first locus are equal to 1;', '2) assuming, whenever possible, that shared common alleles at the first locus are shared in the proportion of the non-common alleles sharing the common allele,', '3) ensuring that minimum peak height defined in step (c) is maintained across all alleles at the first locus;', '4) calculating the proportion of each allele combination at the first locus to the sum calculated at step (f);', '5) calculating a peak height ratio for each allele combination at the first locus;', '6) presenting the transformed quantitative allele peak data in a machine readable form, said transformed data comprising allele combinations;, '(g) transforming the quantitative allele peak data using a machine to produce individual DNA profiles from the DNA mixture, said transformation comprising the steps of(h) limiting allele combinations presented after the transforming step by applying the at least one reference sample from step (e) resulting in a ...

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Номер записи: 69
09-07-2020 дата публикации

COMBINATORIAL BARCODE SEQUENCES, AND RELATED SYSTEMS AND METHODS

Номер: US20200216898A1
Автор: HUBBELL Earl
Принадлежит: LIFE TECHNOLOGIES CORPORATION

A kit for use with a nucleic acid sequencing instrument can include a plurality of combinatorial barcodes sequences meeting the following criteria: each of the combinatorial barcode sequences comprise a plurality of iterations of a sequence motif, where the sequence motif comprises a first nucleotide base from a first group of nucleotide bases followed by a second nucleotide base from a second group of nucleotide bases, the first group and the second group differing from each other; and the plurality of combinatorial barcode sequences is at least 1,000,000 different barcode sequences. 1. A kit for use with a nucleic acid sequencing instrument , the kit comprising:a plurality of containers each containing an oligonucleotide comprising a combinatorial barcode sequence, at least some oligonucleotides contained in the plurality of containers comprising differing combinatorial barcode sequences,each of the combinatorial barcode sequences comprising a plurality of iterations of a sequence motif,the sequence motif comprising a first nucleotide base from a first group of nucleotide bases followed by a second nucleotide base from a second group of nucleotide bases, the first group and the second group differing from each other,each of the combinatorial barcode sequences being one of at least eight potential combinatorial barcode sequences, the potential combinatorial barcode sequences being synchronized in flow space based on a predetermined order of nucleotide flows, andeach of the oligonucleotides configured to be incorporated into a polynucleotide to be sequenced.2. The kit of claim 1 , wherein one or both of the first and second groups comprise at least two different nucleotide bases.3. The kit of claim 1 , wherein each combinatorial barcodes sequence has a length comprising a length for the sequence motif multiplied by a number of iterations for the sequence motif.4. The kit of claim 1 , wherein one of the first and second groups contains at least two different ...

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Номер записи: 70
09-07-2020 дата публикации

Automated instrumentation for production of t-cell receptor peptide libraries

Номер: US20200216977A1
Автор: Deanna Church, Michael Graige, Stephen Federowicz
Принадлежит: Inscripta Inc

The present disclosure provides instrumentation and automated methods for creating cell surface display libraries, where the cells of the library display engineered peptides on their cell surfaces for identification of antigens that bind to T-cell receptors. The engineered peptides may be putative antigens or binding regions of the T-cell receptors.

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Номер записи: 71
16-08-2018 дата публикации

METHOD AND COMPOSITIONS FOR DETECTING PATHOGENIC ORGANISMS

Номер: US20180230458A1
Автор: Bruinsma Stephen Paul, Grunenwald Haiying Li, Khanna Anupama, Vaidyanathan Ramesh
Принадлежит:

Some embodiments of the present invention relate to the enrichment of non-host nucleic acids in a mixture of host and non-host nucleic acids. Some embodiments include methods for detecting pathogenic organisms from a nucleic acid sample comprising host nucleic acids and nucleic acids indicative of the pathogenic organism. 1. A method for the enrichment of non-host RNAs in a nucleic acid sample comprising host RNAs and non-host RNAs , comprising:(a) obtaining a plurality of capture probes, wherein each capture probe comprises an affinity tag and a nucleic acid complementary to a host RNA;(b) contacting the nucleic acid sample with the plurality of capture probes; and(c) removing capture probes hybridized to the host RNAs, thereby obtaining a population of nucleic acids enriched for non-host RNAs.2. The method of claim 1 , wherein the plurality of capture probes comprises capture probes prepared by a method comprising:(i) obtaining single-stranded target nucleic acids;(ii) obtaining double-stranded target nucleic acids from the single-stranded target nucleic acids, wherein the double-stranded target nucleic acids comprise an RNA polymerase promoter; and(iii) contacting the double-stranded nucleic acids with an RNA polymerase to obtain RNAs complementary to the single-stranded target nucleic acids.3. (canceled)4. The method of claim 2 , wherein step (ii) comprises linking the double-stranded target nucleic acids with a primer comprising the RNA polymerase promoter or complement thereof.5. (canceled)6. The method of claim 2 , wherein step (ii) comprises linking the single-stranded target nucleic acids with a primer comprising the RNA polymerase promoter or a complement thereof.7. The method of claim 2 , wherein step (ii) comprises linking the single-stranded nucleic acids with an adapter primer claim 2 , and hybridizing a primer comprising the RNA polymerase promoter or a complement thereof to the adapter primer.8. The method of claim 2 , wherein step (ii) comprises ...

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Номер записи: 72
01-08-2019 дата публикации

Concealing information present within nucleic acids

Номер: US20190233877A1
Автор: Sterling SAWAYA
Принадлежит: Geneinfosec Inc

Methods related to concealment of genetic information present within nucleic acid sequences, wherein individual nucleic acid molecules are barcoded. In some embodiments barcoding occurs before, after, or during enrichment. Barcoded nucleic acids are then combined with control barcoded nucleic acids. Different methods are provided for barcoding and pooling to conceal different types of genetic information present within nucleic acids.

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Номер записи: 73
09-09-2021 дата публикации

FUNCTIONALIZED GEL BEADS

Номер: US20210277444A1
Автор: Bent Zachary, Gagnon Alex, Meer Elliott, RIORDAN Daniel, Ryvkin Paul, Srinivas Niranjan, Terry Jessica
Принадлежит:

The present disclosure provides methods of generating supports (e.g., beads) comprising barcode molecules coupled thereto. A barcode molecule coupled to a support may comprise a barcode sequence and a functional sequence. A barcode molecule may be generated using two or more ligation reactions in a combinatorial fashion. A support comprising two or more different barcode molecules may be useful for analyzing or processing one or more analytes such as nucleic acid molecules, proteins, and/or perturbation agents. 130.-. (canceled)31. A method for generating nucleic acid barcode molecules , comprising:(a) providing a plurality of nucleic acid molecules coupled to a support;(b) coupling one or more additional nucleic acid molecules to nucleic acid molecules of said plurality of nucleic acid molecules coupled to said support, thereby assembling a plurality of nucleic acid barcode molecules coupled to said support; and(c) digesting a subset of said plurality of nucleic acid barcode molecules with an exonuclease.32. The method of claim 31 , wherein said exonuclease comprises an Exo1 exonuclease claim 31 , an Exo111 nuclease claim 31 , a heat-labile DNAse claim 31 , or a combination thereof.33. The method of claim 31 , wherein nucleic acid molecules of said subset of said plurality of nucleic acid molecules are free of an exonuclease-resistant bond.34. The method of claim 31 , wherein said one or more additional nucleic acid molecules comprise an exonuclease-resistant bond.35. The method of claim 34 , wherein said exonuclease-resistant bond is a phosphorothioate bond.36. The method of claim 34 , wherein fewer than 50% of said one or more additional nucleic acid molecules comprising an exonuclease-resistant bond are digested with said exonuclease.37. The method of claim 34 , wherein said exonuclease-resistant bond is disposed between a final 3′ base of said one or more additional nucleic acid molecules and a base immediately preceding said final 3′ base of said one or more ...

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Номер записи: 74
15-08-2019 дата публикации

FUNCTIONALIZED GEL BEADS

Номер: US20190249226A1
Автор: Bent Zachary, Gagnon Alex, Meer Elliott, RIORDAN Daniel, Ryvkin Paul, Srinivas Niranjan, Terry Jessica
Принадлежит:

The present disclosure provides methods of generating supports (e.g., beads) comprising barcode molecules coupled thereto. A barcode molecule coupled to a support may comprise a barcode sequence and a functional sequence. A barcode molecule may be generated using two or more ligation reactions in a combinatorial fashion. A support comprising two or more different barcode molecules may be useful for analyzing or processing one or more analytes such as nucleic acid molecules, proteins, and/or perturbation agents. 1. A method for generating a plurality of barcode molecules , comprising:(a) providing a plurality of molecules coupled to a plurality of supports; and(b) combinatorially assembling said plurality of barcode molecules coupled to said plurality of supports by coupling one or more molecules each comprising one or more segments to each of said plurality of molecules,wherein said plurality of barcode molecules comprises (i) a first set of barcode molecules coupled to a support of said plurality of supports and (ii) a second set of barcode molecules coupled to said support, wherein each barcode molecule of said first set of barcode molecules comprises a first nucleic acid sequence and each barcode molecule of said second set of barcode molecules comprises a second nucleic acid sequence, wherein said first nucleic acid sequence is different than said second nucleic acid sequence.2. The method of claim 1 , wherein said barcode molecules of said first set of barcode molecules and said barcode molecules of said second set of barcode molecules comprise barcode sequences that are different from barcode sequences of barcode molecules coupled to other supports of said plurality of supports.3. The method of claim 1 , wherein said first nucleic acid sequence and said second nucleic acid sequence comprise an identical barcode sequence.4. The method of claim 1 , wherein said plurality of supports is a plurality of beads claim 1 , and wherein said barcode molecules of said ...

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Номер записи: 75
15-08-2019 дата публикации

CONVERTIBLE ADAPTERS

Номер: US20190249239A1
Автор: FROHME Marcus, Glökler Jörn
Принадлежит: TECHNISCHE HOCHSCHULE WILDAU

The invention relates to a method and a kit comprising single or at least partially double-stranded adapters comprising sequences with base modifications that are ligated to nucleic acid fragments and converted to generate asymmetric ends for specific recognition. The method is based on two main sequence conversion mechanisms, a direct conversion of specific bases and an indirect conversion by copying, wherein both mechanisms may be combined. 2. The method according to claim 1 , wherein said at least one convertible base and said at least one converted base preferably pair with different bases.3. The method according to claim 1 , comprisingsaid first extension step, wherein said complementary strand is extended yielding said first antisense adapter nucleic acid molecule ligated to said complementary strand, and said template strand is extended yielding said second antisense adapter nucleic acid molecule ligated to said template strand, and a primer is annealed to said second antisense nucleic acid molecule and extended yielding a nucleic acid strand comprising said primer, a nucleic acid complementary to said complementary strand, and a third antisense adapter nucleic acid molecule,', 'a primer is annealed to said first antisense adapter nucleic acid molecule, and extended yielding a nucleic acid strand comprising said primer, a nucleic acid complementary to said template strand, and a fourth antisense adapter nucleic acid molecule, and', 'said third antisense adapter nucleic acid molecule is essentially complementary to said first adapter nucleic acid molecule and said second antisense adapter nucleic acid molecule is essentially complementary said second adapter nucleic acid except at positions opposite to said at least one universal, convertible or converted base comprised within said first and second adapter nucleic acid molecule, and said third and fourth antisense adapter nucleic acid molecule are characterized by different base sequences, thereby yielding an ...

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Номер записи: 76
15-08-2019 дата публикации

Methods and Kits for Detecting Contamination and Sample Misidentification

Номер: US20190249334A1
Автор: Josh Kinman, Shannon Piehl
Принадлежит: Bioo Scientific Corp, PerkinElmer Health Sciences Inc

The disclosed methods and kits are useful in processing and analyzing a multiplicity of samples in molecular biology workflows where there is an increased chance for sample cross-contamination or misidentification. Some embodiments of the methods and kits utilize at least one spike in control and at least one barcode per sample.

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Номер записи: 77
21-09-2017 дата публикации

METHOD OF ALIGNING HIGH-DENSITY BIOCHEMICAL ARRAY CHIPS WITH ASYNCHRONOUS TRACKS BY MOIRÉ AVERAGING

Номер: US20170268999A1
Автор: Staker Bryan P.
Принадлежит: Complete Genomics, Inc.

An array chip useful for biochemical assays is provided wherein the chip includes a field region arranged with attachment sites according to a first pitch and at least one track region having a one-dimensional spot pattern arranged according to a second pitch that is less dense and is a non-integer multiple of the first pitch so that one-dimensional Moiré averaging may be applied in the track region, thereby to attain alignment of the chip to the optical instrumentation with a higher density of attachment sites.

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Номер записи: 78
29-08-2019 дата публикации

HIGH RESOLUTION SPATIAL GENOMIC ANALYSIS OF TISSUES AND CELL AGGREGATES

Номер: US20190262831A1
Автор: Hukari Kyle, West Jason A.A.
Принадлежит:

Provided are devices and methods for capturing template sample nucleic acids in a spatially specific manner that is coordinated with the original location of the templates in a tissue sample. By preserving spatial information regarding a given sample, the disclosed technology allows for improved diagnostics as well as improved therapeutic decision making for patient care and therapy. 1. A system , comprising:a plurality of base polynucleotides disposed on an amplification substrate;an optionally porous cellular support substrate,the optionally porous cellular support substrate and the amplification substrate defining a chamber therebetween;an inlet, the inlet optionally comprising an aperture formed in the cover,the inlet optionally placing the optionally porous layer into fluid communication with the environment exterior to the optionally porous cellular support substrate.2. The system of claim 1 , further comprising a frame disposed between the amplification substrate and the optionally porous cellular support substrate claim 1 , the frame defining the chamber between the optionally porous cellular support substrate and the amplification substrate.3. The system of any of - claim 1 , wherein one or both of the substrate and the optionally porous cellular support substrate is a conductive material.4. The system of any of - claim 1 , further comprising a voltage source configured to supply an electrical gradient between the optionally porous cellular support substrate and the substrate claim 1 , the electrical gradient being configured so as to translocate nucleic acids in a direction essentially perpendicular to the optionally porous cellular support substrate and toward the amplification substrate.5. The system of any of - claim 1 , further comprising an imager in optical communication with the chamber.6. The system of any of - claim 1 , further comprising a source of reagents in fluid communication with the inlet.7. The system of any of - claim 1 , wherein the ...

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Номер записи: 79
27-08-2020 дата публикации

OLIGONUCLEOTIDE PROBES AND USES THEREOF

Номер: US20200270610A1
Автор: DOMENYUK Valeriy, MIGLARESE Mark, Spetzler David, STARK Adam, XIAO Nianqing, ZHONG Zhenyu
Принадлежит: Caris Science, Inc.

Methods and compositions are provided for oligonucleotide probes and oligonucleotide probe libraries that recognize targets of interest. The targets include circulating biomarkers such as microvesicles, including those derived from various diseases. 169-. (canceled)70. A method of generating a single-stranded DNA (ssDNA) molecule comprising: [ (1) a 5′ leader region comprising a lengthener region, a terminator region, and a forward primer region;', '(2) a variable region positioned 3′ of the leader region; and', '(3) a tail region positioned 3′ of the variable region and comprising a reverse primer region; and, '(i) a nucleic acid molecule comprising, '(ii) forward and reverse primers configured to amplify the nucleic acid molecule from the forward primer region and reverse primer region, respectively; and, '(a) providing a mixture comprising(b) performing asymmetric polymerase chain reaction (PCR) on the mixture in a) to favorably amplify the reverse strand of the nucleic acid molecule, wherein the forward and reverse primers in the mixture are at a ratio of between about 1:20-1:50 (F/R) in favor of the reverse primers; thereby generating the ssDNA molecule.71. (canceled)72. The method of claim 70 , wherein the ratio is between about 1:37.5 (F/R) in favor of the reverse primers.73. The method of claim 70 , further comprising isolating the amplified reverse strand of the nucleic acid molecule on a native gel.74. The method of claim 70 , further comprising:(c) denaturing the amplified nucleic acid molecules from b); and(d) isolating the denatured reverse strand of the nucleic acid molecules from c).75. The method of claim 74 , wherein the denatured reverse strand of the nucleic acid molecules is isolated on a denaturing gel.76. The method of claim 70 , wherein the mixture in a) further comprises at least one of an enrichment buffer claim 70 , non-target molecules claim 70 , proteins claim 70 , microvesicles claim 70 , and polyethylene glycol.7779-. (canceled)80. The ...

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Номер записи: 80
25-10-2018 дата публикации

COMPOSITIONS AND METHODS FOR IMPROVING SAMPLE IDENTIFICATION IN INDEXED NUCLEIC ACID LIBRARIES

Номер: US20180305753A1
Автор: Bevis-Mott Claire, Boutell Jonathan Mark, Chesney Michael, Kalbande Angela, Smith Vincent Peter
Принадлежит: ILLUMINA CAMBRIDGE LIMITED

The present invention is concerned with compositions and methods for improving the rate of correct sample identification in indexed nucleic acid library preparations for multiplex next generation sequencing by exonuclease treatment and optionally blocking the 3′ ends of pooled indexed polynucleotides from multiple samples prior to amplification and sequencing. 1. A composition comprising: wherein the adapter comprises a first sample-specific universal adapter,', (i) a region of double stranded nucleic acid, and', '(ii) a region of single-stranded non-complementary nucleic acid strands comprising at least one universal primer binding site,, 'wherein the first sample-specific universal adapter comprises'}, 'wherein the first sample-specific universal adapter further comprises a first set of sample-specific tag sequences that differentiates the first plurality of adaptor-target-adapter molecules from adaptor-target-adaptor molecules originating from a different source, the first set of sample-specific tag sequences present in the single stranded non-complementary nucleic acid strands, and, 'a first plurality of adapter-target-adapter molecules comprising double-stranded target fragments isolated from a first source,'}an exonuclease.2. The composition of claim 1 , further comprising first sample-specific universal adapters not attached to a target fragment.3. (canceled)4. The composition of claim 1 , wherein the exonuclease comprises a 5′ to 3′ DNA exonuclease activity that is biased for double stranded DNA that comprises a 5′ phosphate at the 5′ end of the region of double stranded nucleic acid.5. The composition of claim 4 , wherein the exonuclease is lambda exonuclease.6. The composition of claim 1 , wherein the exonuclease comprises a 5′ to 3′ DNA exonuclease activity and 3′ to 5′ DNA exonuclease activity.7. The composition of claim 6 , wherein the adaptor-target-adaptor molecules comprise a modification at each of the 3′ ends to block the 3′ to 5′ DNA exonuclease ...

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Номер записи: 81
24-09-2020 дата публикации

ASSAY FOR EXO-SITE BINDING MOLECULES

Номер: US20200299685A1
Автор: Liu David R., Maianti Juan Pablo
Принадлежит: President and Fellows of Harvard College

Methods for the identification of agents the bind to exo-sites of proteins are provided. Agents identified by the methods described herein and pharmaceutical compositions comprising the identified agents are also provided. Methods of using an identified agent for the treatment or prevention of a disease, disorder, or condition are also provided, including methods of treating or preventing a disease associated with reduced, elevated, or ectopic expression or aberrant activity of a protein comprising an exo-site. 1. A method of identifying an agent that binds an exo-site of a protein , the method comprising:providing a first variant of the protein, wherein the protein comprises an exo-site;providing a second variant of the protein, wherein the exo-site of the second variant comprises at least one different amino acid than the exo-site of the first variant;contacting a library of candidate agents with each of the first and second variants;determining an enrichment-dependent parameter of each candidate agent to each of the first and second variants by a library binding or enrichment assay;comparing, for each candidate agent, the binding to the first variant with the binding to the second variant, wherein if the enrichment-based parameter using the first protein variant is greater than the enrichment-based parameter using the second protein variant, then the candidate agent is identified as an agent that binds an exo-site of the protein.2. The method of claim 1 , wherein the exo-site comprises a binding pocket that modulates the interactions of the protein with one or more substrates claim 1 , one or more metabolites claim 1 , or one or more native partners of the protein when an agent is bound to the exo-site.3. The method of or claim 1 , wherein the exo-site comprises a binding pocket that modulates the substrate selectivity or binding preferences of the protein when an agent is bound to the exo-site.4. The method of any one of - claim 1 , wherein the exo-site ...

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Номер записи: 82
03-10-2019 дата публикации

METHODS FOR DETERMINING LYMPHOCYTE RECEPTOR CHAIN PAIRS

Номер: US20190300954A1
Автор: DA COSTA Daniel Jay, Hansen Carl Lars Genghis, HEYRIES Kevin Albert, MEWIS Georgia Elizabeth, RICICOVA Marketa, VANINSBERGHE Michael Andrew
Принадлежит:

Provided herein are high-throughput sequencing methods to study the diversity and functionality of lymphocyte receptor chains and pairing of the same. Specifically, the methods provided herein are used to identify with confidence one or more lymphocyte receptor chain pairs in a sample, for example one or more functional chain pairs. 1. A method for identifying a functional immunoglobulin (Ig) heavy and Ig light chain pair in a sample comprising antibody secreting cells (ASCs) , comprising ,partitioning the sample into a plurality of individual vessels to provide a plurality of sample subpopulations;performing a functional assay on one or more of the sample subpopulations, wherein the functional assay measures a property of an Ig heavy and Ig light chain pair;identifying one or more functional subpopulations based on the results of the functional assay;lysing the ASCs in the one or more functional subpopulations;amplifying the Ig heavy and Ig light chain nucleic acid;attaching a unique DNA barcode to the Ig heavy and Ig light chain nucleic acid in each functional subpopulation, wherein the unique DNA barcode sequence identifies the functional subpopulation from which the Ig heavy and Ig light chain nucleic acid originated;pooling the barcoded Ig heavy and Ig light chain nucleic acid;sequencing the barcoded Ig heavy and Ig light chain nucleic acid to determine the identity of the Ig heavy and Ig light chains in each functional subpopulation;determining the observed distribution of each of the Ig heavy and Ig light chains across the functional subpopulations and calculating statistical probabilities that the observed distributions of Ig heavy and light chain pairs in the sample subpopulations are independent from one another; andidentifying the functional Ig heavy and light chain pair based on the calculated statistical probabilities.2. The method of claim 1 , further comprising claim 1 , prior to the partitioning the sample step claim 1 , subjecting the sample to ...

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Номер записи: 83
01-11-2018 дата публикации

ENCRYPTED OPTICAL MARKERS FOR SECURITY APPLICATIONS

Номер: US20180312998A1
Автор: Liang MingHwa Benjamin, Szczepanik Maciej B.
Принадлежит: APDN (B.V.I.) INC.

Encrypted markers that are not readily detectable can be revealed by treatment with a specific reagent used as a developer to reveal a readily detectable physical property of the marker, such as a characteristic fluorescence emission after excitation with a particular excitation wavelength, or to reveal a visible color. The encrypted marker can be developed in situ, or a sample can be removed by brushing, scraping, swabbing or scratching the marked object or item and developing the encrypted marker or a sample thereof with the appropriate developer to reveal an overt marker or optical signal. The encrypted marker may include a DNA taggant. 1. A method for cryptically marking an item , the method comprising:providing a marker wherein the marker is a pro-fluorophore, a chromogen, or a combination thereof being capable of producing a readily detectable fluorophore, chromophore, or combination thereof upon reaction with a developer, andcombining the DNA taggant and the marker to form a DNA security marker;attaching the DNA security marker to the item; andthereby providing a cryptically marked item.2. The method according to claim 1 , wherein the marker is a pro-fluorophore.3. The method according to claim 1 , wherein the marker is a chromogen.4. The method according to claim 1 , wherein the DNA taggant is double-stranded DNA of a non-naturally occurring sequence that may be readily identifiable.5. The method according to claim 1 , wherein the DNA has a length of between about 80 and 500 base pairs.6. The method according to claim 1 , wherein the DNA is suspended in an aqueous solution or a non-aqueous solution.7. The method according to claim 1 , wherein the ratio of DNA taggant to marker is about 1:100 to about 1:10 claim 1 ,000.8. The method according to claim 7 , wherein the ratio of DNA taggant to marker is about 1:800 to about 1:5 claim 7 ,000.9. The method according to claim 1 , wherein the DNA security marker further comprises a perturbant.10. The method ...

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Номер записи: 84
08-10-2020 дата публикации

AUTOMATED INSTRUMENTATION FOR PRODUCTION OF T-CELL RECEPTOR PEPTIDE LIBRARIES

Номер: US20200318256A1
Автор: Church Deanna, Federowicz Stephen, Graige Michael
Принадлежит:

The present disclosure provides instrumentation and automated methods for creating cell surface display libraries, where the cells of the library display engineered peptides on their cell surfaces for identification of antigens that bind to T-cell receptors. The engineered peptides may be putative antigens or binding regions of the T-cell receptors. 1. An automated method of creating a cell library co-expressing engineered proteins and MHC molecules , the method comprising:providing a population of cells;processing the population of cells using an instrument for multiplexed nuclease-directed genome editing using introduced nucleic acids and a nucleic acid-directed nuclease, wherein the introduced nucleic acids comprise nucleic acids that encode engineered proteins and MHC molecules configured to be displayed on a surface of the cells, and wherein transcription of at least one component of a nucleic acid-guided nuclease editing system is under the control of an inducible promoter;incubating the processed cells to facilitate nucleic acid editing in the cells, wherein the edited cells co-express the engineered proteins and MHC molecules in the cells; andallowing the cells to display the engineered proteins and MHC molecules on the surface of the cells.2. The method of claim 1 , wherein expression of the nucleic acid-directed nuclease is under control of an inducible promoter.3. The method of claim 1 , wherein expression of a guide RNA is under control of an inducible promoter.4. The method of claim 1 , wherein the population of cells are yeast cells.5. The method of claim 1 , wherein the population of cells are mammalian cells.6. The method of claim 1 , wherein the nuclease comprises an RNA-directed nuclease.7. The method of claim 6 , wherein the nuclease comprises Cas 9.8. The method of claim 6 , wherein the nuclease comprises Cas 12/Cpf I.9. The method of claim 6 , wherein the RNA-directed nuclease comprises MAD7.10. (canceled)11. An automated method of creating a ...

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Номер записи: 85
23-11-2017 дата публикации

Methods for Modulating ATRX-Dependent Gene Repression

Номер: US20170335317A1
Автор: Lee Jeannie T., Sarma Kavitha
Принадлежит:

Methods and compositions for modulation of the activity of alpha thalassemia/mental retardation syndrome X-linked (ATRX), e.g., modulation of DNA-ATRX or RNA-ATRX interactions, and methods for identifying and using compounds that modulate DNA-ATRX or RNA-ATRX interactions, as well as the compounds themselves. 1. A method of preparing a library of nuclear ribonucleic acids (nRNAs) that specifically bind ATRX , the method comprising:(a) contacting a sample containing nRNAs with (i) ATRX protein and (ii) a ATRX binding agent, under conditions sufficient to form complexes between the nRNA, ATRX protein and the ATRX binding agent, and(b) isolating the complexes.2. The method of claim 1 , further comprising:(c) synthesizing cDNA complementary to the nRNA, and(d) selecting cDNAs that (i) have RPKM above a desired threshold or (ii) are enriched compared to a control library, or both (i) and (ii).3. A method of preparing a plurality of cDNAs complementary to a pool of nuclear ribonucleic acids (nRNAs) claim 1 , the method comprising:(a) providing a sample comprising nRNAs bound to nuclear proteins;(b) contacting the sample with an agent, preferably an antibody, that binds specifically to ATRX protein, under conditions sufficient to form complexes between the agent and ATRX proteins, such that the nRNAs remain bound to the ATRX proteins;(c) isolating the complexes;(d) synthesizing DNA complementary to the nRNAs to provide an initial population of cDNAs;(e) optionally PCR-amplifying the cDNAs using strand-specific primers;purifying the initial population of cDNAs to obtain a purified population of cDNAs that are at least about 20 nucleotides (nt) in length;(f) sequencing at least part of substantially all of the purified population of cDNAs; comparing the high-confidence sequences to a reference genome, and selecting those sequences that have a high degree of identity to sequences in the reference genome; and(g) selecting those cDNAs that have (i) reads per kilobase per ...

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Номер записи: 86
01-12-2016 дата публикации

DISEASE-ASSOCIATED GENETIC VARIATIONS AND METHODS FOR OBTAINING AND USING SAME

Номер: US20160348178A1
Автор: Bueno Raphael, Sugarbaker David J.
Принадлежит:

The invention provides a comprehensive, rapid, unbiased, and accurate method for identifying and/or discovering disease-associated genetic variations, e.g., disease-associated variations. The present invention further provides novel disease-associated genetic variations for use as genetic markers of disease, e.g., cancer. The invention further provides methods for assessing an individual's risk for developing a disease, e.g., cancer, by detecting the presence the novel disease-associated genetic variations of the invention. 1. An isolated nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NOS: 1 , 3 , 5 , 7 , 9 , 11 , 13 , 15 , 17 , 19 , 21 , 23 , 25 , 27 , 29 and a homolog thereof , wherein the nucleotide sequences each further comprise at least one genetic variation which predisposes a person to malignant pleural mesothelioma.2. The isolated nucleic acid molecule of claim 1 , wherein the at least one genetic variation is a single nucleotide polymorphism.3. The isolated nucleic acid molecule of claim 1 , wherein the at least one genetic variation is a single nucleotide polymorphism claim 1 , a somatic mutation claim 1 , an inversion claim 1 , a deletion claim 1 , an insertion claim 1 , or an LOH mutation.4. The isolated nucleic acid molecule of claim 3 , wherein the LOH mutation is due to a deletion claim 3 , epigenetic silencing claim 3 , X inactivation claim 3 , or RNA editing.5. The isolated nucleic acid molecule of claim 1 , wherein the homolog has at least about 85% sequence identity.6. The isolated nucleic acid molecule of claim 1 , wherein the homolog has at least about 90% sequence identity.7. The isolated nucleic acid molecule of claim 1 , wherein the homolog has at least about 95% sequence identity.8. An isolated nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NOS: 2 claim 1 , 4 claim 1 , 6 claim 1 , 8 claim 1 , 10 claim 1 , 12 claim 1 , 14 claim 1 , 16 ...

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Номер записи: 87
31-10-2019 дата публикации

METHOD FOR GENERATING HIGHER ORDER GENOME EDITING LIBRARIES

Номер: US20190330616A1
Автор: DE BRUYN Rahel, DIEHL Valentina, ERNST Andreas, KAULICH Manuel, Wegner Martin, WIECHMANN Svenja
Принадлежит:

The present invention pertains to a novel method for the generation of highly diverse RNA expressing vectors and vector libraries for use in targeted gene knock out, knock down and genome modification approaches. The invention pertains to a method for generating such higher order libraries without the need of classical cloning technologies. This is particularly useful for libraries based on large vectors wherein a sequence cannot be easily mutated with classical mutagenesis methods. The vectors and libraries generated according to the methods of the invention are in particular for RNA assisted silencing technologies such as RNA interference, and for targeted genome editing using the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system or similar RNA/DNA-encoded gene perturbation systems which use small guide RNAs to target the CRISPR complex to a specific genomic sequence. The invention provides also kits comprising the materials for performing the methods of the invention.

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Номер записи: 88
15-12-2016 дата публикации

Pooled method for high throughput screening of trans factors affecting rna levels

Номер: US20160362684A1
Автор: Calvin H. JAN, Jonathan Weissman, Luke A. GILBERT, Onn Brandman
Принадлежит: UNIVERSITY OF CALIFORNIA

Provided herein are methods directed to multiplexed detection of the modulation of transcriptional activity using perturbation elements.

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Номер записи: 89
14-11-2019 дата публикации

METHODS FOR IDENTIFYING T-CELL RECEPTOR ANTIGENS

Номер: US20190345484A1
Автор: Church Deanna, Federowicz Stephen, Graige Michael
Принадлежит:

The present disclosure automated methods for creating cell surface display libraries, where the cells of the library display engineered peptides on their cell surfaces for identification of antigens that bind to T-cell receptors. The engineered peptides are preferably expressed in the cells under conditions that provide both secretion and display of the engineered peptides on the cell surfaces, thus providing access of the engineered peptide antigens to identify potential TCR binding targets. The cell libraries cab be engineered using an automated editing system that provides for one or more targeted edits per cell. 1. An automated method of creating a cell library expressing engineered peptides using instrumentation , the method comprising:providing a population of cells;processing the population of cells using an instrument for multiplexed nuclease-directed genome editing using introduced nucleic acids and a nucleic acid-directed nuclease to create cells comprising nucleic acids that encode engineered peptides configured to be displayed on a surface of the cells;incubating the processed cells to facilitate nucleic acid editing in the cells, wherein the editing provides nucleic acids that encode engineered peptide antigens in the cells; andallowing the cells to express and display the engineered peptides on the surface of the cells.2. The method of claim 1 , wherein the engineered peptides are putative TCR binding antigens3. The method of claim 1 , wherein the engineered peptides comprise predicted TCR binding regions.4. The method of claim 1 , wherein the population of cells are yeast cells.5. The method of claim 1 , wherein the nuclease is an RNA-directed nuclease.6. An automated method of creating a cell library expressing engineered putative T-cell receptor (TCR) antigens on the surface of the cells claim 1 , the method comprising:providing a population of cells;processing the population of cells using an instrument for multiplexed nuclease-directed genome ...

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Номер записи: 90
14-11-2019 дата публикации

METHODS AND SYSTEMS FOR MOLECULAR LIBRARY GENERATION

Номер: US20190345636A1
Автор: McDermott Geoffrey, Price Andrew, Schnall-Levin Michael
Принадлежит:

The present disclosure provides methods and systems for generation of libraries comprising two or more sets of molecules. Libraries may comprise sets of molecules of different types. Sets of molecules may be generated by attachment to supports. Different types of molecules may be generated combinatorially, thereby generating a large number of unique molecules. One or more types of molecules may be used to identify one or more additional types of molecules. Libraries of the present disclosure may be used in, for example, nucleic acid sequencing. 1. A method for generating a plurality of molecules , comprising:(a) providing a plurality of supports; and(b) combinatorially generating a plurality of molecules coupled to and/or comprised in each of said plurality of supports, wherein a support of said plurality of supports comprises a first set of molecules and a second set of molecules of said plurality of molecules, which first set of molecules and second set of molecules are of different types.2. The method of claim 1 , wherein said first set of molecules comprises deoxyribonucleic acid (DNA) molecules and said second set of molecules comprises molecules selected from the group consisting of (i) ribonucleic acid (RNA) molecules claim 1 , (ii) peptide molecules claim 1 , (iii) chemical compounds each having a molecular weight less than 900 Daltons claim 1 , and (iv) molecules that are not DNA and RNA.3. (canceled)4. The method of claim 1 , wherein said first set of molecules comprises ribonucleic acid (RNA) molecules and said second set of molecules comprises molecules selected from the group consisting of (i) peptide molecules claim 1 , (ii) chemical compounds each having a molecular weight less than 900 Daltons claim 1 , and (iii) molecules that are not DNA and RNA.57.-. (canceled)8. The method of claim 1 , wherein said first set of molecules comprises one or more nucleic acid barcode sequences.9. The method of claim 1 , wherein said plurality of supports is a ...

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Номер записи: 91
21-12-2017 дата публикации

METHOD FOR IN SITU DETERMINATION OF NUCLEIC ACID PROXIMITY

Номер: US20170362649A1
Автор: DUDCHENKO Olga, Lander Eric, LIEBERMAN-AIDEN Erez, Rao Suhas, STAMENOVA Elena
Принадлежит:

Disclosed is an in situ method for detecting spatial proximity relationships between nucleic acid sequences, such as DNA, in a cell. The method includes: providing a sample of one or more cells comprising nucleic acids; fragmenting the nucleic acids present in the cells that leaves 5′ overhanging ends; filling in the overhanging ends with at least one labeled nucleotide; joining the filled in end of the fragmented nucleic acids that are in close physical proximity to create one or more end joined nucleic acid fragments having a junction; isolating the one or more end joined nucleic acid fragments using the labeled nucleotide; and determining the sequence at the junction of the one or more end joined nucleic acid fragments. 1. An in situ method for detecting spatial proximity relationships between nucleic acid sequences in a cell , comprising:providing a sample of one or more cells, cellular nuclei, or an acellular nucleic acid system comprising nucleic acids;fragmenting the nucleic acids present in the cells, wherein the fragmented nucleic acids are fragmented to create overhanging ends;filling in the overhanging ends with at least one labeled nucleotide, wherein the labeled nucleotide is used to isolate the nucleic acids;joining the filled in end of the fragmented nucleic acids that are in close physical proximity to create one or more end joined nucleic acid fragments having one or more junctions, wherein the site of the one or more junctions comprises one or more labeled nucleic acids;isolating the one or more end joined nucleic acid fragments using the labeled nucleotide; anddetermining the sequence at the one or more junctions of the one or more end joined nucleic acid fragments, thereby detecting spatial proximity relationships between nucleic acid sequences in a cell.2. The method of claim 1 , wherein the nucleic acids are held in a fixed position relative to one another.3. The method of or claim 1 , wherein determining the sequence at the one or more ...

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Номер записи: 92
05-11-2020 дата публикации

METHODS AND COMPOSITIONS FOR POLYPEPTIDE ANALYSIS

Номер: US20200348307A1
Автор: Beierle John M., Gunderson Kevin, JAMES Robert C., LEBL Michael, Monfregola Luca, Shi Lei
Принадлежит: Encodia, Inc.

The present disclosure relates to methods and kits for analysis of polypeptides. In some embodiments, the present methods and kits employ barcoding and nucleic acid encoding of molecular recognition events, and/or detectable labels. 2. The method of claim 1 , wherein:step (a) comprises providing the polypeptide and an associated recording tag joined to a support (e.g., a solid support);step (a) comprises providing the polypeptide joined to an associated recording tag in a solution;step (a) comprises providing the polypeptide associated indirectly with a recording tag; orthe polypeptide is not associated with a recording tag in step (a).35-. (canceled)78-. (canceled)9. The method of claim 6 , further comprising the steps of:functionalizing the new NTAA of the polypeptide with a chemical reagent to yield a newly functionalized NTAA;(g) contacting the polypeptide with a second (or higher order) binding agent comprising a second (or higher order) binding portion capable of binding to the newly functionalized NTAA and (g1) a second coding tag with identifying information regarding the second (or higher order) binding agent, or (g2) a second detectable label; '(h2) detecting the second detectable label, and', '(h) (h1) transferring the information of the second coding tag to the first extended recording tag to generate a second extended recording tag and analyzing the second extended recording tag, or'}(i) eliminating the functionalized NTAA to expose a new NTAA;wherein step (f) is conducted before step (g), after step (g) and before step (h), or after step (h).1012-. (canceled)13. The method of claim 1 , wherein the polypeptide is obtained by fragmenting a protein from a biological sample.14. The method of claim 1 , wherein the recording tag and/or coding tag comprises a nucleic acid claim 1 , an oligonucleotide claim 1 , a modified oligonucleotide claim 1 , a DNA molecule claim 1 , a DNA with pseudo-complementary bases claim 1 , a DNA with protected bases claim 1 , an ...

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Номер записи: 93
21-11-2019 дата публикации

Display of molecules on silently genetically encoded nanoscale carriers for determining synergistic molecular interactions

Номер: US20190352636A1
Автор: Nicholas Bennett, Ratmir Derda, Susmita SARKAR
Принадлежит: University of Alberta

The present application provides a method of producing a “liquid” array of ligand (such as glycan) modified bacteriophage where the ligand modification is encoded genetically within the bacteriophage genome. This method will allow for the determination of the ligand binding profile of biomacromolecules and cells. Furthermore the method allows the elucidation of ligand-protein interactions where ligand binding is co-operative and synergistic.

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Номер записи: 94
20-12-2018 дата публикации

METHOD OF SYNTHESIS AND TESTING OF COMINATORIAL LIBRARIES USING MICROCAPSULES

Номер: US20180361346A1
Автор: Abell Chris, Griffiths Andrew David, Hollfelder Florian, Mastrobattista Enrico
Принадлежит:

Methods for use in the synthesis and identification of molecules which bind to a target component of a biological system or modulate the activity of a target are described. 1. A method of forming a small molecule compound comprising:providing at least a first aqueous fluid comprising at least a first small molecule compound having a molecular weight of less than 500 Daltons;providing at least a second aqueous fluid comprising at least a second small molecule compound having a molecular weight of less than 500 Daltons;encapsulating said at least first small molecule compound into at least a first aqueous microcapsule within an immiscible fluorocarbon oil;encapsulating said at least second small molecule compound into at least a second aqueous microcapsule within an immiscible fluorocarbon oil; andfusing said at least first and said at least second aqueous microcapsules, thereby causing a chemical reaction between said at least first and at least second small molecule compounds to form at least a third small molecule compound.2. The method of claim 1 , wherein said at least first small molecule compound is attached to a microbead.3. The method of claim 1 , wherein said at least second small molecule compound is attached to a microbead.4. The method of claim 1 , further comprising isolating said at least third small molecule compound.5. The method of claim 1 , further comprising identifying said at least third small molecule compound.6. The method of claim 5 , wherein said at least third small molecule compound is identified by its optical properties.7. The method of claim 1 , further comprising identifying said at least first and at least second small molecule compounds which reacted to form said at least third small molecule compound.8. The method of claim 1 , wherein said at least first small molecule compound comprises at least a first label and said at least second small molecule compound comprises at least a second label claim 1 , wherein said at least first and ...

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Номер записи: 95
28-11-2019 дата публикации

HIGH-THROUGHPUT SINGLE-CELL POLYOMICS

Номер: US20190360121A1
Автор: Dura Burak, Fan Rong
Принадлежит: YALE UNIVERSITY

Provided herein, in some embodiments, are devices, systems and methods for high-throughput single-cell polyomics (e.g., genomic, epigenomic, proteomic and/or phenotypic profile) analyses. 1. A polyomic multiplexing device , comprising:a substrate comprising X rows intersecting Y columns to form X*Y patches, wherein each of the X*Y patches comprises a unique nucleic acid barcode that is immobilized to the substrate and comprises a polyT sequence,wherein each row comprises a different subset of barcoded nucleic acid strands of a first set of nucleic acid strands, and each column comprises a different subset of barcoded nucleic acid strands of a second set of nucleic acid strands, andwherein the nucleic acid strands of the first set are bound to nucleic acid strands of the second set to form a unique nucleic acid barcode.2. The polyomic multiplexing device of claim 1 , wherein X is at least 10.3. The polyomic multiplexing device of claim 1 , wherein X is at least 20 claim 1 , at least 50 claim 1 , at least 100 claim 1 , at least 1000 claim 1 , at least 10000 claim 1 , or at least 20000.4. The polyomic multiplexing device of claim 1 , wherein X is 10 to 20000.5. The polyomic multiplexing device of any one of - claim 1 , wherein Y is at least 10.6. The polyomic multiplexing device of any one of - claim 1 , wherein Y is at least 20 claim 1 , at least 50 claim 1 , at least 100 claim 1 , at least 1000 claim 1 , at least 10000 claim 1 , or at least 20000.7. The polyomic multiplexing device of any one of - claim 1 , wherein Y is 10 to 20000.8. The polyomic multiplexing device of any one of - further comprising Zcolumns intersecting the X rows to form X*Z patches claim 1 , wherein each of the X*Z patches comprises a molecular binding partner immobilized to the substrate claim 1 , and wherein n is zero or greater.9. The polyomic multiplexing device of claim 8 , wherein n is at least 1 claim 8 , and each of the Zcolumns comprises a different molecular binding partner.10. The ...

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Номер записи: 96
26-11-2020 дата публикации

Engineering aav

Номер: US20200370137A1
Автор: David S. OJALA, Kyle McGovern
Принадлежит: Sangamo Therapeutics Inc

The present disclosure provides methods and compositions to develop AAV capsids with a desired characteristic compared to a natural AAV serotype. These capsids are useful, for example, for the delivery of genome engineering molecules and gene therapy molecules for the treatment of a subject in need thereof.

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Номер записи: 97
26-11-2020 дата публикации

METHODS, SYSTEMS, COMPUTER READABLE MEDIA, AND KITS FOR SAMPLE IDENTIFICATION

Номер: US20200370202A1
Автор: HUBBELL Earl
Принадлежит: LIFE TECHNOLOGIES CORPORATION

A method for sequencing a polynucleotide sample having a barcode sequence, includes: introducing a series of nucleotides to the polynucleotide sample according to a predetermined flow ordering; obtaining a series of signals resulting from the introducing of nucleotides to the polynucleotide sample; and resolving the series of signals over the barcode sequence to render a flowspace string, wherein the flowspace string is a codeword of an error-tolerant code capable of distinguishing the barcode sequence from other barcode sequences in the presence of one or more errors. 1. A method for sequencing a polynucleotide sample having a barcode sequence , comprising:introducing a series of nucleotides to the polynucleotide sample according to a predetermined flow ordering;obtaining a series of signals resulting from the introducing of nucleotides to the polynucleotide sample; andresolving the series of signals over the barcode sequence to render a flowspace string, wherein the flowspace string is a codeword of an error-tolerant code capable of distinguishing the barcode sequence from other barcode sequences in the presence of one or more errors.2. The method of claim 1 , wherein the error-tolerant code is an error-correcting code.3. The method of claim 2 , wherein the error-correcting code is a ternary code using a three character alphabet.4. The method of claim 3 , wherein a first character of the alphabet represents a 0-mer signal claim 3 , a second character in the alphabet represents a 1-mer signal claim 3 , and a third character in the alphabet represents a 2-mer signal.5. The method of claim 3 , wherein the ternary code is a ternary Hamming code.6. The method of claim 3 , wherein the ternary code is a ternary Golay code.7. The method of claim 1 , wherein the codeword has a length of 15 or fewer characters.8. The method of claim 2 , wherein the error-correcting code is capable of correcting up to two errors in the flowspace string.9. The method of claim 2 , wherein the ...

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Номер записи: 98
05-12-2019 дата публикации

OLIGONUCLEOTIDE PROBES AND USES THEREOF

Номер: US20190367903A1
Автор: DOMENYUK Valeriy, Hornung Tassilo, MIGLARESE Mark, Spetzler David
Принадлежит:

Methods and compositions are provided to identify oligonucleotides that bind targets of interest. The targets include tissues, cells, circulating biomarkers such as microvesicles, including those derived from various diseases. The oligonucleotides can be used in diagnostic and therapeutic applications. 1. A method of enriching an oligonucleotide probe library comprising a plurality of oligonucleotides , the method comprising:(a) providing a support arrayed with a plurality of samples, wherein a portion of the plurality of samples are cancer samples and another portion of the plurality of samples are non-cancer control samples, and wherein each of the plurality of samples comprises microvesicles;(b) contacting the plurality of samples arrayed on the support with the plurality of oligonucleotides; and(c) recovering members of the oligonucleotide probe library that bound to members of the plurality of samples, thereby enriching the oligonucleotide probe library.2. The method of claim 1 , further comprising repeating steps (a)-(c) at least 5 times.310.-. (canceled)11. The method of claim 1 , wherein the unenriched oligonucleotide probe library comprises at least 10different oligonucleotide sequences.1214.-. (canceled)15. The method of claim 1 , wherein the plurality of cancer samples comprises a bodily fluid or a fraction or derivative thereof.16. The method of claim 15 , wherein the bodily fluid comprises at least one of peripheral blood claim 15 , sera claim 15 , plasma claim 15 , ascites claim 15 , urine claim 15 , cerebrospinal fluid (CSF) claim 15 , sputum claim 15 , saliva claim 15 , bone marrow claim 15 , synovial fluid claim 15 , aqueous humor claim 15 , amniotic fluid claim 15 , cerumen claim 15 , breast milk claim 15 , broncheoalveolar lavage fluid claim 15 , semen claim 15 , prostatic fluid claim 15 , Cowper's fluid claim 15 , pre-ejaculatory fluid claim 15 , female ejaculate claim 15 , sweat claim 15 , fecal matter claim 15 , hair oil claim 15 , tears claim ...

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Номер записи: 99
31-12-2020 дата публикации

AUTOMATED INSTRUMENTATION FOR PRODUCTION OF T-CELL RECEPTOR PEPTIDE LIBRARIES

Номер: US20200407876A1
Автор: Church Deanna, Federowicz Stephen, Graige Michael
Принадлежит:

The present disclosure provides instrumentation and automated methods for creating cell surface display libraries, where the cells of the library display engineered peptides on their cell surfaces for identification of antigens that bind to T-cell receptors. The engineered peptides may be putative antigens or binding regions of the T-cell receptors. 1. A method for expressing T-cell receptors (TCR's) on the surface of cells comprising:providing a population of cells;processing the population of cells using an instrument for multiplexed nuclease-directed genome editing using introduced nucleic acids and a nucleic acid-directed nuclease to create cells, wherein the introduced nucleic acids comprise nucleic acids derived from a T-cell receptor loci;incubating the processed cells to facilitate nucleic acid editing in the cell; andallowing the cells to express and display the engineered T-cell receptor on the surface of the cells.2. The method of claim 1 , wherein the T-cell receptor loci is a TCRα loci.3. The method of claim 1 , wherein the T-cell receptor loci is a TCRβ loci.4. The method of claim 1 , wherein the T-cell receptor loci is a TCRδ loci.5. The method of claim 1 , wherein the T-cell receptor loci is a TCRγ loci.6. The method of claim 1 , wherein the nuclease is an RNA-directed nuclease.7. The method of claim 6 , wherein the RNA-directed nuclease is selected from MAD7 claim 6 , Cas 9 claim 6 , Cas12/Cpf1.8. The method of claim 1 , wherein the cells are mammalian cells.9. A method for identifying cells expressing T-cell receptors (TCR's) on their surface comprising:providing a population of cells;processing the population of cells using an instrument for multiplexed nuclease-directed genome editing using introduced nucleic acids and a nucleic acid-directed nuclease to create cells, wherein the introduced nucleic acids comprise nucleic acids derived from a T-cell receptor loci;incubating the processed cells to facilitate nucleic acid editing in the cell; ...

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Номер записи: 100