Поиск патентов

Настройки

Глубина выборки

Укажите год

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

Подробнее

Мониторинг СМИ

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

Подробнее

Форма поиска

Ключевые слова. Может быть несколько по одной на строку

Поддерживает ввод нескольких поисковых фраз (по одной на строку). При поиске обеспечивает поддержку морфологии русского и английского языка

Автор

Ведите корректный номера.

Владелец

Ведите корректный номера.

Классы IPC

Ведите корректный номера.

Классы CPC

Ведите корректный номера.

Начиная с года

Укажите год

Заканчивая годом

Укажите год

Применить Всего найдено 11730. Отображено 100.

01-03-2012 дата публикации

OLIGONUCLEOTIDES FOR DETECTING E. coli O157:H7 STRAINS AND USE THEREOF

Номер: US20120052494A1

Автор: Jason Opdyke, Jun Li, Win Den Cheung

Принадлежит: Samsung Techwin Co Ltd

Oligonucleotides, a kit, and a method for detecting E. coli O157:H7 strains are provided. According to the kit for detecting E. coli O157:H7 strains and the method of detecting E. coli O157:H7 strains by using the kit, the results of the detection can be rapidly identified with a reduced number of copies of a sample in real-time.

Подробнее

Номер записи: 1

08-03-2012 дата публикации

METHOD OF DIAGNOSING POOR SURVIVAL PROGNOSIS COLON CANCER USING miR-203

Номер: US20120058914A1

Автор: Aaron J. Schetter, Carlo M. Croce, Curtis C. Harris

Принадлежит: Ohio State University Research Foundation, US Department of Health and Human Services

The present invention provides novel methods and compositions for the diagnosis and treatment of colon cancers. In particular, the present invention provides diagnostics and prognostics for colon (including colon adenocarcinoma) cancer patients, wherein the methods related to measuring miR levels can predict poor survival. The invention also provides methods of identifying inhibitors of tumorigenesis.

Подробнее

Номер записи: 2

27-09-2012 дата публикации

Method for detecting microorganisms belonging to mycoplasma pneumoniae and/or mycoplasma genitalium

Номер: US20120244544A1

Автор: Atsuko Minagawa, Hatsue Itagaki, Kazuyuki Sugiyama, Toyomasa Hiroshima, Yasushi Shimada, Yuki Mitobe

Принадлежит: LSI Medience Corp

A detection method and a detection kit for rapidly and specifically diagnosing Mycoplasma pneumoniae and/or Mycoplasma genitalium infections are provided. The DnaK of Mycoplasma pneumoniae or Mycoplasma genitalium is used as an indicator.

Подробнее

Номер записи: 3

04-10-2012 дата публикации

Method for analyzing rna

Номер: US20120253687A1

Автор: Hitoshi Nobumasa, Osamu Nomura, Toshihiko Kuroda

Принадлежит: TORAY INDUSTRIES INC

A method of analyzing RNA extracted from a tissue or cell(s) fixed with a fixative, said method comprising a determining step of whether said RNA satisfies: B/A≦1 wherein A represents the weight ratio (%) of RNA within the range of 1000 to 4000 nucleotides with respect to the total weight of RNA as determined by electrophoresis; and B represents the weight ratio (%) of RNA within the range of more than 4000 nucleotides with respect to the total weight of RNA as determined by electrophoresis.

Подробнее

Номер записи: 4

18-10-2012 дата публикации

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

Номер: US20120264630A1

Автор: Ismaïl Cisse, Ulrich Bockelmann, Virgile Viasnoff

Принадлежит: Individual

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

Подробнее

Номер записи: 5

01-11-2012 дата публикации

Kit for detection of microorganism

Номер: US20120277121A1

Автор: Shinichi Yoshida, Takashi Soejima

Принадлежит: Morinaga Milk Industry Co Ltd

A kit for use in a method for detecting live cells, injured cells and dead cells of a microorganism in a test sample by a nucleic acid amplification method is disclosed. The kit includes a cross-linker capable of cross-linking DNA by irradiation with light having a wavelength of 350 nm to 700 nm, medium, and a primer (s) for amplifying a DNA target region in the microorganism by a nucleic acid amplification method. The nucleic acid amplification method may be a PCR, LAMP, SDA, LCR or DNA microarray method. A cross-linker may be included in the kit such as ethidium monoazide, ethidium diazide, psoralen, 4,5′,8-trimethyl psoralen, or 8-methoxy psoralen.

Подробнее

Номер записи: 6

22-11-2012 дата публикации

Determining codon distribution and/or base pair distance between codons in a nucleic acid

Номер: US20120295251A1

Автор: Adrian John Gibbs, Andrew Franklin, Christian Samundsett, Mark John Gibbs, Murali Nayudu, Sheba Khan, Terry John Murphy, Yafei Zhang

Принадлежит: EZYGENE Pty Ltd

The present invention relates to methods for the design and/or production of a probe or primer that is capable of hybridizing to a plurality of sites in a sample comprising nucleic acid. Furthermore, the present invention provides methods for detecting and amplifying nucleic acid using such a probe or primer, for example, for identification of a strain, species or genera. Probe or primer sequences are determined by reference to codon usage bias of a target nucleic acid. In addition, the present invention provides methods for determining codon distribution and/or base pair distance between codons in a nucleic acid.

Подробнее

Номер записи: 7

24-01-2013 дата публикации

Use of Ribozymes in the Detection of Adventitious Agents

Номер: US20130022964A1

Автор: Matthew C. Coffey

Принадлежит: Oncolytics Biotech Inc

The present invention provides a method of detecting adventitious agents in a composition comprising a microorganism by using ribozyme-expressing indicator cells, as well as indicator cells useful in such detection.

Подробнее

Номер записи: 8

28-03-2013 дата публикации

Probe, and polymorphism detection method using the same

Номер: US20130078631A1

Автор: Mariko Komori

Принадлежит: Arkray Inc

The present disclosure relates to a probe for detecting a polymorphism, a method of detecting a polymorphism, a method of evaluating the efficacy of a drug, and a reagent kit for detecting a polymorphism.

Подробнее

Номер записи: 9

11-04-2013 дата публикации

Detection of Immune Cells, In Particular T Cells Through DNA-Methylation Analysis of the Genes CCR6 and BLR1

Номер: US20130089861A1

Автор: Sven Olek

Принадлежит: Individual

The present invention relates to a method, in particular an in vitro method, for identifying certain immune cells of a mammal, comprising analysing the methylation status of at least one CpG position in the gene CCR6 and/or BLR1 or an orthologous or paralogous gene thereof, and the use of DNA-methylation analysis of the genes of the proteins CCR6 and/or BLR1 for a detection and quality assurance and control of certain immune cells. In particular, the present invention relates to analysing the methylation status of at least one CpG position in the gene CCR6 in T cells. Furthermore, the present invention relates to a kit for performing the above methods, as well as to respective uses.

Подробнее

Номер записи: 10

02-05-2013 дата публикации

Real-time redox sequencing

Номер: US20130109577A1

Автор: Jonas Korlach, Lei Sun, Stephen Turner

Принадлежит: Pacific Biosciences of California Inc

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

Подробнее

Номер записи: 11

16-05-2013 дата публикации

Method for producing rna-containing probe for detecting a target nucleotide

Номер: US20130122503A1

Автор: Hiroyuki Mukai, Junko Yamamoto, Kiyozo Asada, Tomoo Inden, Tooru Suzuki, Toshiharu Ohba

Принадлежит: Takara Bio Inc

An object of the present invention is to provide a simple and useful method for producing an RNA-containing probe for detecting a target nucleotide, a simple and useful method, device, and system for processing nucleotide sequence information, and a simple and useful method for detecting a target nucleotide. The present invention provides a method for processing nucleotide sequence information, the method comprising the step of generating partial nucleotide sequences which has 7 to 14 nucleotides and a Tm value of 25 to 40° C. and in which a target nucleotide or a nucleotide adjacent to the target nucleotide is located at a position between 3 and 5 nucleotides from the 3′ or 5′ end. The method according to the present invention is useful for simply and efficiently producing an RNA-containing probe for detecting a target nucleic acid, without the basis of researchers' experiences or guess, and are extremely useful not only in the field of genetic engineering, but also in the field of medical research.

Подробнее

Номер записи: 12

16-05-2013 дата публикации

Methods and devices for manipulation of target cells using a combined application of acoustical and optical radiations

Номер: US20130122564A1

Автор: Boris Krasovitski, Eitan Kimmel, Shy Shoham

Принадлежит: Technion Research and Development Foundation Ltd

A method of applying a nondestructive mechanical force on one or more cells in aqueous environment by inducing heat generated acoustic pressure pulses. The method comprises providing an energy transmission pattern to induce the applying of a desired nondestructive mechanical force on a at least one cell by forming a plurality of heat generated acoustic pressure pulses in an aqueous environment and instructing the radiation of a target area in proximity to the at least one cell in the aqueous environment with light energy according to the energy transmission pattern.

Подробнее

Номер записи: 13

30-05-2013 дата публикации

Rapid nucleic acid purification

Номер: US20130137107A1

Автор: Minsu KO, Siseok Lee, Young Cheol Kim

Принадлежит: Nanohelix Co Ltd

Provided is a method for rapid nucleic acid purification, and the method for rapid nucleic acid isolation according to the present invention is very useful in diagnosing causes of disease or detecting a target gene; can be used in molecular diagnosis of causes of disease more rapidly and conveniently, as compared with the existing nucleic acid isolation method requiring complicated and special equipment; does not require skills therefor, thereby allowing an ordinary person to personally conduct isolation of nucleic acid for analyzing causes of disease and further solving the existing inconvenience caused by directly going to the hospitals or health clinical centers; and can analyze causes of disease more promptly.

Подробнее

Номер записи: 14

04-07-2013 дата публикации

Method of dna sequencing by polymerisation

Номер: US20130171636A1

Автор: David Bensimon, Fang-Yuan Ding, Jean-Francois Allemand, Maria Manosas, Vincent Croquette

Принадлежит: ECOLE NORMALE SUPERIEURE

Described herein is a method for determining a nucleic acid sequence, said method comprising: a) denaturing a double-stranded nucleic acid molecule corresponding to the said nucleic acid sequence; b) hybridizing a single-stranded nucleic acid molecule, the primer, with the said denatured double-stranded nucleic acid molecule; c) applying a tension to the hybridized primer/double-stranded nucleic acid molecule obtained in b); d) incubating the hybridized primer/double-stranded nucleic acid molecule obtained in b) with a polymerase in conditions which will lead to at least one pause in replication; and e) determining a position of the said pause in replication with respect to one end of the double-stranded nucleic acid.

Подробнее

Номер записи: 15

05-09-2013 дата публикации

Capture of target dna and rna by probes comprising intercalator molecules

Номер: US20130230856A1

Автор: Jan Gorm Lisby, Nina JØHNK, Uffe Vest Schneider

Принадлежит: Quantibact AS

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

Подробнее

Номер записи: 16

19-09-2013 дата публикации

Compositions and methods for sequencing nucleic acids

Номер: US20130244886A1

Автор: Jason Richard Betley, Jonathan Mark Boutell, Niall Anthony Gormley, Roberto Rigatti

Принадлежит: Illumina Inc

Disclosed herein are compositions and methods for sequencing nucleic acids.

Подробнее

Номер записи: 17

31-10-2013 дата публикации

Nanoscale calorimeter on chip and related methods and devices

Номер: US20130288386A1

Автор: Chung Wah Fon, Michael L. Roukes

Принадлежит: California Institute of Technology CalTech

An article comprising: an array of calorimeter devices, wherein the device comprises: at least one fluidic enclosure disposed on a microfluidic chip, wherein the fluidic enclosure is substantially gas impermeable; at least one first chamber and at least one second chamber, wherein the first chamber and the second chamber are disposed within and enclosed by the fluidic enclosure, wherein the first chamber and the second chamber are not vacuum encapsulated; at least two microfluidic channels connected to the first chamber and at least two microfluidic channels connected to the second chamber; and at least one thermal sensor disposed between the chip and the first and second chambers, wherein the thermal sensor is adapted to measure a temperature differential between the first and second chambers. Examples include DSC and TSA devices. Biological binding and melting experiments can be done with high sensitivity.

Подробнее

Номер записи: 18

12-12-2013 дата публикации

Method for predicting the outcome of colon cancer by analysing mirna expression

Номер: US20130331291A1

Автор: Bernhard Mlecnik, Franck Pages, Herve Fridman, Jérôme GALON

Принадлежит: Assistance Publique Hopitaux de Paris APHP, Universite Paris Descartes Paris 5

The present invention relates to a method for predicting the outcome of a cancer. More particularly, the present invention relates to a method for predicting the outcome of a cancer in a patient comprising a step consisting of determining the expression level of a miRNA cluster in a sample obtained from said patient, wherein said miRNA cluster comprises: -miR.609 or, -miR.518c or, -miR.520f or, -miR.220a or, -miR.362 or, -miR.29a or, -miR.660 or, -miR.603 or, -miR.558 or, -miR519b or, -miR.494 or, -miR.130a or, -miR.639.

Подробнее

Номер записи: 19

26-12-2013 дата публикации

Nucleic Acid Analysis by Random Mixtures of Non-Overlapping Fragments

Номер: US20130345070A1

Автор: Radoje Drmanac

Принадлежит: Callida Genomics Inc

The invention provides methods and kits for ordering sequence information derived from one or more target polynucleotides. In one aspect, one or more tiers or levels of fragmentation and aliquoting are generated, after which sequence information is obtained from fragments in a final level or tier. Each fragment in such final tier is from a particular aliquot, which, in turn, is from a particular aliquot of a prior tier, and so on. For every fragment of an aliquot in the final tier, the aliquots from which it was derived at every prior tier is known, or can be discerned. Thus, identical sequences from overlapping fragments from different aliquots can be distinguished and grouped as being derived from the same or different fragments from prior tiers. When the fragments in the final tier are sequenced, overlapping sequence regions of fragments in different aliquots are used to register the fragments so that non-overlapping regions are ordered. In one aspect, this process is carried out in a hierarchical fashion until the one or more target polynucleotides are characterized, e.g. by their nucleic acid sequences, or by an ordering of sequence segments, or by an ordering of single nucleotide polymorphisms (SNPs), or the like.

Подробнее

Номер записи: 20

09-01-2014 дата публикации

Nucleic Acid Analysis by Random Mixtures of Non-Overlapping Fragments

Номер: US20140011688A1

Автор: Radoje Drmanac

Принадлежит: Callida Genomics Inc

The invention provides methods and kits for ordering sequence information derived from one or more target polynucleotides. In one aspect, one or more tiers or levels of fragmentation and aliquoting are generated, after which sequence information is obtained from fragments in a final level or tier. Each fragment in such final tier is from a particular aliquot, which, in turn, is from a particular aliquot of a prior tier, and so on. For every fragment of an aliquot in the final tier, the aliquots from which it was derived at every prior tier is known, or can be discerned. Thus, identical sequences from overlapping fragments from different aliquots can be distinguished and grouped as being derived from the same or different fragments from prior tiers. When the fragments in the final tier are sequenced, overlapping sequence regions of fragments in different aliquots are used to register the fragments so that non-overlapping regions are ordered. In one aspect, this process is carried out in a hierarchical fashion until the one or more target polynucleotides are characterized, e.g. by their nucleic acid sequences, or by an ordering of sequence segments, or by an ordering of single nucleotide polymorphisms (SNPs), or the like.

Подробнее

Номер записи: 21

06-01-2022 дата публикации

TAGMENTATION-ASSOCIATED MULTIPLEX PCR ENRICHMENT SEQUENCING

Номер: US20220002793A1

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

Принадлежит:

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

Подробнее

Номер записи: 22

07-01-2016 дата публикации

Methods and compositions for tagging and analyzing samples

Номер: US20160001248A1

Автор: Edward A. Hutchins, Hei-Mun Christina Fan

Принадлежит: Lineage Biosciences Inc USA

The invention relates to methods of tagging analytes in a sample.

Подробнее

Номер записи: 23

04-01-2018 дата публикации

METHODS OF CONSTRUCTING A CIRCULAR TEMPLATE AND DETECTING DNA MOLECULES

Номер: US20180002731A1

Автор: Tien Meng-Tsung, WU Mengchu

Принадлежит: Personal Genomics, Inc.

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

Подробнее

Номер записи: 24

04-01-2018 дата публикации

METHODS FOR MOLECULAR DETECTION

Номер: US20180002734A1

Автор: Jackson George, Quan Christopher

Принадлежит:

This invention relates to methods for molecular detection, particularly to methods utilizing target-specific molecular probes. In exemplary embodiments, target-specific molecular probes include single-stranded deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) aptamers. In general, the molecular probe may bind with relatively high specificity to a given target. In one aspect, a method for molecular detection comprises a molecular probe paired to a reporter molecule wherein the molecular probe impairs the amplification of the reporter molecule in the absence of the target molecule. 2. The method of claim 1 , wherein said at least one molecular probe is added prior to the addition of either of said at least one reporter molecule claim 1 , primer or both.3. The method of claim 2 , wherein said at least one molecular probe claim 2 , at least one reporter molecule and at least one primer are added simultaneously.4. The method of claim 1 , wherein said primer extension reaction comprises rolling circle amplification.5. The method of claim 1 , wherein said at least one molecule probe comprises a 3′ modification which is non-extensible by said primer extension reaction.6. The method of claim 1 , wherein said at least one primer shares at least a portion of its sequence in common with said at least one molecule probe.7. The method of claim 4 , wherein said rolling circle amplification is performed with a polymerase enzyme having strand displacement activity.8. The method of claim 1 , wherein said single-stranded nucleic acid product comprises a concatenation of the complement of said reporter molecule.9. The method of claim 1 , further comprising performing a quantification of said single-stranded nucleic acid product claim 1 , wherein said quantification correlates to the amount of said target present in said sample.10. The method of claim 9 , wherein said quantification comprises adding a dye that binds to said single-stranded nucleic acid product.11. The method of ...

Подробнее

Номер записи: 25

02-01-2020 дата публикации

NUCLEIC ACID DETECTION METHOD

Номер: US20200002756A1

Автор: Egan Christopher, LAMBLE Henry John, LLOYD David

Принадлежит: Sense Biodetection Limited

The present invention relates to methods for the detection of nucleic acids of defined sequence and kits for use in said methods. The methods employ nicking agent(s), polymerase and oligonucleotide probes to produce probe fragments in the presence of a target nucleic acid. 2. A method according to wherein the second oligonucleotide probe (P2) is attached to a solid material or to a moiety that permits its attachment to a solid material.3. (canceled)4. A method according to wherein the probe (P1) derived strand of the double-stranded nucleic acid amplifier comprises one or more modifications that render it resistant to cleavage by the first and/or second nicking agent(s) claim 1 , such as a phosphorothioate linkage.5. (canceled)6. A method according to wherein the double-stranded nucleic acid amplifier contains two or more nicking agent cleavage sites.7. A method according to wherein the sample is also contacted with a target nucleic acid primer which optionally contains the cleavage site for the first nicking agent.8. A method according to wherein the first oligonucleotide probe (P1) comprises a complementarity region at its 3′ end that is capable of sequence specific hybridisation to the target nucleic acid; whereby claim 1 , following hybridisation of probe (P1) to the target nucleic acid claim 1 , extension of probe (P1) by the polymerase forms a double-stranded nucleic acid that contains the cleavage site for the first nicking agent within the target nucleic acid strand; and whereby the first nicking agent specifically recognises said double-stranded nucleic acid and cleaves said target nucleic acid strand to produce a primer that remains hybridised to the probe (P1) derived strand; and the polymerase extends the primer to produce the double-stranded nucleic acid amplifier which is cleaved in step a) (A).9. A method according to wherein probe (P1) contains the recognition sequence for a probe (P1) nicking agent and whereby the double-stranded nucleic acid ...

Подробнее

Номер записи: 26

02-01-2020 дата публикации

METHOD AND SYSTEM FOR SEQUENCING NUCLEIC ACIDS

Номер: US20200002762A1

Автор: BERNARD Mark A., DAMBACHER Corey M., ROKICKI Joseph, Tu Eugene, Vijayan Kandaswamy, WILSON Kerry

Принадлежит:

Provided are compositions, methods and systems for determining the sequence of a template nucleic acid using a polymerase-based, sequencing-by-binding procedure. An examination step involves monitoring the interaction between a polymerase and template nucleic acid in the presence of one or more nucleotides. Identity of the next correct nucleotide in the sequence is determined without incorporation of any nucleotide into the structure of the primer by formation of a phosphodiester bond. An optional incorporation step can be used after the examination step to extend the primer by one or more nucleotides, thereby incrementing the template nucleotides that can be examined in a subsequent examination step. The sequencing-by-binding procedure does not require the use of labeled nucleotides or polymerases, but optionally can employ these reagents. 1. A method of determining the identity of the next correct nucleotide for a primed template nucleic acid , the method comprising:(a) contacting the primed template nucleic acid with a plurality of nucleotide mixtures in serial fashion while precluding incorporation of any nucleotide into the primer, wherein each of the nucleotide mixtures comprises a combination of nucleotides that is complementary to two, three or four different types of bases in the template nucleic acid, thereby forming stabilized ternary complexes that comprise the primed template nucleic acid molecule, a polymerase and a next correct nucleotide from each of the nucleotide mixtures; and(b) detecting the stabilized ternary complexes while precluding incorporation of any nucleotide into the primer, thereby determining the identity of the next correct nucleotide for the primed template nucleic acid.2. The method of claim 1 , wherein the plurality of nucleotide mixtures comprises a plurality of different nucleotide mixtures.3. The method of claim 2 , wherein the plurality of different nucleotide mixtures comprises four different nucleotide mixtures.4. The method ...

Подробнее

Номер записи: 27

02-01-2020 дата публикации

METHODS AND SYSTEMS FOR PROCESSING POLYNUCLEOTIDES

Номер: US20200002763A1

Автор: "OKeeffe Christopher Joachim", BELGRADER Phillip, Bent Zachary, Bharadwaj Rajiv, Boutet Stephane Claude, Gopalan Vijay Kumar Sreenivasa, Harada Josephine, Hindson Christopher, Lenji Mohammad Rahimi, Lucero Michael Ybarra, McDermott Geoffrey, Meer Elliott, Mikkelsen Tarjei Sigurd, Pfeiffer Katherine, Price Andrew D., Ryvkin Paul, Saxonov Serge, Srinivas Niranjan, Stuelpnagel John R., Taylor Sarah, Terry Jessica Michele, Wheeler Tobias Daniel, Wu Indira, Ziraldo Solongo Batjargal

Принадлежит:

The present disclosure provides compositions, methods, systems, and devices for polynucleotide processing and analyte characterization. Such polynucleotide processing may be useful for a variety of applications, including analyte characterization by polynucleotide sequencing. The compositions, methods, systems, and devices disclosed herein generally describe barcoded oligonucleotides, which can be bound to a bead, such as a gel bead, useful for characterizing one or more analytes including, for example, protein (e.g., cell surface or intracellular proteins), genomic DNA, and RNA (e.g., mRNA or CRISPR guide RNAs). Also described herein, are barcoded labelling agents and oligonucleotide molecules useful for “tagging” analytes for characterization. 130-. (canceled)31. A method for analyzing a tissue sample comprising:(a) delivering a plurality of spatial oligonucleotides to a location in a tissue sample comprising cells, wherein a spatial oligonucleotide of the plurality of spatial oligonucleotides comprises (i) a spatial barcode sequence and (ii) a cell labeling agent configured to deliver the spatial oligonucleotide to a cell at the location in the tissue sample;(b) dissociating the tissue sample into a plurality of labeled cells, wherein a labeled cell of the plurality of labeled cells comprises: (i) the spatial oligonucleotide and (ii) a plurality of analytes;(c) partitioning the labeled cell and a plurality of cell barcode nucleic acid molecules into a partition, wherein each cell barcode nucleic acid molecule of the plurality of cell barcode nucleic acid molecules (i) comprises a common cell barcode sequence and (ii) is configured to couple to the spatial oligonucleotide and analytes of the plurality of analytes;(d) in the partition, generating (i) a spatial barcoded nucleic acid molecule comprising the spatial barcode sequence or complement thereof, (ii) a first barcoded nucleic acid molecule corresponding to a first analyte of the plurality of analytes, and ( ...

Подробнее

Номер записи: 28

02-01-2020 дата публикации

METHODS AND SYSTEMS FOR PROCESSING POLYNUCLEOTIDES

Номер: US20200002764A1

Автор: "OKeeffe Christopher Joachim", BELGRADER Phillip, Bent Zachary, Bharadwaj Rajiv, Boutet Stephane Claude, Gopalan Vijay Kumar Sreenivasa, Harada Josephine, Hindson Christopher, Lenji Mohammad Rahimi, Lucero Michael Ybarra, McDermott Geoffrey, Meer Elliott, Mikkelsen Tarjei Sigurd, Pfeiffer Katherine, Price Andrew D., Ryvkin Paul, Saxonov Serge, Srinivas Niranjan, Stuelpnagel John R., Taylor Sarah, Terry Jessica Michele, Wheeler Tobias Daniel, Wu Indira, Ziraldo Solongo Batjargal

Принадлежит:

The present disclosure provides compositions, methods, systems, and devices for polynucleotide processing and analyte characterization. Such polynucleotide processing may be useful for a variety of applications, including analyte characterization by polynucleotide sequencing. The compositions, methods, systems, and devices disclosed herein generally describe barcoded oligonucleotides, which can be bound to a bead, such as a gel bead, useful for characterizing one or more analytes including, for example, protein (e.g., cell surface or intracellular proteins), genomic DNA, and RNA (e.g., mRNA or CRISPR guide RNAs). Also described herein, are barcoded labelling agents and oligonucleotide molecules useful for “tagging” analytes for characterization. 130-. (canceled)31. A method for analyzing a tissue sample comprising:(a) contacting a plurality of cells with a plurality of labelling molecules to generate a plurality of labelled cells, wherein said plurality of labelling molecules comprise a plurality of cell barcode sequences, and wherein a labelled cell of said plurality of labeled cells comprises (i) a cell barcode sequence that is different from cell barcode sequences of other labelled cells of said plurality of labelled cells and (ii) a plurality of analytes;(b) generating a plurality of partitions comprising said plurality of labelled cells and a plurality of partition nucleic acid barcode molecules, wherein said plurality of partition nucleic acid barcode molecules comprise a plurality of partition barcode sequences, wherein a partition of said plurality of partitions comprises a partition barcode sequence that is different from partition barcode sequences of other partitions of said plurality of partitions, wherein a partition nucleic acid barcode molecule of said plurality of partition nucleic acid barcode molecules is configured to couple to an analyte from said plurality of analytes, wherein said analyte comprises messenger ribonucleic acid (mRNA), and wherein ...

Подробнее

Номер записи: 29

03-01-2019 дата публикации

NUCLEOTIDE-SPECIFIC RECOGNITION SEQUENCES FOR DESIGNER TAL EFFECTORS

Номер: US20190002824A1

Автор: Cong Le, Zhang Feng

Принадлежит:

The invention relates to methods of altering expression of a genomic locus of interest or specifically targeting a genomic locus of interest in an animal cell, which may involve contacting the genomic locus with a non-naturally occurring or engineered composition that includes a deoxyribonucleic acid (DNA) binding polypeptide having a N-terminal capping region, a DNA binding domain comprising at least five or more Transcription activator-like effector (TALE) monomers and at least one or more half-monomers specifically ordered to target the genomic locus of interest, and a C-terminal capping region, wherein the polypeptide includes at least one or more effector domains, and wherein the polypeptide is encoded by and translated from a codon optimized nucleic acid molecule so that the polypeptide preferentially binds to the DNA of the genomic locus. 110-. (canceled)11. A non-naturally occurring or engineered composition comprising a deoxyribonucleic acid (DNA) binding polypeptide comprising:(a) a N-terminal capping region,(b) a DNA binding domain comprising at least five or more Transcription activator-like effector (TALE) monomers and at least one or more half-monomers specifically ordered to target a genomic locus of interest, and(c) a C-terminal capping region,wherein (a), (b) and (c) are arranged in a predetermined N-terminus to C-terminus orientation,wherein the polypeptide includes at least one or more mSin interaction domain (SID) repressor domain having the sequence MNIQMLLEAADYLERREREAEHGYASMLP (SEQ ID NO: 49),{'sub': 1-11', '12', '13', '14-33 or 34 or 35', 'z, 'wherein the DNA binding domain comprises (X-XX-X),'}{'sub': '1-11', 'wherein Xis a chain of 11 contiguous amino acids,'}{'sub': 12', '13, 'wherein XXis a repeat variable diresidue (RVD),'}{'sub': '14-33 or 34 or 35', 'wherein Xis a chain of 21, 22 or 23 contiguous amino acids,'}wherein z is at least 5 to 40, and{'sub': '13', 'wherein at least one RVD is selected from the group consisting of HH, KH, NH, ...

Подробнее

Номер записи: 30

03-01-2019 дата публикации

RNase H-Based Assays Utilizing Modified RNA Monomers

Номер: US20190002865A1

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

Принадлежит:

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

Подробнее

Номер записи: 31

03-01-2019 дата публикации

METHOD AND COMPOSITION

Номер: US20190002870A1

Автор: Rogers Jan, TORO RUEDA Carlos

Принадлежит:

A method of extracting DNA and/or RNA from a cell or capsid, the method comprising steps of: (a) contacting a composition comprising the cell or capsid with a composition comprising (ii) a quaternary ammonium compound or a precursor thereof; and (b) contacting the composition obtained in step (a) with a composition comprising a proteinaceous washing agent. 1. A method of extracting DNA and/or RNA from a cell or capsid , the method comprising steps of: '(i) a quaternary ammonium compound or a precursor thereof; and', '(a) contacting a composition comprising the cell or capsid with a composition comprising'}(b) contacting the composition obtained in step (a) with a composition comprising a proteinaceous washing agent.2. A method according to wherein the composition comprising the cell or capsid is a blood sample.3. A method according to wherein the composition comprising the cell or capsid is a whole blood sample.6. A method according to wherein the composition contacted with the cell or capsid in step (a) further comprises (ii) a non-ionic surfactant.7. A method according to wherein the non-ionic surfactant is an alkyl polyglucoside (APG).9. A method according to wherein the composition contacted with the composition comprising the cell or capsid in step (a) has a pH of from 6 to 8.10. A method according to wherein there are no purification steps between step (a) and step (b).11. A method according to wherein the proteinaceous washing agent is selected from bovine serum albumin and casein.12. A kit comprising: (i) a quaternary ammonium; and optionally', '(ii) a non-ionic surfactant; and, '(a) a first composition comprising'}(b) a second composition comprising an proteinaceous washing agent.13. A method of identifying a component of genetic material claim 1 , the method comprising the steps of: (i) a quaternary ammonium compound; and optionally', '(ii) a non-ionic surfactant;, '(a) contacting a composition comprising a cell or capsid with a composition comprising(b) ...

Подробнее

Номер записи: 32

07-01-2021 дата публикации

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

Номер: US20210002718A1

Автор: HARTMAN Wyatt, Zwirko Zachary

Принадлежит: Trace Genomics, Inc.

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

Подробнее

Номер записи: 33

03-01-2019 дата публикации

A novel method for the preparation of bar-coded primer sets

Номер: US20190002953A1

Автор: Can SÖNMEZER, Christian KRENDL, Florian OPPERER, Micha Drukker

Принадлежит: Helmholtz Zentrum Muenchen Deutsches Forschungszentrum fuer Gesundheit und Umwelt GmbH, Helmholtz Zentrum Muenchendeutsches Forschungszentrum fur Gesundheit und Umwelt

The present invention relates to a method of producing a set of primers suitable for the reverse transcription and/or amplification of a plurality (N) of nucleic acid molecules of interest, wherein for each nucleic acid molecule of interest at least one primer is produced and wherein the primers carry a bar-code, the method comprising the steps of: (a)(i) combining (1) a first oligonucleotide, wherein said first oligonucleotide comprises a first bar-code nucleic acid sequence linked at its 3′ end to a first adapter nucleic acid sequence with (2) a plurality (N) of second oligonucleotides, wherein each second oligonucleotide comprises the reverse complementary sequence of a forward primer specific for a nucleic acid molecule of interest, wherein said reverse complementary sequence of the forward primer is linked at its 3′ end to the reverse complementary sequence of the first adapter nucleic acid sequence; and/or (a)(ii) combining (1) a third oligonucleotide, wherein said third oligonucleotide comprises a second bar-code nucleic acid sequence linked at its 3′ end to a second adapter nucleic acid sequence with (2) a plurality (N) of fourth oligonucleotides, wherein each fourth oligonucleotide comprises the reverse complementary sequence of a reverse primer specific for said nucleic acid molecule of interest, wherein said reverse complementary sequence of the reverse primer is linked at its 3′ end to the reverse complementary sequence of the second adapter nucleic acid sequence; wherein steps (a)(i) and (a)(ii) are carried out under conditions that enable the annealing of the first and second adapter nucleic acid sequences to the respective reverse complementary sequences thereof; (b) extending the oligonucleotides of (a)(i) and (a)(ii) by polymerase-mediated oligonucleotide synthesis; and (c) optionally, removing the second and fourth oligonucleotides. The present invention further relates to methods of producing a plurality (M) of nucleic acid amplification products ...

Подробнее

Номер записи: 34

03-01-2019 дата публикации

NUCLEIC ACID AMPLIFICATION

Номер: US20190002957A1

Автор: Erlander Mark G., SALUNGA Ranelle C.

Принадлежит:

The present invention provides methods for the amplification of nucleic acid molecules. Methods for amplifying target polynucleotides, including mRNA, using oligonucleotides, DNA and RNA polymerases are provided. The invention further provides compositions and kits for practicing the methods, as well as methods which use the amplification products. 120.-. (canceled)21. A method of amplifying RNA sequences , comprising:annealing a single-stranded target polynucleotide with a first oligonucleotide that includes a primer region comprising an oligo dT sequence of about 18-21 T residues and a promoter region comprising a T7 promoter sequence to form a first complex;synthesizing a first strand cDNA by reverse transcription of the first complex to form an RNA/cDNA heteroduplex;separating the first strand cDNA from the RNA/cDNA heteroduplex;annealing the first strand cDNA with a plurality of random primers that hybridize at a plurality of positions on the first strand cDNA to form a second complex;forming a double-stranded cDNA template from the second complex using a combination of DNA dependent polymerase enzymes including exonuclease deficient Klenow and Taq polymerase; andtranscribing the double-stranded cDNA template with an RNA polymerase capable of initiating transcription via said promoter region to produce amplified RNA (aRNA) containing a sequence complementary to the single-stranded target polynucleotide.22. The method of claim 21 , wherein the first oligonucleotide further comprises an anchor sequence between the primer region and the promoter region.23. The method of claim 21 , further comprising degrading the first oligonucleotide remaining after synthesizing a first strand cDNA with an exonuclease.24. The method of claim 23 , wherein the exonuclease is exonuclease I.25. The method of claim 21 , wherein separating the first strand cDNA from the RNA/cDNA heteroduplex comprises denaturing the RNA/cDNA heteroduplex.26. The method of claim 25 , wherein denaturing ...

Подробнее

Номер записи: 35

03-01-2019 дата публикации

POLYPEPTIDE TAGGED NUCLEOTIDES AND USE THEREOF IN NUCLEIC ACID SEQUENCING BY NANOPORE DETECTION

Номер: US20190002968A1

Автор: Bergmann Frank, Gremyachinskiy Dmitriy, HILLRINGHAUS Lars, KUCHELMEISTER Hannes, SEIDEL Christoph, TRANS Andrew

Принадлежит:

The present disclosure relates to compositions and methods based on polypeptide-tagged nucleotide, and the use of such polypeptide-tagged nucleotides in nanopore devices and methods. 1. A compound of structural formula (I){'br': None, 'N-P-L-T\u2003\u2003 (I)'} N is a nucleoside;', 'P is an oligophosphate covalently attached to a 5′-O group of the nucleoside,', 'wherein the oligophosphate consists of 3 to 12 phosphate groups;', 'L is a linker covalently attached to a terminal phosphate group of the oligophosphate; and', 'T is a polypeptide tag covalently attached to the linker, wherein the polypeptide has an overall charge and comprises at least one helical structure., 'wherein,'}3. The compound of claim 1 , wherein the length of the polypeptide tag is at least 16 amino acid residues claim 1 , optionally wherein the length of the polypeptide tag is at least 20 amino acid residues claim 1 , at least 25 amino acid residues claim 1 , at least 30 amino acid residues claim 1 , at least 40 amino acid residues claim 1 , at least 50 amino acid residues claim 1 , at least 60 amino acid residues claim 1 , at least 70 amino acid residues claim 1 , at least 80 amino acid residues claim 1 , or at least 90 amino acid residues.4. The compound of claim 1 , wherein the helical structure comprises is at least 8 amino acid residues claim 1 , optionally wherein the polypeptide helical structure comprises at least 16 amino acid residues claim 1 , at least 20 amino acid residues claim 1 , at least 25 amino acid residues claim 1 , at least 30 amino acid residues claim 1 , at least 40 amino acid residues claim 1 , at least 50 amino acid residues claim 1 , or at least 60 amino acid residues.5. The compound of claim 1 , wherein the helical structure is an α-helix claim 1 , optionally wherein the length of the α-helix is at least 10 amino acid residues claim 1 , at least 16 amino acid residues claim 1 , at least 20 amino acid residues claim 1 , at least 25 amino acid residues claim 1 , at ...

Подробнее

Номер записи: 36

01-01-2015 дата публикации

METHOD FOR SUPPRESSING THE EFFECTS OF ASCORBIC ACID

Номер: US20150004635A1

Автор: Soya Haruyo

Принадлежит:

Provided is a method for suppressing an effect of ascorbic acid on a measurement of glycated hemoglobin in a glycated hemoglobin-containing sample. The method of the present invention is a method for suppressing an effect of ascorbic acid on the measurement of glycated hemoglobin in a glycated hemoglobin-containing sample, comprising reacting the glycated hemoglobin-containing sample with a proteolytic enzyme, reacting a generated glycated peptide with a glycated peptide oxidase, and then measuring a generated hydrogen peroxide, wherein the reaction of the glycated peptide with the glycated peptide oxidase is performed in the presence of a halogen oxide and a substance selected from the group consisting of a substance represented by formula (I), wherein Rrepresents substituted or unsubstituted alkyl and a substance represented by the general formula (II), wherein Rrepresents substituted or unsubstituted alkyl; and represents a single bond or a double bond. 2. The method according to claim 1 , wherein the halogen oxide is selected from the group consisting of iodic acid or a salt thereof and bromic acid or a salt thereof.3. The method according to claim 1 , wherein the glycated hemoglobin is hemoglobin A1c.5. The reagent according to claim 4 , wherein the halogen oxide is selected from the group consisting of iodic acid or a salt thereof and bromic acid or a salt thereof.6. The reagent according to claim 4 , wherein the glycated hemoglobin is hemoglobin A1c.7. The reagent according to claim 4 , wherein the reagent suppresses an effect of ascorbic acid on the measurement of glycated hemoglobin in the sample.8. The method according to claim 2 , wherein the glycated hemoglobin is hemoglobin A1c.9. The reagent according to claim 5 , wherein the glycated hemoglobin is hemoglobin A1c.10. The reagent according to claim 5 , wherein the reagent suppresses an effect of ascorbic acid on the measurement of glycated hemoglobin in the sample.11. The reagent according to claim 6 , ...

Подробнее

Номер записи: 37

04-01-2018 дата публикации

Methods and kits for measuring and quantifying dna double-stranded breaks using gamma-h2ax and h2ax

Номер: US20180003720A1

Автор: Christophe E. Redon, Jiuping Ji, William M. Bonner, Yiping Zhang

Принадлежит: US Department of Health and Human Services

Disclosed are methods of measuring the amount of exposure of a host to a DNA double-stranded break (DSB)-causing agent by determining the ratio of the quantity of γ-H2AX to the quantity of total H2AX in a biological sample from the host as compared to the ratio of the quantity of γ-H2AX to the quantity of total H2AX in a positive control biological sample that has been exposed to a known amount of a DSB-causing agent. Related kits and methods of quantifying DSBs in a test biological sample are also disclosed.

Подробнее

Номер записи: 38

03-01-2019 дата публикации

METHOD AND APPARATUS FOR ANALYTE DETECTION USING AN ELECTROCHEMICAL BIOSENSOR

Номер: US20190004005A1

Автор: Feldman Benjamin, Oja Stephen M.

Принадлежит:

A method for sensing an analyte utilizing a sensor having a working electrode, the method includes providing the working electrode with an analyte-specific enzyme and a redox mediator, providing the working electrode to the analyte, accumulating charge derived from the analyte reacting with the analyte-specific enzyme and the redox mediator for a set period of time, connecting the working electrode to circuit after the set period of time, and measuring the signal from the accumulated charge. 1. A method for sensing an analyte utilizing a sensor , the sensor including a working electrode , the method comprising:providing the working electrode with an analyte-specific enzyme and a redox mediator;providing the working electrode to the analyte;accumulating charge derived from the analyte reacting with the analyte-specific enzyme and the redox mediator for a set period of time;connecting the working electrode to a circuit after the set period of time; andmeasuring a signal from the accumulated charge.2. The method of claim 1 , wherein prior to providing the working electrode to an analyte claim 1 , the method further comprises connecting the working electrode to the circuit claim 1 , and prior to providing the working electrode to the analyte claim 1 , the method further comprises disconnecting the working electrode from the circuit.3. The method of claim 1 , wherein the working electrode is connected to the circuit prior to providing the working electrode to the analyte claim 1 , the method further comprises disconnecting the working electrode from the circuit prior to providing the working electrode to the analyte.4. The method of claim 1 , wherein the sensor is an enzymatic electrochemical biosensor.5. The method of claim 1 , wherein the redox mediator is an immobilized redox polymer.6. The method of claim 1 , wherein the analyte is selected from the group consisting of cortisol claim 1 , glucose claim 1 , lactate claim 1 , 3-hydroxy butyrate claim 1 , alcohol claim 1 ...

Подробнее

Номер записи: 39

03-01-2019 дата публикации

CONTROL OF pH IN AQUEOUS UREA-CONTAINING SOLUTIONS UTILIZING AMINO ACID-CONTAINING COMPOSITIONS

Номер: US20190004072A1

Автор: Horan Kevin, Uretsky Laura

Принадлежит: SIEMENS HEALTHCARE DIAGNOSTICS INC.

Aqueous calibration or quality control reagents that include urea are disclosed; the reagents may further include at least one amino acid-containing composition to provide pH stability thereto. Methods of production and use thereof are also disclosed. 1. A composition comprising an aqueous diagnostic quality control or calibration reagent disposed in a closed system , the composition comprising:urea present in a concentration in a range of from about 0.1 mM to about 150 mM;at least one buffer; andat least one amino acid-containing composition having at least one available primary or secondary amine, the at least one amino acid-containing composition consisting of ornithine, wherein a ratio of urea to the at least one amino acid-containing composition is in a range of from about 0.1:1 to about 100:1, and wherein the pH of the aqueous quality control or calibration reagent varies by +/−1.0 or less of the original pH following storage of the composition, and further wherein formation of ammonia in the aqueous diagnostic quality control or calibration reagent is substantially reduced by at least 5% following storage of the composition when compared to a composition prepared in the absence of the at least one amino acid-containing composition and following storage thereof; andwherein the aqueous diagnostic quality control or calibration reagent is for utilization with a diagnostic sensor.2. The composition of claim 1 , wherein the formation of ammonia in the aqueous diagnostic quality control or calibration reagent is substantially reduced by at least 10% when compared to a composition prepared in the absence of the at least one amino acid-containing composition following storage thereof.3. The composition of claim 1 , wherein the formation of ammonia in the aqueous diagnostic quality control or calibration reagent is substantially reduced by at least 15% when compared to a composition prepared in the absence of the at least one amino acid-containing composition ...

Подробнее

Номер записи: 40

03-01-2019 дата публикации

DIGITAL MEASUREMENTS FROM TARGETED SEQUENCING

Номер: US20190005193A1

Автор: Amorese Douglas A., HUELGA Stephanie C., SCHROEDER Benjamin G., Scolnick Jonathan

Принадлежит:

Disclosed herein are methods, compositions and kits for quantitating one or more specific nucleic acids within a plurality of nucleic acids. In some embodiments, a sequencing library is constructed from enriched probe extension products specific for the specific nucleic acids and sequenced. In some embodiments, the resulting reads are used for removing duplicate reads. In some embodiments, counting of verified probes is used to quantitate or determine the number of specific nucleic acid molecules in the starting nucleic acid sample. 1. A method for quantitating a plurality of specific nucleic acid molecules in a composition comprising: a. generating a plurality of probe extension products , wherein each probe extension product comprises a probe sequence that is complementary to a probe target region within a specific nucleic acid molecule; b. sequencing the plurality of probe extension products to generate a sequence for each of the plurality of probe extension products; c. aligning the sequence of each of the plurality of probe extension products to a reference sequence database , wherein the reference sequence database comprises probe sequences; and d. determining the number of alignments for the sequence of each probe extension product with a sequence in the reference sequence database , wherein the number of alignments indicates the quantity of each of the specific nucleic acid molecule that the probe of the probe extension product is complementary to.2. A method for quantitating a plurality of specific nucleic acid molecules comprising: a. generating a plurality of probe extension products , wherein each probe extension product comprises (i) a first adapter , and (ii) a probe sequence complementary to a probe target region within a specific nucleic acid molecule; b. sequencing the plurality of probe extension products to generate sequence data comprising a sequence for each of the plurality of probe extension products; c. identifying the presence of the probe ...

Подробнее

Номер записи: 41

27-01-2022 дата публикации

Methods for non-invasive prenatal ploidy calling

Номер: US20220025456A1

Автор: Allison Ryan, Bernhard Zimmermann, George Gemelos, Johan Baner, Matthew Hill, Matthew Rabinowitz, Milena Banjevic, Styrmir Sigurjonsson, Zachary Demko

Принадлежит: Natera Inc

The present disclosure provides methods for determining the ploidy status of a chromosome in a gestating fetus from genotypic data measured from a mixed sample of DNA comprising DNA from both the mother of the fetus and from the fetus, and optionally from genotypic data from the mother and father. The ploidy state is determined by using a joint distribution model to create a plurality of expected allele distributions for different possible fetal ploidy states given the parental genotypic data, and comparing the expected allelic distributions to the pattern of measured allelic distributions measured in the mixed sample, and choosing the ploidy state whose expected allelic distribution pattern most closely matches the observed allelic distribution pattern. The mixed sample of DNA may be preferentially enriched at a plurality of polymorphic loci in a way that minimizes the allelic bias, for example using massively multiplexed targeted PCR.

Подробнее

Номер записи: 42

08-01-2015 дата публикации

Method for the synthesis of a bifunctional complex

Номер: US20150011434A1

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

Принадлежит: Nuevolution AS

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

Подробнее

Номер записи: 43

14-01-2021 дата публикации

Labelled nucleotides

Номер: US20210009623A1

Автор: John Milton, Silke Ruediger, Xiaohai Liu, Xiaolin Wu

Принадлежит: Illumina Cambridge Ltd

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

Подробнее

Номер записи: 44

11-01-2018 дата публикации

ORGANISM IDENTIFICATION PANEL

Номер: US20180010167A1

Автор: Blaschke-Bonkowsky Anne Jeannette, Poritz Mark Aaron

Принадлежит:

Methods and containers are provided for identifying a species, illustratively a bacterial species. Illustrative methods comprise amplifying various genes in the nucleic acid from the bacterial species in a single reaction mixture using pairs of outer first-stage primers designed to hybridize to generally conserved regions of the respective genes to generate a plurality of first-stage amplicons, dividing the reaction mixture into a plurality of second-stage reactions, each using a unique pair of second-stage primers, each pair of second-stage primers specific for a target bacterial species or subset of bacterial species, detecting which of the second-stage reactions amplified, and identifying the bacterial species based on second-stage amplification. Methods for determining antibiotic resistance are also provided, such methods also using first-stage primers for amplifying genes known to affect antibiotic resistance a plurality of the second-stage reactions wherein each pair of second-stage primers specific for a specific gene for conferring antibiotic resistance. 131-. (canceled)32. A method for identifying a bacterial species comprising the steps ofobtaining a sample containing the bacterial species,amplifying, in a single reaction mixture containing nucleic acid from the sample, a plurality of bacterial genes using pairs of outer first-stage primers designed to hybridize to generally conserved regions of the bacterial genes to generate a plurality of first-stage amplicons,dividing the reaction mixture into a plurality of second-stage reactions,subjecting each of a plurality of the second-stage reactions to amplification conditions, wherein each of the plurality of second-stage reactions uses a pair of second-stage primers, each pair of second-stage primers specific for a different target bacterial species or group of bacterial species,generating an amplification curve for each of the second-stage reactions that amplified,determining a crossing point for each ...

Подробнее

Номер записи: 45

11-01-2018 дата публикации

METHODS AND SYSTEMS FOR SEQUENCING LONG NUCLEIC ACIDS

Номер: US20180010180A1

Автор: Matsuzaki Hajime, Mei Rui, Zhou Wei

Принадлежит:

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

Подробнее

Номер записи: 46

10-01-2019 дата публикации

FLUORESCENT POLYMERASE ENZYME SUBSTRATES HAVING PROTEIN SHIELDS

Номер: US20190010183A1

Автор: Bjornson Keith, BROGLEY Louis, Hanes Jeremiah, Kamtekar Satwik, Miller Erik, SEBO Lubomir

Принадлежит:

Compositions, methods, and systems are provided for fluorescent polymerase enzyme substrates comprising protein shields for improving enzyme photostability in single molecule real time sequencing. Fluorescent polymerase enzyme substrates of the invention have a protein shield between the fluorescent dye moieties and nucleotide moieties of the polymerase enzyme substrate. The polymerase enzyme substrates have a nucleotide component and a dye component, each attached to a protein. The attachments can be covalent. The protein can, for example, prevent the direct interaction of the fluorescent dye moiety with the enzyme when carrying out nucleotide synthesis, preventing photodamage to the enzyme. The polymerase enzyme substrates of the invention can have multiple dyes and multiple nucleotide moieties. 1. A polymerase enzyme substrate comprising:a protein comprising at least 60 amino acids;a nucleotide component comprising at least one nucleoside polyphosphate attached through its phosphate portion to a first position on the protein;a dye component comprising at least one fluorescent dye moiety attached to a second position on the protein.2. The polymerase enzyme substrate of wherein the substrate is connected through covalent attachments.3. The polymerase enzyme substrate of wherein the protein comprises 60 to 1 claim 1 ,000 amino acids.4. The polymerase enzyme substrate of wherein the protein comprises 80 to 600 amino acids.5. The polymerase enzyme substrate of wherein the nucleotide component and dye component are covalently attached to the protein.6. The polymerase enzyme substrate of wherein the nucleotide component comprises two or more nucleoside phosphates.7. The polymerase enzyme substrate of wherein the substrate has 2 claim 1 , 3 claim 1 , 4 claim 1 , 5. 6 claim 1 , 7 claim 1 , or 8 nucleotide phosphates.8. The polymerase enzyme substrate of wherein the dye component comprises two or more fluorescent dye moieties.9. The polymerase enzyme substrate of wherein ...

Подробнее

Номер записи: 47

14-01-2021 дата публикации

Method for Introducing Mutations

Номер: US20210010008A1

Автор: BURKE Catherine M., DARLING Aaron E., IMELFORT Michael, MONAHAN Leigh G., To Joyce

Принадлежит:

The present invention relates to a method for introducing mutations into at least one target nucleic acid molecule comprising (a) providing at least one sample comprising at least one target nucleic acid molecule; and (b) amplifying the at least one target nucleic acid molecule using a low bias DNA polymerase. The present further relates to a use of a low bias DNA polymerase in a method for introducing mutations into one or more nucleic acid molecule(s), a group of sample tags, a method for designing the group of sample tags, a computer readable medium, and a method for preferentially amplifying target nucleic acid molecules. 1. A method for introducing mutations into at least one target nucleic acid molecule comprising:a. providing at least one sample comprising at least one target nucleic acid molecule; andb. amplifying the at least one target nucleic acid molecule using a low bias DNA polymerase.2. Use of a low bias DNA polymerase in a method for introducing mutations into at least one target nucleic acid molecule.3. The use of claim 2 , wherein the method for introducing mutations into at least one target nucleic acid molecule comprises:a. providing at least one sample comprising at least one target nucleic acid molecule; andb. amplifying the at least one target nucleic acid molecule using a low bias DNA polymerase.4. The method or use of any one of the preceding claims claim 2 , wherein the mutations are substitution mutations.5. The method or use of any one of the preceding claims claim 2 , wherein the low bias DNA polymerase mutates adenine claim 2 , thymine claim 2 , guanine claim 2 , and cytosine nucleotides in the at least one target nucleic acid molecule at a rate ratio of 0.5-1.5:0.5-1.5:0.5-1.5:0.5-1.5 claim 2 , 0.6-1.4:0.6-1.4:0.6-1.4:0.6-1.4 claim 2 , 0.7-1.3:0.7-1.3:0.7-1.3:0.7-1.3 claim 2 , 0.8-1.2:0.8-1.2:0.8-1.2:0.8-1.2 claim 2 , or around 1:1:1:1 respectively.6. The method or use of any one of the preceding claims claim 2 , wherein the low bias ...

Подробнее

Номер записи: 48

14-01-2021 дата публикации

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

Номер: US20210010061A1

Автор: Mendez Pedro, Ruff David

Принадлежит:

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

Подробнее

Номер записи: 49

14-01-2016 дата публикации

Nanopore sequencing methods

Номер: US20160011169A1

Автор: Jonas Korlach, Stephen Turner

Принадлежит: Pacific Biosciences of California Inc

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

Подробнее

Номер записи: 50

10-01-2019 дата публикации

HIGH THROUGHPUT GENOME SEQUENCING ON DNA ARRAYS

Номер: US20190010542A1

Автор: Callow Matthew J., Drmanac Radoje, Drmanac Snezana

Принадлежит: Complete Genomics Inc.

The present invention is directed to methods and compositions for acquiring nucleotide sequence information of target sequences using adaptors interspersed in target polynucleotides. The sequence information can be new, e.g. sequencing unknown nucleic acids, re-sequencing, or genotyping. The invention preferably includes methods for inserting a plurality of adaptors at spaced locations within a target polynucleotide or a fragment of a polynucleotide. Such adaptors may serve as platforms for interrogating adjacent sequences using various sequencing chemistries, such as those that identify nucleotides by primer extension, probe ligation, and the like. Encompassed in the invention are methods and compositions for the insertion of known adaptor sequences into target sequences, such that there is an interruption of contiguous target sequence with the adaptors. By sequencing both “upstream” and “downstream” of the adaptors, identification of entire target sequences may be accomplished. 1. (canceled)2. A method of preparing an array of fragments of a target nucleic acid to be sequenced , the method comprising:(a) providing a plurality of double-stranded DNA molecules each containing a target sequence from the target nucleic acid;(b) introducing a nick into one of the strands in each of the double-stranded DNA molecules at an initial site, exposing a free 5′ strand and a free 3′ strand at the site;(c) treating the nicked DNA molecules with a DNA polymerase so as to extend the free 3′ strand and displace or degrade the free 5′ strand, thereby moving the nick along the nicked strand to a new position downstream from the initial site;(d) disabling the DNA polymerase;(e) cleaving the DNA molecules at the new position, thereby forming a gap in both strands of the DNA molecules;(f) inserting a downstream oligonucleotide adaptor into the gap of each DNA molecule to form a nucleic acid construct; and(g) arraying the constructs on a solid surface for nucleic acid sequencing.3. The ...

Подробнее

Номер записи: 51

10-01-2019 дата публикации

METHODS FOR SIMULTANEOUS AMPLIFICATION OF TARGET LOCI

Номер: US20190010543A1

Автор: BABIARZ Joshua, CONSTANTIN Tudor Pompiliu, EUBANK Lane A., Gemelos George, HILL Matthew Micah, KIRKIZLAR Huseyin Eser, Rabinowitz Matthew, SAKARYA Onur, SIGURJONSSON Styrmir, Zimmermann Bernhard

Принадлежит: Natera, Inc.

The invention provides methods for simultaneously amplifying multiple nucleic acid regions of interest in one reaction volume as well as methods for selecting a library of primers for use in such amplification methods. The invention also provides library of primers with desirable characteristics, such as minimal formation of amplified primer dimers or other non-target amplicons. 1. A method for determining a transplant status , comprising(a) obtaining a nucleic acid sample from a blood, plasma, or serum sample of a transplant recipient, wherein the nucleic acid sample comprises a mixture of cell-free DNA from a transplant and cell-free DNA from the transplant recipient;(b) amplifying at least 100 different target loci from the nucleic acid sample by contacting the nucleic acid sample with a library of at least 100 non-immobilized, non-identical primers that hybridize to the at least 100 different target loci to produce a reaction mixture, and subjecting the reaction mixture to PCR conditions to produce amplification products comprising target amplicons, wherein at least 50% of the amplification products each comprises at least one of the target loci;(c) measuring an absolute or relative amount of transplant cell-free DNA from the amplification products, and determining a transplant status based on the measured amount of transplant cell-free DNA, wherein the transplant status is transplant rejection, tolerance, non-rejection based allograft injury, transplant function, transplant survival, chronic transplant injury, or tittering of pharmacological immunosuppression.2. The method of claim 1 , wherein the length of the target amplicons is less than 100 nucleotides.3. The method of claim 1 , wherein a range of melting temperatures among the different primers in the library is less than 10° C.4. The method of claim 1 , wherein a range of melting temperatures among the different primers in the library is less than 5° C.5. The method of claim 1 , further comprising ...

Подробнее

Номер записи: 52

10-01-2019 дата публикации

MOLECULAR ZIPPER TWEEZERS AND SPRING DEVICES

Номер: US20190010544A1

Автор: Lal Ratneshwar, Landon Preston B., Mo Alexander, Ramachandran Srinivasan

Принадлежит:

Techniques, structures, devices and systems are disclosed for implementing molecular zipper tweezers and springs. In one aspect, a molecular device includes three molecular components including at least a passive side molecular component, a binding side molecular component and a target molecular component adapted to interact together as a zipper that separate two of the molecular components held together by molecular interaction forces. 1. A method of capturing a target molecule , comprising:deploying a double-stranded molecule into a fluid environment, the double-stranded molecule including a binding strand having a sequence of nucleotides that is coupled to a passive strand having a complementary sequence of nucleotides; andattaching a target molecule in the fluid environment to the binding strand, the target molecule including an opening strand having a complement sequence of nucleotides corresponding to the binding strand, wherein the attaching uncouples the passive strand as the nucleotides of the opening strand bond to the corresponding complement nucleotides of the binding strand.2. The method of claim 1 , wherein the fluid environment is within an organism.3. The method of claim 1 , wherein the attaching the target molecule to the binding strand includes the nucleotides of the opening strand forming a bond with the corresponding complement nucleotides of the binding strand at an energy greater than a bond between the passive strand and the binding strand.4. The method of claim 1 , wherein the attaching the target molecule to the binding strand includes detaching the passive strand from the double-stranded molecule.5. The method of claim 1 , wherein the attaching the target molecule to the binding strand uses no external energy.6. The method of claim 1 , wherein the opening strand includes less nucleotides than each of the binding strand and the passive strand.7. The method of claim 6 , wherein the attaching the target molecule to the binding strand does not ...

Подробнее

Номер записи: 53

09-01-2020 дата публикации

METHOD OF IMPROVED SEQUENCING BY STRAND IDENTIFICATION

Номер: US20200010884A1

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

Принадлежит:

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

Подробнее

Номер записи: 54

09-01-2020 дата публикации

METHOD OF NANOPORE SEQUENCING OF CONCATENATED NUCLEIC ACIDS

Номер: US20200010887A1

Автор: Garalde Daniel Ryan, Heron Andrew John, Oxford James White

Принадлежит: Oxford Nanopore Technologies Ltd.

The invention relates to a new method of characterising two or more target polynucleotides using a pore. The method involves sequentially attaching to a first polynucleotide one or more subsequent polynucleotides to form a concatenated polynucleotide. 1. A method of characterising two or more target polynucleotides , comprising:(a) contacting a first target polynucleotide with a transmembrane pore in a membrane such that the first target polynucleotide moves through the pore;(b) sequentially attaching to the first target polynucleotide one or more subsequent target polynucleotides to provide a concatenated polynucleotide within which the target polynucleotides move through the pore in attachment order, wherein a subsequent target polynucleotide is selectively attached to the preceding target polynucleotide in the attachment order when the preceding target polynucleotide moves through the pore; and(c) taking one or more measurements which are indicative of one or more characteristics of the concatenated polynucleotide as it moves with respect to the pore.2. A method according to claim 1 , wherein 2 or more claim 1 , 5 or more claim 1 , 10 or more claim 1 , 20 or more claim 1 , 50 or more claim 1 , 100 or more claim 1 , 500 or more claim 1 , 1 claim 1 ,000 or more claim 1 , 5 claim 1 ,000 or more claim 1 , 10 claim 1 ,000 or more claim 1 , 50 claim 1 ,000 or more claim 1 , 100 claim 1 ,000 or more claim 1 , 500 claim 1 ,000 or more claim 1 , 1 claim 1 ,000 claim 1 ,000 or more or 5 claim 1 ,000 claim 1 ,000 or more subsequent target polynucleotides are attached to the first target polynucleotide.3. A method according to claim 1 , wherein a part of the preceding target polynucleotide is initially protected from attachment to the subsequent target polynucleotide and is revealed for attachment as the preceding target polynucleotide moves through the pore.4. A method according to claim 1 , wherein a part of the subsequent target polynucleotide selectively hybridises to a ...

Подробнее

Номер записи: 55

09-01-2020 дата публикации

APTAMER AS BIOMARKERS

Номер: US20200010897A1

Автор: PENNER Gregory

Принадлежит: NEONEURO

The use of the change in relative frequency of aptamers in a library selected against at least one sample from at least one reference subject of known outcome, prior to and following selection of said library against a sample from a subject, as a means of diagnosing a medical state, disease or condition in said subject. Also, methods for diagnosing a medical state, disease or condition in a subject, involving correlating changes in relative frequency between aptamers to a medical state, disease or condition and diagnosing the subject based on the determined correlations. 1. Use of the change in relative frequency of aptamers in a library selected against at least one sample from at least one reference subject of known outcome , prior to and following selection of said library against a sample from a subject , as a means of diagnosing a medical state , disease or condition in said subject.2. A method for diagnosing a medical state , disease or condition in a subject , comprising the steps of:providing a selected aptamer library, wherein the selected aptamer library comprises a collection of aptamer sequences selected and optionally counter-selected against a bodily tissue or fluid from at least one reference subject,contacting the selected aptamer library with a biological sample from at least one subject with known outcome for the medical state, disease or condition,selecting aptamers from the selected aptamer library that bind to the biological sample, thereby obtaining a subject-specific aptamer library,determining the change in relative frequency between aptamers from the selected aptamer library and the subject-specific aptamer library,correlating changes in relative frequency between aptamers from the selected aptamer library and the subject-specific aptamer library to the medical state, disease or condition of the at least one subject, andapplying the selected aptamer library to subjects for which the medical state, disease or condition is unknown, thereby ...

Подробнее

Номер записи: 56

03-02-2022 дата публикации

AMPLIFICATION WITH PRIMERS OF LIMITED NUCLEOTIDE COMPOSITION

Номер: US20220033891A1

Автор: Chen Xin, WANG Youxiang, Yang Zhijie

Принадлежит:

The invention provides methods of amplification from a single primer or a pair of forward and reverse primers of limited nucleotide composition. Limited nucleotide composition means that the primers are underrepresented in at least one nucleotide type. Such primers have much reduced capacity to prime from each other or to extend initiated by mispriming from other than at their intended primer binding sites in a target nucleic acid. 1. A method of amplifying a segment of a target nucleic acid comprising:contacting a sample comprising a target nucleic acid with forward and reverse primers; andconducting an amplification reaction wherein an amplified segment of the target nucleic acid is formed by extension of the forward and reverse primers with the target nucleic acid serving as a template; whereinthe primers are underrepresented in one or more of the four standard nucleotide types, the underrepresented nucleotide type(s) being the same in the primers, and the amplified segment is the predominant amplification product formed from by extension of the forward and/or reverse primers.2. The method of wherein the target nucleic acid has a strand comprising a complement of a forward primer binding site and a reverse primer binding site.3. The method of wherein the target nucleic acid has a strand comprising a forward primer binding site and a reverse primer binding site.4. The method of claim 1 , wherein the amplified segment constitutes at least 99% of all amplification products formed by extension of the forward and reverse primers.5. The method of claim 1 , wherein the forward and reverse primers have greater complementarity to the forward and reverse primer bindings sites than to any other pair of primer binding sites supporting amplification in the sample.6. The method of claim 1 , wherein the forward and reverse primers have one and only one of the four standard nucleotide types underrepresented.7. The method of claim 1 , wherein the forward primer binding site and ...

Подробнее

Номер записи: 57

03-02-2022 дата публикации

Orthogonal deblocking of nucleotides

Номер: US20220033898A1

Автор: Elaine H. Trepagnier, Tarun Khurana

Принадлежит: Illumina Inc

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

Подробнее

Номер записи: 58

19-01-2017 дата публикации

Nucleic Acid Probe and Method of Detecting Genomic Fragments

Номер: US20170016065A1

Автор: Carl Oscar Fredrik Dahl, Olof John Ericsson

Принадлежит: Vanadis Diagnostics AB

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

Подробнее

Номер записи: 59

18-01-2018 дата публикации

Methods and systems for processing polynucleotides

Номер: US20180016634A1

Автор: Benjamin Hindson, Christopher Hindson, Grace Zheng, Kevin Ness, Michael Schnall-Levin, Mirna Jarosz, Paul Hardenbol, Phillip Belgrader, Rajiv Bharadwaj, Serge Saxonov

Принадлежит: 10X Genomics Inc

The present disclosure provides compositions, methods, systems, and devices for polynucleotide processing. Such polynucleotide processing may be useful for a variety of applications, including polynucleotide sequencing.

Подробнее

Номер записи: 60

21-01-2021 дата публикации

METHOD OF PRODUCING AN IMMUNOLIGAND/PAYLOAD CONJUGATE

Номер: US20210015936A1

Автор: Beerli Roger Renzo, Grawunder Ulf

Принадлежит:

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

Подробнее

Номер записи: 61

17-01-2019 дата публикации

METHOD FOR USING HEAT-RESISTANT MISMATCH ENDONUCLEASE

Номер: US20190017037A1

Автор: Matsumura Kiyoyuki, Mukai Hiroyuki, TAKATSU Nariaki, Uemori Takashi

Принадлежит: TAKARA BIO INC.

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

Подробнее

Номер записи: 62

17-01-2019 дата публикации

METHOD FOR ANALYSIS OF AN RNA MOLECULE

Номер: US20190017100A1

Автор: EBER Fabian Johannes, WOCHNER Aniela

Принадлежит:

The present invention relates to the field of RNA analysis. In particular, the invention concerns the use of a catalytic nucleic acid molecule for the analysis of an RNA molecule and/or of a population of RNA molecules. In one aspect, the invention concerns methods for analyzing RNA molecules having at least one cleavage site for at least one catalytic nucleic acid molecule. In particular, the invention concerns a method for determining a physical property of an RNA molecule by analyzing a 5′ terminal fragment, a 3′ terminal fragment and/or at least one optional central RNA fragment obtained by cleavage of the RNA molecule by at least one catalytic nucleic acid molecule. Moreover, the present invention provides novel uses of a catalytic nucleic acid molecule for analyzing RNA molecules. In particular, the invention relates to the use of a catalytic nucleic acid molecule in a method for analyzing RNA molecules, wherein the resulting 5′ terminal RNA fragment, the 3′ terminal RNA fragment and/or the at least one optional central RNA fragment are analyzed. 1. A method for analyzing an RNA molecule having at least one cleavage site for at least one catalytic nucleic acid molecule , the method comprising the steps of:a) providing an RNA molecule having at least one cleavage site for at least one catalytic nucleic acid molecule,b) cleaving the RNA molecule with the at least one catalytic nucleic acid molecule into a 5′ terminal RNA fragment, a 3′ terminal RNA fragment and optionally into at least one central RNA fragment by contacting the RNA molecule with the at least one catalytic nucleic acid molecule under conditions allowing the cleavage of the RNA molecule,c) determining a physical property of the RNA molecule by analyzing the 3′ terminal RNA fragment and/or the at least one optional central RNA fragment obtained in step b).2. A method for analyzing a population of RNA molecules , wherein the population comprises at least one RNA molecule that has at least one ...

Подробнее

Номер записи: 63

17-01-2019 дата публикации

MATRIX ARRAYS AND METHODS FOR MAKING SAME

Номер: US20190017107A1

Автор: CICERO Ronald, GLAZER Marc, GRAY Jeremy, Gremyachinskiy Dmitriy, Hinz Wolfgang, INMAN Christina, KENNEY Paul, Light David, MASTROIANNI Alexander, ROZHKOV Roman, WANG Yufang

Принадлежит:

A method of forming a polymer matrix array includes applying an aqueous solution into wells of a well array. The aqueous solution includes polymer precursors. The method further includes applying an immiscible fluid over the well array to isolate the aqueous solution within the wells of the well array and polymerizing the polymer precursors isolated in the wells of the well array to form the polymer matrix array. An apparatus includes a sensor array, a well array corresponding to the sensor array, and an array of polymer matrices disposed in the well array. 1. A method of forming a polymer matrix array , the method comprising:applying an aqueous solution into wells of a well array, the aqueous solution comprising polymer precursors;applying an immiscible fluid over the well array to isolate the aqueous solution within the wells of the well array; andpolymerizing the polymer precursors isolated in the wells of the well array to form the polymer matrix array.2. The method of claim 1 , wherein the well has a characteristic diameter in a range of 0.1 micrometers to 2 micrometers.3. The method of claim 1 , wherein the well has a thickness in a range of 0.01 micrometers to 10 micrometers.4. The method of claim 1 , wherein the wells of the well array overlie sensors of a sensor array claim 1 , the wells corresponding to ion sensitive material of the sensors of the sensor array.5. The method of claim 1 , wherein a polymer matrix of the polymer matrix array is conformal with walls of a well of the well array.6. The method of claim 1 , wherein the polymer precursors include a radically polymerizable monomer.7. The method of claim 6 , wherein the radically polymerizable monomer includes a vinyl-based monomer.8. The method of claim 7 , wherein the vinyl-based monomer includes acrylamide claim 7 , vinyl acetate claim 7 , hydroxyalkylmethacrylate claim 7 , variations or derivatives thereof or any combination thereof.9. The method of claim 1 , wherein the polymer precursors ...

Подробнее

Номер записи: 64

17-01-2019 дата публикации

DETERMINATION OF NUCLEIC ACID METHYLATION

Номер: US20190017110A1

Автор: Ying Jackie Y., Zu Yanbing

Принадлежит:

The invention relates to methods and kits for determining the methylation status of a target nucleic acid molecule in a sample comprising (a) bisulfite treatment of the target nucleic acid, (b) amplifying the treated target nucleic acid with methylation-specific primers, (c) contacting the amplicons with a methylation-specific plasmonic nanoprobe and (d) determining the methylation status based on melting temperature Tof the hybrid probe and the target nucleic acid. In particular, a plasmonic gold nanoparticle covalently coupled to a morpholino oligonucleotide probe. Also claimed are methods and kits for determining methylation status of the Septin 9 (SEPT9) gene promoter. 1. A method of determining the methylation status of a target nucleic acid molecule in a sample , wherein the method comprises the steps of:(a) bisulfite treatment of the sample for converting the unmethylated cytosine bases in the target nucleic acid molecule to uracil;(b) amplifying at least part of the converted target nucleic acid molecule, said part comprising a target region the methylation status of which is to be analyzed, in a polymerase chain reaction (PCR), using a pair of methylation-specific primers under conditions allowing such amplification to generate PCR amplification products (amplicons);(c) contacting the amplicons with a methylation-specific plasmonic nanoprobe comprising a plasmonic nanoparticle and a non-ionic oligonucleotide analog probe covalently coupled thereto, the oligonucleotide analog probe comprising a base sequence that is complementary to the target region, under conditions that allow the oligonucleotide analog probe and the amplicons comprising the target region to hybridize to each other, wherein the probe generates a detectable signal if hybridized to the target nucleic acid molecule that is distinguishable from the signal of the unhybridized probe;{'sub': 'm', '(d) determining the methylation status of the target nucleic acid molecule based on the ...

Подробнее

Номер записи: 65

17-01-2019 дата публикации

Methods and systems for processing polynucleotides

Номер: US20190017115A9

Автор: Benjamin Hindson, Christopher Hindson, Kevin Ness, Michael Schnall-Levin, Mima Jarosz, Paul Hardenbol, Phillip Belgrader, Rajiv Bharadwaj, Serge Saxonov, Xinying Zheng

Принадлежит: 10X Genomics Inc

The present disclosure provides compositions, methods, systems, and devices for polynucleotide processing. Such polynucleotide processing may be useful for a variety of applications, including polynucleotide sequencing.

Подробнее

Номер записи: 66

21-01-2021 дата публикации

Methods for controlling gene expression

Номер: US20210017514A1

Автор: Alexander Morgan Jones, Bo Larsen

Принадлежит: University of Cambridge

The invention relates to methods for precisely controlling expressions of a target gene in an organism using a light-inducible kinase and a response regulator. The invention also relates to nucleic acid constructs and nucleic acids encoding the light-inducible kinase and response regulator, as well as organisms expressing these constructs.

Подробнее

Номер записи: 67

21-01-2021 дата публикации

Sequential probing of molecular targets based on pseudo-color barcodes with embedded error correction mechanism

Номер: US20210017587A1

Автор: Chee Huat Eng, Long Cai, Sheel SHAH

Принадлежит: California Institute of Technology CalTech

The present invention, among other things, provides technologies for detecting and/or quantifying nucleic acids in cells, tissues, organs or organisms. Pre-designed barcodes are associated specific molecular targets through sequential hybridization experiments. A pseudo-color based barcoding scheme is developed to overcome limitations in the previous generation of the technology such as lack of visual signals that can be associated with the probes or small internal within cell when carrying out in situ experiments. The current method can be applied to both in vitro and in situ analysis. According to the method, each barcoding round comprises multiple serial hybridizations where a small number of colored signals (that are associated with probes) are used in each hybridization experiment within a serial hybridization round. Images from each serial hybridization experiment within the same serial hybridization round are combined to form a composite image for each barcoding round. In each barcoding round, the same set of molecular targets are analyzed. After all barcoding rounds are completed, associated of the barcode with these molecular targets is completed.

Подробнее

Номер записи: 68

21-01-2021 дата публикации

AMPLIFICATION OF NUCLEIC ACIDS

Номер: US20210017588A1

Автор: MINTER Stephen John, Patsos Georgios

Принадлежит:

There is provided a method of amplifying a nucleic acid sequence. The method comprises providing an amplification mixture comprising an exonuclease capable of digestion of a strand of a double stranded nucleic acid molecule from the 5′-end towards the 3′-end, a strand displacing polymerase, a double stranded nucleic acid molecule comprising first and second nucleic acid strands, a first nucleic acid primer, and nucleotides as appropriate to provide for amplification of the first nucleic acid sequence to be amplified. The method further comprises effecting the amplification reaction under conditions permitting digestion, exonuclease digestion and strand displacement polymerisation thereby producing a product mixture comprising an amplified amount of said first nucleic acid sequence. There is also provided a method of determining the presence or quantity of target nucleic acid sequence in a biological sample using a double stranded probe having a fluorophore on one strand and its quencher on the other and using denaturation and re-hybridisation to detect the target. 1. A method of amplifying a nucleic acid sequence comprising: (i) an exonuclease capable of effecting digestion of a strand of a double stranded nucleic acid molecule with the digestion being from the 5′-end of the strand towards the 3′-end,', '(ii) a strand displacing polymerase,', '(iii) a double stranded nucleic acid molecule comprising first and second nucleic acid strands hybridised to each other, said first strand incorporating a first nucleic acid sequence to be amplified and having a first 5′-end region which remote from its 5′-end has a nucleotide sequence resistant to digestion (under the conditions of the method) by the exonuclease defined as (i),', '(iv) a first nucleic acid primer having the same nucleotide sequence as said first end region of the first nucleic acid, and incorporating the same digestion resistant region, and', '(v) nucleotides as appropriate to provide for amplification of the ...

Подробнее

Номер записи: 69

21-01-2021 дата публикации

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

Номер: US20210017589A1

Автор: LIPINSKI Kamil Andrzej

Принадлежит: DNAE DIAGNOSTICS LIMITED

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

Подробнее

Номер записи: 70

21-01-2021 дата публикации

Methods for Microbial DNA Analysis

Номер: US20210017590A1

Автор: Benjamin Alan Katchman, Candy Mavis Rivas, Michael Edward Hogan, Yasmine Eve Baca

Принадлежит: PathogenDX Inc

Provided herein are methods for isolating nucleic acids from intact cells in a sample of intact cells, contamination dead cells, cell debris, and biofilm using two separation steps, either by centrifugation or filtration, performed in sequentially. Also provided is a method for isolating nucleic acids from intact cells using a first separation step followed by treatment with a nuclease and then a second separating step. Provided herein is a related method for isolating DNA from intact cells using a nuclease that produces DNA cuts on double stranded DNA, followed by a second separating step.

Подробнее

Номер записи: 71

16-01-2020 дата публикации

MODIFIED NUCLEOTIDES

Номер: US20200017908A1

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

Принадлежит:

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

Подробнее

Номер записи: 72

21-01-2021 дата публикации

MEMORY WRITING

Номер: US20210017595A1

Автор: Cai Qing, Chen Chang, Covens Kris, Fauvart Maarten, Stakenborg Tim

Принадлежит:

The present invention relates to a method for writing data comprising a sequence of bits, the data being written in a form of nucleic acid, by in-vitro enzymatically producing memory nucleic acid from a strand of memory writing substrate nucleic acid, wherein the strand of memory writing substrate nucleic acid comprises a plurality of spacer sections and memory writing sections sandwiched between the spacer sections. Each of the spacer sections comprises one or more nucleobases, and each of the memory writing sections comprises a nucleobase other than the nucleobases of an adjacent spacer section upstream of the memory writing section in a travel direction of an enzyme along the strand of memory writing substrate nucleic acid. The method comprising: repeating of: synthesising, in liquid medium comprising the strand of memory writing substrate nucleic acid contacted with the enzyme, a spacer portion of the memory nucleic acid from a spacer section by the enzyme by contacting with a solution of spacer nucleotides compatible with the nucleobases of the spacer section; halting the synthesising of the spacer portion in a position where the enzyme is reaching the memory writing section resulting from incompatibility between spacer nucleotides and nucleobases of the portion of the memory nucleic acid from the memory writing section; receiving a sub-sequence of the sequence of bits, said sub-sequence comprising at least one bit; selecting a memory nucleotide compatible with the nucleobase of the memory writing section, and comprising a first label or first modification, on a condition that said sub-sequence comprises a predetermined first sequence of bit-values, and selecting a memory nucleotide compatible with the nucleobase of the memory writing section, and comprising a second label or second modification, on a condition that said sub-sequence comprises a predetermined second sequence of bit-values; and subsequent to the halting, synthesising, in the liquid medium ...

Подробнее

Номер записи: 73

25-01-2018 дата публикации

Methods for Preparative In Vitro Cloning

Номер: US20180023120A1

Автор: Jacobson Joseph, Kung Li-Yun A., Schindler Daniel

Принадлежит:

Methods and devices relate to the isolation of nucleic acids of interest from within a population of nucleic acids such as libraries of nucleic acid sequences. 1. A method of isolating a target nucleic acid , the method comprising:(a) providing a population of nucleic acid molecules comprising a plurality of distinct nucleic acid molecules;(b) providing a plurality of oligonucleotide tags;(c) attaching the oligonucleotide tags to one terminus of the plurality nucleic acid molecules thereby generating a subpopulation of nucleic acid-oligonucleotide tag molecules;(d) amplifying the plurality of nucleic acid-oligonucleotide tag molecules using primers complementary to the plurality of oligonucleotide tags; and(e) isolating at least one target nucleic acid-oligonucleotide tag molecule of the subpopulation of nucleic acid-oligonucleotide tag molecules.2. The method of further sequencing the at least one isolated target nucleic acid- oligonucleotide tag molecule.3. The method of further identifying the target nucleic acid.4. The method of wherein the sequencing step is by high throughput sequencing.5. The method of further amplifying the at least one target nucleic acid-oligonucleotide tag molecule.6. The method of wherein the step of isolating is by limiting dilution.7. The method of further amplifying the target nucleic acid-oligonucleotide tag molecule.8. The method of wherein the target nucleic acid has a predefined sequence.9. A method of isolating a target nucleic acid claim 1 , the method comprising:(a) providing a population comprising a plurality of different nucleic acid molecules;(b) providing a plurality of microparticles, wherein each microparticle has an oligonucleotide sequence complementary to a portion of the nucleic acid molecules immobilized on its surface;(c) forming a population of nucleic acid molecules hybridized to the complementary oligonucleotide sequence on the microparticles; and(d) isolating at least one target nucleic acid molecule.10. The ...

Подробнее

Номер записи: 74

25-01-2018 дата публикации

ADDITIVE FOR ACCELERATING HYBRIDIZATION

Номер: US20180023126A1

Автор: ASIOLI Sofia, DI OTO Enrico, MONTI Valentina

Принадлежит:

The present invention describes an additive for accelerating hybridization comprising: a) an aqueous solution of sodium dextran sulphate, b) a salt, c) a buffer system, d) a strong mineral base possibly mixed with at least one polar aprotic solvent, or at least one polar aprotic solvent. 1. An additive for accelerating hybridization comprisinga) an aqueous solution of sodium dextran sulphate,b) a salt,c) a buffer system, andd) a strong mineral base possibly mixed with at least one polar aprotic solvent, or at least one polar aprotic solvent.2. The additive of claim 1 , wherein said mineral base is a hydroxide of a metal.3. The additive of claim 2 , wherein said metal hydroxide is selected from the group consisting of sodium hydroxide claim 2 , lithium hydroxide claim 2 , potassium hydroxide claim 2 , rubidium hydroxide claim 2 , caesium hydroxide claim 2 , beryllium hydroxide claim 2 , magnesium hydroxide claim 2 , calcium hydroxide claim 2 , strontium hydroxide claim 2 , barium hydroxide claim 2 , aluminium hydroxide claim 2 , gallium hydroxide claim 2 , indium hydroxide and tin hydroxide.4. The additive of claim 1 , wherein said polar aprotic solvent is an organic solvent having a dipole moment of at least approximately 2 Debye units claim 1 , a solubility in water of at least approximately 5% by volume at around room temperature and that does not undergo significant hydrogen exchange at around neutral pH.5. The additive of claim 4 , wherein said solvent is selected from the group consisting of ethylene carbonate claim 4 , γ-butyrolactone claim 4 , tetramethylene sulfone sulphur dioxide claim 4 , acetonitrile claim 4 , glycol sulphite/ethylene sulphite claim 4 , propylene carbonate claim 4 , ethylene trithiocarbonate claim 4 , ε-caprolactone claim 4 , N-methyl pyrrolidinone claim 4 , acetanilide claim 4 , N-acetylpyrrolidone claim 4 , 4-amminopyridine claim 4 , benzamide claim 4 , benzimidazole claim 4 , 1 claim 4 ,2 claim 4 ,3-benzotriazole claim 4 , butadiene ...

Подробнее

Номер записи: 75

25-01-2018 дата публикации

MULTIPLEXED ANALYSIS OF NUCLEIC ACID HYBRIDIZATION THERMODYNAMICS USING INTEGRATED ARRAYS

Номер: US20180023129A1

Автор: Hassibi Arjang, JIRAGE Kshama, Manickam Arun, MILANINIA Kaveh

Принадлежит:

The present disclosure provides methods and devices for simultaneous identification of a plurality of target nucleic acid sequences in a single sample chamber that includes an addressable array of nucleic acid probes attached to a solid surface. Addressable signals can be generated and measured, in real-time, upon hybridization of target sequences at the individual probe locations within the array while the temperature of the system is varied. Such generated signals, as a function temperature, can then be used to compute the properties of nucleic acid hybridization at each addressable location which is ultimately utilized to estimate the sequence of the target nucleic acids. In particular, an integrated semiconductor biosensor array device can be used to measure the addressable signals. 153.-. (canceled)54. A method for assaying a presence of a target nucleic acid molecule in a sample , comprising:(a) providing (i) a surface comprising a probe coupled to an energy donor, wherein said probe is configured to selectively couple to said target nucleic acid molecule, and (ii) an optical detector below said surface, wherein said optical detector is configured to detect at least one optical signal generated upon energy transfer between an energy acceptor coupled to said target nucleic acid molecule and said energy donor;(b) bringing said sample containing or suspected of containing said target nucleic acid molecule in contact with said surface under conditions sufficient to permit said probe to selectively couple to said target nucleic acid molecule;(c) using said optical detector to measure said at least one optical signal while subjecting said surface to a temperature change; and(d) generating signal versus temperature data using measurements of said at least one optical signal with said temperature change, thereby assaying said presence of said target nucleic acid molecule in said sample.55. The method of claim 54 , wherein said at least one optical signal is not ...

Подробнее

Номер записи: 76

25-01-2018 дата публикации

SEQUENCE BASED GENOTYPING BASED ON OLIGONUCLEOTIDE LIGATION ASSAYS

Номер: US20180023135A1

Автор: Hogers René Cornelis Josephus, Van Eijk Michael Josephus Theresia

Принадлежит: Keygene N.V.

The invention relates to a method for the detection of a target nucleotide sequence in a sample based on an oligonucleotide ligation assay wherein probes are used that contain (a combination of) sequence-based identifiers that can identify the sample and the target sequence (i.e. locus and/or allele combination) wherein after the ligation step, the ligated probes, or after amplification, the amplified ligated probes, are restricted using restriction enzymes to cut of part of the probes and continue with those parts (identifiers and target sequence) that contain the relevant information in the sequencing step. 125-. (canceled)26. A method for detection of one or more polymorphisms in at least one target nucleotide sequence in a sample comprising: wherein the first probe comprises a first target specific section and a first tag section that is non-complementary to the target nucleotide sequence and that optionally comprises a first primer binding sequence;', 'wherein the second probe comprises a second target specific section and a second tag section that is non-complementary to the target nucleotide sequence and that comprises an optional second primer binding sequence;, '(a) providing for each target nucleotide sequence a first probe and a second probe,'}(b) allowing the first and second target specific section of the respective first and second probe to hybridize to the target sequence;(c) ligating the first and second probe when the respective target specific sections of the probes are hybridized to essentially adjacent sections on the target sequence to provide ligated probes;(d) amplifying the ligated probes with a first and/or an optional second primer wherein the first primer comprises an identifier to provide amplicons containing an identifier;(e) subjecting the amplicons to a high throughput sequencing to determine at least part of the target nucleotide sequence and the identifier contained in the amplicons; and(f) identifying one or more polymorphism in the ...

Подробнее

Номер записи: 77

10-02-2022 дата публикации

Flexible and high-throughput sequencing of targeted genomic regions

Номер: US20220042096A1

Автор: Adam Payton, Leandro Gomide Neves

Принадлежит: Rapid Genomics LLC

The disclosure pertains to materials and methods for capturing a target genomic region, comprising hybridizing an extension probe and a ligation probe to target sequences that flank the target genomic region; elongating the 3′ end of the extension probe until the 3′ end of the elongated extension probe is adjacent to the 5′ end of the ligation probe; and ligating the 3′ end of the elongated extension probe with the 5′ end of the ligation probe to produce a ligated probe. The ligated probe can be PCR amplified to produce copies of the target genomic region that can be detected or sequenced. Certain embodiments of the invention also provide methods of producing double stranded probes suitable for capturing and analyzing both strands of a target genomic region in a double stranded genomic DNA. The invention also provides kits for performing the methods disclosed herein.

Подробнее

Номер записи: 78

10-02-2022 дата публикации

METHODS FOR NON-INVASIVE PRENATAL PLOIDY CALLING

Номер: US20220042103A1

Автор: Baner Johan, Banjevic Milena, Demko Zachary, Gemelos George, Hill Matthew, Rabinowitz Matthew, Ryan Allison, Zimmermann Bernhard

Принадлежит: Natera, Inc.

The present disclosure provides methods for determining the ploidy status of a chromosome in a gestating fetus from genotypic data measured from a sample of DNA from the mother of the fetus and from the fetus, and from genotypic data from the mother and optionally also from the father. The ploidy state is determined by using a joint distribution model to create a set of expected allele distributions for different possible fetal ploidy states given the parental genotypic data, and comparing the expected allelic distributions to the pattern of measured allelic distributions measured in the mixed sample, and choosing the ploidy state whose expected allelic distribution pattern most closely matches the observed allelic distribution pattern. In an embodiment, the mixed sample of DNA may be preferentially enriched at a plurality of polymorphic loci in a way that minimizes the allelic bias. 1. A method for preparing a preparation of amplified and enriched DNA derived from a biological sample comprising cell-free DNA of maternal origin and cell-free DNA of fetal origin useful for fetal fraction analysis , comprising:(a) extracting cell-free DNA from the biological sample, wherein the extracted DNA comprises a mixture of cell-free DNA of maternal origin and cell-free DNA of fetal origin;(b) preparing a preparation of amplified and enriched DNA by: ligating adaptors comprising at least one universal amplification sequence to the extracted DNA to obtain adaptor-ligated DNA, amplifying the adaptor-ligated DNA using at least one universal primer to obtain amplified DNA, performing targeted enrichment of 100 to 20,000 polymorphic loci from the amplified DNA; and(c) analyzing the amplified and enriched DNA by: performing next-generation sequencing to obtain quantitative measurements of different alleles at each polymorphic locus, and estimating the fetal fraction of the cell-free DNA in the biological sample using the quantitative measurements.2. The method of claim 1 , wherein ...

Подробнее

Номер записи: 79

24-01-2019 дата публикации

EDITING MITOCHONDRIAL DNA

Номер: US20190024073A1

Автор: Campbell Jarryd M., Ekker Stephen C.

Принадлежит: Mayo Foundation for Medical Education and Research

Materials and methods for making targeted changes (e.g., deletions) in mitochondrial DNA are described. 1. A method for modifying mitochondrial DNA within a cell , comprising introducing into the cell a recombinant DNA endonuclease targeted to a selected sequence within the mitochondrial DNA.2. The method of claim 1 , wherein the recombinant DNA endonuclease comprises a mutation that results in attenuated endonuclease activity claim 1 , as compared to a corresponding DNA endonuclease that lacks the mutation.3. The method of claim 1 , wherein the recombinant DNA endonuclease has nickase activity.4. The method of claim 1 , wherein the recombinant DNA endonuclease comprises:(a) a first portion comprising a mitochondrial targeting sequence;(b) a second portion comprising an amino acid sequence targeting the DNA endonuclease to the selected sequence within the mitochondrial DNA; and(c) a third portion comprising a nuclease domain that cleaves the mitochondrial DNA at or near the selected sequence.5. The method of claim 4 , wherein the first portion comprises a mitochondrial targeting sequence from isocitrate dehydrogenase 2.6. The method of claim 4 , wherein the second portion comprises a transcriptional activator-like effector (TALE) backbone and a plurality of tandem repeat sequences comprising repeat variable dinucleotides that claim 4 , in combination claim 4 , are targeted to the selected mitochondrial DNA sequence.7. The method of claim 4 , wherein the nuclease domain is from a modified FokI endonuclease claim 4 , or a portion thereof.8. The method of claim 7 , wherein the modified FokI endonuclease or portion thereof comprises an aspartic acid to alanine substitution at the amino acid position corresponding to position 483 of an unmodified FokI endonuclease.9. The method of claim 7 , wherein the modified FokI endonuclease or portion thereof comprises an aspartic acid to alanine substitution at the amino acid position corresponding to position 450 of an unmodified ...

Подробнее

Номер записи: 80

24-01-2019 дата публикации

Methods and compositions for target detection

Номер: US20190024075A1

Автор: Eric HARNESS, Meredith L. CARPENTER, Stephane B. Gourguechon

Принадлежит: ARC Bio LLC

Provided herein are methods and compositions for the detection and identification of targets in a sample, using target-specific guide nucleic acids and nucleic acid-guided nuclease system proteins.

Подробнее

Номер записи: 81

24-01-2019 дата публикации

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

Номер: US20190024127A1

Автор: YEH Chen-Hsiung

Принадлежит:

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

Подробнее

Номер записи: 82

24-01-2019 дата публикации

Methods and compositions for isolating asymmetric nucleic acid complexes

Номер: US20190024140A1

Автор: David Christopher Scherer, Lei Sun, Natasha Popovich, Sassan Sheikholeslami

Принадлежит: Pacific Biosciences of California Inc

The present disclosure provides improved methods for isolating asymmetrically-primed and/or asymmetrically-tagged nucleic acid complexes that find use in downstream analytical analyses, including sequence analysis. Compositions comprising such complexes and kits and systems for generating such complexes are also provided.

Подробнее

Номер записи: 83

24-01-2019 дата публикации

Direct Capture, Amplification and Sequencing of Target DNA Using Immobilized Primers

Номер: US20190024141A1

Автор: Hanlee P. Ji, Jason D. BUENROSTRO, Samuel Myllykangas

Принадлежит: Leland Stanford Junior University

Certain embodiments provide a method for capturing a genomic fragment. The method may comprise: obtaining a substrate comprising a first population of surface-bound oligonucleotides and a second population of surface-bound oligonucleotides; hybridizing a first member of the first population of surface-bound oligonucleotides to a selection oligonucleotide comprising a region that hybridizes with the first member and a region that contains a genomic sequence; extending the first member of the first population of surface-bound oligonucleotides to produce a support-bound selection primer that comprises a sequence that is complementary to the genomic sequence; hybridizing the support-bound selection primer to a nucleic acid fragment comprising the genomic sequence; extending the support-bound selection primer to produce an extension product that contains a sequence that flanks the genomic sequence, e.g., in a genome; and amplifying the extension product on the substrate.

Подробнее

Номер записи: 84

24-01-2019 дата публикации

NUCLEIC ACID AMPLIFICATION BIOSENSOR FOR USE IN ROLLING CIRCLE AMPLIFICATION (RCA)

Номер: US20190024148A1

Автор: Brennan John, Li Yingfu, LIU MENG

Принадлежит: MCMASTER UNIVERSITY

The present application describes a biosensor for detecting target nucleic acid. The biosensor's mode of operation is based on binding of the target nucleic acid to another nucleic acid sequence and a circular template which triggers rolling circle amplification and detection of the amplified product as the indicator of the presence of the target nucleic acid. The biosensor is immobilized on a solid support, such as paper. 1. A nucleic acid sensor probe for detection of target nucleic acid comprising:a) a capture oligonucleotide that comprises a nucleic acid sequence that is complementary to a first nucleic acid sequence of the target oligonucleotide;b) a solid support immobilizing the capture oligonucleotide; andc) reagents for performing rolling-circle amplification (RCA) of the target oligonucleotide.2. The nucleic acid sensor probe of claim 1 , wherein the reagents for performing RCA are comprised in a stabilized composition.3. The nucleic acid sensor probe of claim 2 , wherein the reagents are encapsulated in a stabilizing matrix or are freeze dried.4pullulan.. The nucleic acid sensor probe of claim 3 , wherein the reagents for performing RCA are encapsulated with5. The nucleic acid sensor probe of claim 1 , wherein the capture oligonucleotide is biotinylated and bound with streptavidin on a membrane surface.6. The nucleic acid sensor probe of claim 1 , wherein the solid support is paper or a paper-based material.7. The nucleic acid sensor probe of claim 1 , wherein the reagents for performing RCA comprise a circular template comprising a nucleic acid sequence that is complementary to a second nucleic acid sequence of the target oligonucleotide and a nucleic acid polymerase having exonuclease activity.8. The nucleic acid sensor probe of claim 7 , wherein the nucleic acid polymerase is a DNA polymerase having 3′ to 5′ exonuclease activity or an RNA polymerase having 3′ to 5′ exonuclease activity.9. The nucleic acid sensor probe of claim 8 , wherein the nucleic ...

Подробнее

Номер записи: 85

24-01-2019 дата публикации

INDEXING BASED DEEP DNA SEQUENCING TO IDENTIFY RARE SEQUENCES

Номер: US20190024150A1

Автор: LI CHAK KAR (ERIC), Liu Lin, WANG GARY P.

Принадлежит: University of Florida Research Foundation, Incorporated

The invention pertains to an assay that is capable of detecting a mutant polynucleotide in a plurality of polynucleotides. In one embodiment, the assay of the invention is capable of detecting one copy of a mutant polynucleotide in about 50,000 to about 100,000 copies of polynucleotides. The assay of the invention can be used to identify a mutant viral quasispecies or a mutant mRNA encoding an oncogenic protein from a tumor sample. The assay of the invention involves producing the single stranded complements of each of a plurality of polynucleotides containing the target sequence, wherein each of the single stranded complements contain a unique tag sequence and amplifying the single stranded complements by PCR using several sets of primers designed to introduce the sequences appropriate for a paired-end sequencing analysis of the amplified polynucleotides. The invention also pertains to kits for carrying out the assays of the invention. 1. A method of producing a single stranded complement of each of a plurality of polynucleotides containing a target sequence , the method comprising conducting one cycle of a polymerase chain reaction (PCR) using a plurality of primers , wherein each copy of the primer in the plurality of primers comprises , from the 5′ end:i) an outer PCR primer motif,ii) an inner PCR primer motif,iii) a tag comprising a sequence unique for each copy of the primer, wherein the unique sequence comprises about 4-20 nucleotides, andiv) a 3′ target sequence which has a sequence that corresponds to the sequence at the 3′ end of the target sequence,wherein each of the single stranded complements of each of the plurality of polynucleotides produced in the PCR comprises, from the 5′ end:i) the outer PCR primer motif,ii) the inner PCR primer motif,iii) the tag comprising a unique sequence of about 4-20 nucleotides,iv) the sequence corresponding to the target sequence.2. An assay to identify , from a plurality of polynucleotides , a polynucleotide having a ...

Подробнее

Номер записи: 86

24-01-2019 дата публикации

Detection of Nucleic Acids

Номер: US20190024160A1

Автор: Donald Cortny, Lonergan Dina, Mokany Elisa, Todd Alison Velyian, Walker Samantha, Yeen Tan Lit

Принадлежит:

The present invention provides modified oligonucleotides and methods for their use in the detection of nucleic acids. The oligonucleotides and methods find particular application in amplifying and/or detecting areas of genetic variation in target nucleic acid sequences. 1. A method for determining the presence or absence of a target polynucleotide in a sample , the method comprising: a first primer component terminating at the 5′ end of the oligonucleotide and capable of hybridising to a first portion of a strand of the target polynucleotide by complementary base pairing, and', 'a second primer component terminating at the 3′ end of the oligonucleotide and capable of hybridising to a second portion of the target polynucleotide strand by complementary base pairing;, 'providing a primer oligonucleotide comprising'}contacting a sample potentially comprising the target polynucleotide with the primer oligonucleotide under conditions suitable for hybridisation of the first primer component and second primer component with the target polynucleotide strand to thereby form a double-stranded duplex, wherein at least one strand of an intermediate section of the duplex comprises a sequence of at least four nucleotides that remains unhybridised to an opposing strand of the intermediate section due to an absence of a sequence of nucleotides in the opposing strand of the intermediate section sharing base pair complementarity with the sequence of at least four nucleotides;contacting the sample with a polymerase enzyme capable of using the target polynucleotide strand as a template to extend the length of the primer oligonucleotide of the duplex and thereby generate an amplicon comprising an internal component intermediate to first and second end components, whereinthe amplicon is generated using a polymerase chain reaction (PCR), strand displacement amplification (SDA), loop-mediated isothermal amplification (LAMP), rolling circle amplification (RCA), transcription-mediated ...

Подробнее

Номер записи: 87

24-01-2019 дата публикации

METHODS AND COMPOSITIONS FOR SEQUENCING MODIFIED NUCLEIC ACIDS

Номер: US20190024162A1

Автор: Clark Tyson A., Korlach Jonas, Turner Stephen

Принадлежит:

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

Подробнее

Номер записи: 88

24-01-2019 дата публикации

MOLECULAR IDENTIFICATION WITH SUBNANOMETER LOCALIZATION ACCURACY

Номер: US20190024165A1

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

Принадлежит:

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

Подробнее

Номер записи: 89

24-01-2019 дата публикации

Methods and Systems for Processing Polynucleotides

Номер: US20190024166A1

Автор: BELGRADER Phillip, Bharadwaj Rajiv, Hardenbol Paul, HINDSON Benjamin, Hindson Christopher, Jarosz Mirna, Ness Kevin, Saxonov Serge, Schnall-Levin Michael, Zheng Xinying

Принадлежит:

The present disclosure provides compositions, methods, systems, and devices for polynucleotide processing. Such polynucleotide processing may be useful for a variety of applications, including polynucleotide sequencing. 1. A method for nucleic acid sequencing , comprising:(a) co-partitioning a plurality of beads and a plurality of primers in a plurality of droplets, wherein a droplet of said plurality of droplets comprises (i) a ribonucleic acid (RNA) molecule comprising a nucleic acid sequence, (ii) a primer from said plurality of primers, and (iii) a bead from said plurality of beads, wherein said bead comprises a nucleic acid barcode molecule coupled thereto, wherein said nucleic acid barcode molecule comprises a barcode sequence;(b) hybridizing said primer to a region at a 3′ end of said RNA molecule;(c) using an enzyme to extend said primer to generate a nucleic acid product comprising a sequence corresponding to said nucleic acid sequence of said RNA molecule, wherein said enzyme incorporates a sequence at a 3′ end of said nucleic acid product that is complementary to said nucleic acid barcode molecule;(d) hybridizing said nucleic acid barcode molecule to said nucleic acid product generated in (c) and extending said nucleic acid product using said nucleic acid barcode molecule as template, to generate a barcoded nucleic acid molecule comprising, from a 5′ end to a 3′ end, (1) said sequence corresponding to said nucleic acid sequence of said RNA molecule and (2) a complement of said barcode sequence; and(e) sequencing said barcoded nucleic acid molecule or a derivative thereof,wherein, after (a), said nucleic acid barcode molecule is released from said bead.2. The method of claim 1 , wherein said RNA molecule is from a cell.3. The method of claim 2 , wherein said droplet comprises said cell.4. The method of claim 3 , further comprising releasing said RNA molecule from said cell prior to (b).5. The method of claim 1 , wherein said bead further comprises a ...

Подробнее

Номер записи: 90

24-01-2019 дата публикации

PRIMERS AND METHODS FOR THE DETECTION AND DISCRIMINATION OF NUCLEIC ACIDS

Номер: US20190024167A1

Автор: Astatke Mekbib, Darfler Marlene, Gebeyehu Gulilat, NAZARENKO Irina, Pires Richard M., Rashtchian Ayoub, Solus Joseph

Принадлежит:

The present invention provides novel primers and methods for the detection of specific nucleic acid sequences. The primers and methods of the invention are useful in a wide variety of molecular biology applications and are particularly useful in allele specific PCR. 174.-. (canceled)75. A composition comprising at least one nucleic acid molecule and at least two oligonucleotides , wherein at least a portion of said first and second oligonucleotides are capable of hybridizing with at least a portion of said nucleic acid molecule and wherein said first and second oligonucleotides each comprise at least one modified nucleotide internally and/or at or near the 3′- and/or 5′-terminal nucleotide.76. The composition of claim 75 , wherein said at least one modified nucleotide of said first and/or said second oligonucleotide is a 2′-O-alkyl ribonucleotide.77. The composition of claim 75 , said composition further comprising at least one component selected from the group consisting of one or more nucleotides claim 75 , one or more DNA polymerases and one or more reverse transcriptases.78. The composition of claim 75 , wherein said modified nucleotide of said first oligonucleotide comprises a detectable label.79. The composition of claim 78 , wherein said detectable label is a fluorescent moiety.80. The composition of claim 79 , wherein said modified nucleotide on said second oligonucleotide is a quencher moiety.81. The composition of claim 80 , wherein said fluorescent moiety and/or said quencher moiety is located at a 3′-terminal nucleotide on said first and/or said second oligonucleotide.82. The composition of claim 80 , wherein said fluorescent moiety and/or said quencher moiety is located at a 5′-terminal nucleotide on said first and/or said second oligonucleotide.83. The composition of claim 75 , wherein said nucleic acid molecule comprises a RNA molecule.84. The composition of claim 75 , wherein said nucleic acid molecule comprises a cDNA molecule.85. The composition of ...

Подробнее

Номер записи: 91

24-01-2019 дата публикации

HIGH INTENSITY LABELED REACTANT COMPOSITIONS AND METHODS FOR SEQUENCING

Номер: US20190024168A1

Автор: DODD David, Huang Haidong, Lackey Jeremy, Reed Brian, Rothberg Jonathan M., Shi Xinghua

Принадлежит: Quantum-Si incorporated

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

Подробнее

Номер записи: 92

24-01-2019 дата публикации

METHOD FOR DETECTION OF A GENETIC VARIANT

Номер: US20190024175A1

Автор: Ying Jackie Y., Zu Yanbing

Принадлежит:

A method and kit for detecting a genetic variant associated with a disease or disorder, including incompatibility with a pharmaceutical. The method and kit using a first nano-particle coupled to at least one morpholino nucleic acid probe comprising a target complimentary region base sequence that is a perfect match to a genetic variant sequence. 1. A detection kit for performing a method of detecting a genetic variant associated with a disease or disorder , including incompatibility with a pharmaceutical , the method comprising the steps of:(i) providing a first nano-particle coupled to at least one morpholino nucleic acid probe comprising a target-complementary region comprising a base sequence that is a perfect match to the genetic variant;(ii) combining a sample containing nucleic acid suspected of containing the genetic variant and the first nano-particle to provide a first mixture under conditions that allow hybridization of the morpholino nucleic acid probe and the nucleic acid suspected of containing the genetic variant;(iii) sequentially heating the first mixture and determining the melting temperature of the hybridization complex between the morpholino nucleic acid probe and the nucleic acid suspected of containing the genetic variant; and(iv) comparing the melting temperature determined in step c) with a standard to determine whether the sample comprises nucleic acid containing the genetic variant,wherein the kit comprises a first nano-particle coupled to at least one morpholino nucleic acid probe comprising a target complimentary region comprising a base sequence that is a perfect match to a genetic variant sequence associated with a disease or disorder, including incompatibility with a pharmaceutical.2. The kit according to claim 1 , further comprising a second nano-particle coupled to at least one morpholino nucleic acid probe comprising a target complimentary region base sequence that is a perfect match to a wildtype sequence associated with a disease ...

Подробнее

Номер записи: 93

23-01-2020 дата публикации

Methods for the epigenetic analysis of dna, particularly cell-free dna

Номер: US20200024643A1

Автор: Chunxiao Song, Damek SPACEK, Patrick A. Arensdorf

Принадлежит: Bluestar Genomics Inc

Methods are provided for the epigenetic analysis of cell-free DNA using organic boranes to convert oxidized 5-methylcytosine residues in the cell-free DNA to dihydrouracil (DHU) residues. Cell-free DNA is contacted with an organic borane selected to successively bring about reduction, deamination, and decarboxylation of oxidized 5-methylcytosine residues such as 5-carboxymethylcytosine and 5-formylcytosine, resulting in DHU residues in place thereof. Following amplification, the treated cell-free DNA is sequenced, with the DHU residues read as thymine residues. Reaction mixtures, kits and additional methods are also provided, as are related methods for the epigenetic analysis of DNA, including cell-free DNA.

Подробнее

Номер записи: 94

23-01-2020 дата публикации

USE OF ANTICOAGULANTS IN THE POLYMERASE CHAIN REACTION

Номер: US20200024645A1

Автор: Ainsworth Shaun, Cobb Ben, Miele Gino

Принадлежит:

The invention provides a method and kit for preventing inhibition of a thermal cycling reaction by protein coagulation in a sample. 2. The method of claim 1 , wherein the sample is not significantly diluted prior to the thermal cycling reaction claim 1 , wherein a significant dilution is by a factor of 4 claim 1 , 5 claim 1 , 6 claim 1 , 7 claim 1 , 8 claim 1 , 9 claim 1 , or 10.3. The method of claim 1 , wherein the anticoagulant comprises arginine.4. The method of claim 3 , wherein the sample is mixed with arginine to give an arginine concentration of 2-6 mg/ml claim 3 , preferably 5 mg/ml.5. The method of claim 4 , further comprising the step of diluting the sample claim 4 , wherein the sample is diluted following mixing with arginine and prior to incubating the sample under thermal cycling conditions.6. The method of claim 5 , wherein the concentration of arginine in the sample during incubation of the sample under thermal cycling conditions is 0.5-2 mg/ml claim 5 , preferably 1.7 mg/ml.7. The method of any preceding claim claim 5 , wherein the reagents and anticoagulant are provided in a single solution.8. The method of any preceding claim claim 5 , wherein the anticoagulant comprises MgCl.9. The method of claim 8 , wherein the sample is mixed with MgClto give a MgClconcentration of 2-6 mM claim 8 , preferably 5 mM.10. The method of any preceding claim claim 8 , wherein the anticoagulant comprises Tris claim 8 , preferably Tris (pH 8.5).11. The method of claim 10 , wherein the sample is mixed with Tris to give a Tris concentration of 30-60 mM claim 10 , preferably 50 mM.12. The method of any preceding claim further comprising the step of adding protease to the sample prior to incubating the sample under thermal cycling conditions.13. The method of any preceding claim claim 10 , wherein the sample is subjected to protein denaturing conditions prior to incubating the sample under thermal cycling conditions.14. The method of wherein subjecting the sample to ...

Подробнее

Номер записи: 95

28-01-2021 дата публикации

Microorganism separation and detection

Номер: US20210024877A1

Автор: Andrew Rogers, Daniel Lockhart, James Turner, Matthew Crow, Paul Jay, William Mullen

Принадлежит: Momentum Bioscience LTD

Methods for separating microorganisms from non-microorganism cells in a non-microorganism cell-containing sample comprise incubating the sample with particles to form particle-microorganism complexes and then separating the particle-microorganism complexes from the non-microorganism cells. These methods are used to detect the absence or presence of a microorganism in a sample that also contains non-microorganism cells. Particular reagents and combinations of reagents enhance the selective capture of microorganisms in mixed samples. Corresponding compositions and kits are also provided.

Подробнее

Номер записи: 96

28-01-2021 дата публикации

METHOD FOR INDICATING THE PROGRESS OF AMPLIFICATION OF NUCLEIC ACIDS AND KIT FOR PERFORMING THE SAME

Номер: US20210024978A1

Автор: Basler Norbert, Becker Claus, Cherkasov Dmitry, GRUNWALD Christian, HESS Hans-Joerg, Müller-Hermann Andreas

Принадлежит: AGCT GmbH

It is disclosed a method for the detection of an amplification of nucleic acids in which substantially use is made of the fact that a pre-defined nucleic acid chain (target sequence) can be multiplied/amplified in the presence of a target sequence-specific activator oligonucleotide. The target sequence-specific activator oligonucleotide causes the separation of re-synthesized complementary primer extension products by means of strand displacement, so that a new primer oligonucleotide can attach to the respective template strand. The thus formed complex of a primer oligonucleotide and a template strand can initiate a new primer extension reaction. The thus formed primer extension products in turn function as templates, so that an exponential amplification reaction results. Amplification is detected by a detection system. 1. A method for amplification of a nucleic acid comprising the following steps:a) hybridizing a first primer oligonucleotide to the 3′ segment of a strand of a nucleic acid chain to be amplified, wherein the nucleic acid chain to be amplified comprises a target sequence, wherein the first primer oligonucleotide comprises:a first primer region in the 3′ segment of the first primer oligonucleotide that can sequence-specifically bind to a strand of a nucleic acid chain to be amplified,a second region that is directly or via a linker linked to the 5′ end of the first primer region of the first primer oligonucleotide and that comprises a polynucleotide tail which is suitable for binding an activator oligonucleotide and supporting strand displacement (step c) by the activator oligonucleotide, wherein the polynucleotide tail remains substantially uncopied by polymerase under the selected reaction conditions,b) extending the first primer oligonucleotide by means of a polymerase to form a first primer extension product comprising a sequence that is complementary to the target sequence of the nucleic acid chain (a) to be amplified, a first single-stranded ...

Подробнее

Номер записи: 97

28-01-2021 дата публикации

Method for detecting nucleic acid

Номер: US20210025012A1

Автор: Kobayashi Shinichiro, Ninomiya Kenji, SHIKATA Masamitsu, TAKAOKA Naoko

Принадлежит: Shimadzu Corp

The present invention relates to a method for detecting an RNA virus in a specimen by reverse transcription-polymerase chain reaction (RT-PCR) and a kit for carrying out the method. More specifically, the present invention relates to a test method, characterized in that the purified water, saline or a buffer for mixing with a specimen to obtain a centrifugation supernatant as an analysis sample previously contain at least one element selected from internal control DNA, forward and reverse primers that specifically hybridize to the DNA, while RT-PCR reaction solution does not contain the above-mentioned element(s) contained in the purified water, saline, or buffer, and a kit for carrying out the method.

Подробнее

Номер записи: 98

24-04-2014 дата публикации

Nucleic acid probes and methods of using the same

Номер: US20140112871A1

Автор: Alexei G. Basnakian, Yevgeniy Apostolov

Принадлежит: University of Arkansas

The present invention provides a composition comprising a fluorescent nucleic acid probe that can be cleaved by enzymatic and non-enzymatic means, and methods of using the same. Advantageously, the nucleic acid probe may be used to detect and quantify nucleic acid cleavage in vitro, in situ, ex vivo, and in vivo.

Подробнее

Номер записи: 99

24-04-2014 дата публикации

Direct nucleic acid amplification kit, reagent and method

Номер: US20140113294A1

Автор: Jeffrey K. Horton, Kathryn L. Lamerton, Peter J. Tatnell

Принадлежит: GE Healthcare UK Ltd

The present invention relates to compositions, methods and kits which can be used to amplify nucleic acids with the advantage of decreasing user time and possible contamination. The dried reagent composition of the invention can be used for easy processing and amplification of nucleic acid samples.

Подробнее

Номер записи: 100