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Небесная энциклопедия

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

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Мониторинг СМИ

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

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Форма поиска

Поддерживает ввод нескольких поисковых фраз (по одной на строку). При поиске обеспечивает поддержку морфологии русского и английского языка
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Применить Всего найдено 6194. Отображено 100.
08-03-2012 дата публикации

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

Номер: US20120058914A1

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.

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04-10-2012 дата публикации

Method for analyzing rna

Номер: US20120253687A1
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.

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18-10-2012 дата публикации

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

Номер: US20120264630A1
Принадлежит: Individual

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

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22-11-2012 дата публикации

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

Номер: US20120295251A1
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.

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04-07-2013 дата публикации

Method of dna sequencing by polymerisation

Номер: US20130171636A1
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.

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05-09-2013 дата публикации

Capture of target dna and rna by probes comprising intercalator molecules

Номер: US20130230856A1
Принадлежит: Quantibact AS

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

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19-09-2013 дата публикации

Compositions and methods for sequencing nucleic acids

Номер: US20130244886A1
Принадлежит: Illumina Inc

Disclosed herein are compositions and methods for sequencing nucleic acids.

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12-12-2013 дата публикации

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

Номер: US20130331291A1

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.

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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.

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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.

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07-01-2016 дата публикации

Methods and compositions for tagging and analyzing samples

Номер: US20160001248A1
Принадлежит: Lineage Biosciences Inc USA

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

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04-01-2018 дата публикации

METHODS OF CONSTRUCTING A CIRCULAR TEMPLATE AND DETECTING DNA MOLECULES

Номер: US20180002731A1
Принадлежит: Personal Genomics, Inc.

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

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04-01-2018 дата публикации

METHODS FOR MOLECULAR DETECTION

Номер: US20180002734A1
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 ...

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02-01-2020 дата публикации

METHODS AND SYSTEMS FOR PROCESSING POLYNUCLEOTIDES

Номер: US20200002763A1
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 ( ...

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02-01-2020 дата публикации

METHODS AND SYSTEMS FOR PROCESSING POLYNUCLEOTIDES

Номер: US20200002764A1
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 ...

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03-01-2019 дата публикации

RNase H-Based Assays Utilizing Modified RNA Monomers

Номер: US20190002865A1
Принадлежит:

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

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03-01-2019 дата публикации

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

Номер: US20190002953A1

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 ...

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03-01-2019 дата публикации

NUCLEIC ACID AMPLIFICATION

Номер: US20190002957A1
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 ...

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03-01-2019 дата публикации

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

Номер: US20190002968A1
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 ...

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03-01-2019 дата публикации

DIGITAL MEASUREMENTS FROM TARGETED SEQUENCING

Номер: US20190005193A1
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 ...

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27-01-2022 дата публикации

Methods for non-invasive prenatal ploidy calling

Номер: US20220025456A1
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.

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14-01-2021 дата публикации

Labelled nucleotides

Номер: US20210009623A1
Принадлежит: Illumina Cambridge Ltd

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

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11-01-2018 дата публикации

ORGANISM IDENTIFICATION PANEL

Номер: US20180010167A1
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 ...

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11-01-2018 дата публикации

METHODS AND SYSTEMS FOR SEQUENCING LONG NUCLEIC ACIDS

Номер: US20180010180A1
Принадлежит:

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

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14-01-2021 дата публикации

Method for Introducing Mutations

Номер: US20210010008A1
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 ...

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14-01-2021 дата публикации

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

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

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

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10-01-2019 дата публикации

HIGH THROUGHPUT GENOME SEQUENCING ON DNA ARRAYS

Номер: US20190010542A1
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 ...

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10-01-2019 дата публикации

MOLECULAR ZIPPER TWEEZERS AND SPRING DEVICES

Номер: US20190010544A1
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 ...

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09-01-2020 дата публикации

METHOD OF NANOPORE SEQUENCING OF CONCATENATED NUCLEIC ACIDS

Номер: US20200010887A1
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 ...

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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 ...

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03-02-2022 дата публикации

AMPLIFICATION WITH PRIMERS OF LIMITED NUCLEOTIDE COMPOSITION

Номер: US20220033891A1
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 ...

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03-02-2022 дата публикации

Orthogonal deblocking of nucleotides

Номер: US20220033898A1
Принадлежит: Illumina Inc

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

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19-01-2017 дата публикации

Nucleic Acid Probe and Method of Detecting Genomic Fragments

Номер: US20170016065A1
Принадлежит: Vanadis Diagnostics AB

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

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18-01-2018 дата публикации

Methods and systems for processing polynucleotides

Номер: US20180016634A1
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.

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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 ...

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17-01-2019 дата публикации

Methods and systems for processing polynucleotides

Номер: US20190017115A9
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.

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21-01-2021 дата публикации

Methods for controlling gene expression

Номер: US20210017514A1
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.

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21-01-2021 дата публикации

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

Номер: US20210017587A1

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.

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21-01-2021 дата публикации

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

Номер: US20210017589A1
Принадлежит: DNAE DIAGNOSTICS LIMITED

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

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16-01-2020 дата публикации

MODIFIED NUCLEOTIDES

Номер: US20200017908A1
Принадлежит:

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

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21-01-2021 дата публикации

MEMORY WRITING

Номер: US20210017595A1
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 ...

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25-01-2018 дата публикации

Methods for Preparative In Vitro Cloning

Номер: US20180023120A1
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 ...

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25-01-2018 дата публикации

SEQUENCE BASED GENOTYPING BASED ON OLIGONUCLEOTIDE LIGATION ASSAYS

Номер: US20180023135A1
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 ...

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10-02-2022 дата публикации

Flexible and high-throughput sequencing of targeted genomic regions

Номер: US20220042096A1
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.

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10-02-2022 дата публикации

METHODS FOR NON-INVASIVE PRENATAL PLOIDY CALLING

Номер: US20220042103A1
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 ...

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24-01-2019 дата публикации

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

Номер: US20190024127A1
Автор: YEH Chen-Hsiung
Принадлежит:

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

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24-01-2019 дата публикации

Methods and compositions for isolating asymmetric nucleic acid complexes

Номер: US20190024140A1
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.

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24-01-2019 дата публикации

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

Номер: US20190024141A1
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.

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24-01-2019 дата публикации

INDEXING BASED DEEP DNA SEQUENCING TO IDENTIFY RARE SEQUENCES

Номер: US20190024150A1

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 ...

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24-01-2019 дата публикации

Detection of Nucleic Acids

Номер: US20190024160A1
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 ...

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24-01-2019 дата публикации

Methods and Systems for Processing Polynucleotides

Номер: US20190024166A1
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 ...

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24-01-2019 дата публикации

PRIMERS AND METHODS FOR THE DETECTION AND DISCRIMINATION OF NUCLEIC ACIDS

Номер: US20190024167A1
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 ...

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24-01-2019 дата публикации

HIGH INTENSITY LABELED REACTANT COMPOSITIONS AND METHODS FOR SEQUENCING

Номер: US20190024168A1
Принадлежит: Quantum-Si incorporated

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

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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 ...

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24-04-2014 дата публикации

Nucleic acid probes and methods of using the same

Номер: US20140112871A1
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.

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29-01-2015 дата публикации

Ligase-assisted nucleic acid circularization and amplification

Номер: US20150031086A1
Принадлежит: General Electric Co

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

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04-02-2016 дата публикации

Modified nucleotides

Номер: US20160032378A1
Принадлежит: Illumina Cambridge Ltd

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

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01-02-2018 дата публикации

METHODS AND COMPOSITIONS FOR DETECTING TARGET NUCLEIC ACIDS

Номер: US20180030520A1
Принадлежит:

The present invention provides compositions, apparatuses and methods for detecting one or more nucleic acid targets present in a sample. Methods of the invention include utilizing two or more ligation probes that reversibly bind a target nucleic acid in close proximity to each other and possess complementary reactive ligation moieties. When such probes have bound to the target in the proper orientation, they are able to undergo a spontaneous chemical ligation reaction that yields a ligation product that is directly detected or that is amplified to produce amplicons that are then detected. The present invention also provides methods to stabilize sample RNA so that degradation does not significantly affect the results of the analysis. 132.-. (canceled)33. A method for detecting a plurality of different target nucleic acids in a blood sample , wherein each target nucleic acid comprises a first target domain adjacent to a second target domain , said method comprising:(a) contacting the blood sample comprising the different target nucleic acids with a lysis buffer to form a reaction mixture; [ (1) a first probe domain complementary to the first target domain of one target nucleic acid in the plurality of target nucleic acids;', '(2) a first primer sequence; and', '(3) a 5′ ligation moiety; and, '(i) a first ligation probe comprising, (1) a second probe domain complementary to the second target domain of the one target nucleic acid in the plurality of target nucleic acid;', '(2) a second primer sequence; and', '(3) a 3′ ligation moiety;, '(ii) a second ligation probe comprising], '(b) contacting the reaction mixture directly with a plurality of different probe sets, each probe set comprising(c) ligating said first and second ligation probes without the use of a ligase enzyme to form a plurality of different ligation products;(d) amplifying the ligation different ligation products; and(e) detecting the presence of the ligation products34. A method for detecting a plurality ...

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01-02-2018 дата публикации

AMPLIFICATION AND ANALYSIS OF WHOLE GENOME AND WHOLE TRANSCRIPTOME LIBRARIES GENERATED BY A DNA POLYMERIZATION PROCESS

Номер: US20180030522A1
Принадлежит:

The present invention regards a variety of methods and compositions for whole genome amplification and whole transcriptome amplification. In a particular aspect of the present invention, there is a method of amplifying a genome comprising a library generation step followed by a library amplification step. In specific embodiments, the library generating step utilizes specific primer mixtures and a DNA polymerase, wherein the specific primer mixtures are designed to eliminate ability to self-hybridize and/or hybridize to other primers within a mixture but efficiently and frequently prime nucleic acid templates. 151-. (canceled)52. A kit comprising:a container; anda population of polynucleotide primers present in the container, wherein the primers of the population:(a) have sequences configured to be non-self-complementary and non-complementary to each other;(b) comprise a variable region and a constant region, wherein the constant region is positioned 5′ to the variable region; and(c) have sequences that are 70% or more comprised of two non-complementary and non-self-complementary nucleotides.53. The kit according to claim 52 , wherein the primers have sequences configured to render the primers incapable of self-hybridization.54. The kit according to claim 52 , wherein the primers have sequences configured to render the primers incapable of self-priming.55. The kit according to claim 52 , wherein the primers have sequences configured to render the primers incapable of hybridization to another primer of the kit.56. The kit according to claim 52 , wherein the primers of the population have sequences that are 75% or more comprised of two non-complementary and non-self-complementary nucleotides.57. The kit according to claim 52 , wherein the primers of the population have sequences that are 80% or more comprised of two non-complementary and non-self-complementary nucleotides.58. The kit according to claim 52 , wherein the primers of the population have sequences that are ...

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01-02-2018 дата публикации

METHODS AND COMPOSITIONS FOR GENERATING AND AMPLIFYING DNA LIBRARIES FOR SENSITIVE DETECTION AND ANALYSIS OF DNA METHYLATION

Номер: US20180030527A1
Принадлежит:

The present invention regards a variety of methods and compositions for obtaining epigenetic information, such as DNA methylation patterns, through the preparation, amplification and analysis of Methylome libraries. In particular, the method employs preparation of a DNA molecule by digesting the DNA molecule with at least one methylation-sensitive restriction enzyme; incorporating a nucleic acid molecule into at least some of the digested DNA molecules by either (1) incorporating at least one primer from a plurality of primers that have a 5′ constant sequence and a 3′ variable sequence, wherein the primers are substantially non-self-complementary and substantially non-complementary to other primers in the plurality; or (2) incorporating an oligonucleotide having an inverted repeat and a loop under conditions wherein the oligonucleotide becomes blunt-end ligated to one strand of the digested DNA molecule, followed by polymerization from a 3′ hydroxyl group present in a nick in the oligonucleotide-linked molecule; and amplifying one or more of the DNA molecules 173-. (canceled)74. A method for preparing a DNA molecule , the method comprising:a) providing a sample comprising methylated nucleic acids and non-methylated nucleic acids;b) generating a nucleic acid library from the methylated nucleic acids and the non-methylated nucleic acids by attaching hairpin adaptors;c) digesting the nucleic acid library with a methylation-sensitive restriction enzyme; andb) detecting methylation sites within nucleic acids of the digested nucleic acid library;wherein the detecting comprises detecting methylated nucleic acids that comprise less than 10% of the nucleic acids in the sample.75. The method of claim 74 , wherein the detecting comprises detecting methylated nucleic acids that comprise less than 1% of the nucleic acids in the sample.76. The method of claim 74 , wherein the detecting comprises detecting methylated nucleic acids that comprise less than 0.1° A of the nucleic ...

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01-02-2018 дата публикации

Single Cell Nucleic Acid Detection and Analysis

Номер: US20180030533A1
Принадлежит: Harvard College

Methods and compositions for digital profiling of nucleic acid sequences present in a sample are provided.

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17-02-2022 дата публикации

CHEMICALLY-ENHANCED PRIMER COMPOSITIONS, METHODS AND KITS

Номер: US20220049289A1
Автор: LEE Linda, MA Peter, WOO Sam
Принадлежит:

A chemically-enhanced primer is provided comprising a negatively charged moiety (NCM), an oligonucleotide sequence having a) non-nuclease resistant inter-nucleotide linkages or b) at least one nuclease resistance inter-nucleotide linkage. The chemically-enhanced primer can be used for sequencing and fragment analysis. Methods for synthesizing the chemically-enhanced primer as well as a method of preparing DNA for sequencing, a method of sequencing DNA, and kits containing the chemically-enhanced primer are also provided. The method of sequencing DNA can comprise contacting amplification reaction products with the composition wherein excess amplification primer is degraded by the nuclease and the chemically-enhanced primer is essentially non-degraded. 115-. (canceled)16. A chemically-enhanced primer , comprising:an oligonucleotide sequence having a) non-nuclease resistant inter-nucleotide linkages or b) at least one nuclease resistance inter-nucleotide linkage; anda negatively charged moiety (NCM), the NCM including one or more alkyl phosphate monomers.17. The chemically-enhanced primer of claim 16 , wherein the NCM is attached at a 5′ end of the chemically-enhanced primer.18. A chemically-enhanced primer claim 16 , comprising:a negatively charged moiety (NCM) disposed at a 5′ end of the chemically-enhanced primer, the NCM having a branched arrangement.1932-. (canceled)33. A composition for sequencing nucleic acid claim 16 , comprising:{'claim-ref': {'@idref': 'CLM-00016', '#text': 'claim 16'}, '#text': 'the chemically-enhanced primer according to .'}34. The composition of claim 33 , wherein the oligonucleotide sequence of the chemically-enhanced primer comprises a universal primer.35. The composition of wherein the universal primer is selected from M13 claim 34 , US1 claim 34 , T7 claim 34 , SP6 claim 34 , and T3.36. (canceled)37. The composition of claim 33 , further comprising a polymerase claim 33 , a nuclease claim 33 , a deoxynucleotide triphosphates claim 33 , ...

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17-02-2022 дата публикации

METHODS FOR DETERMINING A LOCATION OF A BIOLOGICAL ANALYTE IN A BIOLOGICAL SAMPLE

Номер: US20220049293A1
Принадлежит: 10x Genomics, Inc.

Provided herein are methods of determining a location of a biological analyte in a biological sample. 1212-. (canceled)213. A method for determining the location of an analyte in a biological sample , the method comprising:(a) providing an array, wherein the array comprises a plurality of capture probes, wherein a capture probe of the plurality of capture probes comprises (i) a spatial barcode and (ii) a capture domain;(b) contacting the array with the biological sample, wherein the capture domain of the probe binds to an analyte from the biological sample;(c) contacting a plurality of detectable probes with the analyte bound to the capture probe such that a detectable probe of the plurality of detectable probes interacts with the analyte bound to the capture probe; and(d) determining the location of the detectable probe bound to the analyte, thereby identifying the location of the analyte in the biological sample.214. The method of claim 213 , wherein the capture probe comprises one or more functional domains claim 213 , a cleavage domain claim 213 , a unique molecular identifier claim 213 , and combinations thereof.215. The method of claim 213 , wherein the array comprises one or more features.216. The method of claim 213 , wherein the biological sample is permeabilized under conditions sufficient to allow the analyte to bind the capture probe.217. The method of claim 213 , wherein the detectable probe is a fluorescently labeled detectable probe.218. The method of claim 217 , wherein determining the location of the fluorescently labeled detection probe comprises fluorescence detection.219. The method of claim 218 , wherein a fluorescently labeled probe of the plurality of fluorescently labeled probes comprises nucleic acids.220. The method of claim 219 , wherein the fluorescently labeled probe hybridizes to the analyte.221. The method of claim 220 , wherein the analyte interacts with at least 2 or more fluorescently labeled probes.222. The method of claim 220 , ...

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31-01-2019 дата публикации

MASSIVE PARALLEL METHOD FOR DECODING DNA AND RNA

Номер: US20190031704A1

This invention provides methods for attaching a nucleic acid to a solid surface and for sequencing nucleic acid by detecting the identity of each nucleotide analogue after the nucleotide analogue is incorporated into a growing strand of DNA in a polymerase reaction. The invention also provides nucleotide analogues which comprise unique labels attached to the nucleotide analogue through a cleavable linker, and a cleavable chemical group to cap the —OH group at the 3′-position of the deoxyribose. This application claims the benefit of U.S. Provisional Application No. 60/300,894, filed Jun. 26, 2001, and is a continuation-in-part of U.S. Ser. No. 09/684,670, filed Oct. 6, 2000, the contents of both of which are hereby incorporated by reference In their entireties into this application.Throughout this application, various publications are referenced in parentheses by author and year. Full citations for these references may be found at the end of the specification immediately preceding the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains.The ability to sequence deoxyribonucleic acid (DNA) accurately and rapidly is revolutionizing biology and medicine. The confluence of the massive Human Genome Project is driving an exponential growth in the development of high throughput genetic analysis technologies. This rapid technological development involving chemistry, engineering, biology, and computer science makes it possible to move from studying single genes at a time to analyzing and comparing entire genomes.With the completion of the first entire human genome sequence map, many areas in the genome that are highly polymorphic in both exons and introns will be known. The pharmacogenomics challenge is to comprehensively identify the genes and functional polymorphisms associated with the variability in drug response (Roses, 2000 ...

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31-01-2019 дата публикации

MASSIVE PARALLEL METHOD FOR DECODING DNA AND RNA

Номер: US20190031705A1

This invention provides methods for attaching a nucleic acid to a solid surface and for sequencing nucleic acid by detecting the identity of each nucleotide analogue after the nucleotide analogue is incorporated into a growing strand of DNA in a polymerase reaction. The invention also provides nucleotide analogues which comprise unique labels attached to the nucleotide analogue through a cleavable linker, and a cleavable chemical group to cap the —OH group at the 3′-position of the deoxyribose. This application claims the benefit of U.S. Provisional Application No. 60/300,894, filed Jun. 26, 2001, and is a continuation-in-part of U.S. Ser. No. 09/684,670, filed Oct. 6, 2000, the contents of both of which are hereby incorporated by reference in their entireties into this application.Throughout this application, various publications are referenced in parentheses by author and year. Full citations for these references may be found at the end of the specification immediately preceding the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains.The ability to sequence deoxyribonucleic acid (DNA) accurately and rapidly is revolutionizing biology and medicine. The confluence of the massive Human Genome Project is driving an exponential growth in the development of high throughput genetic analysis technologies. This rapid technological development involving chemistry, engineering, biology, and computer science makes it possible to move from studying single genes at a time to analyzing and comparing entire genomes.With the completion of the first entire human genome sequence map, many areas in the genome that are highly polymorphic in both exons and introns will be known. The pharmacogenomics challenge is to comprehensively identify the genes and functional polymorphisms associated with the variability in drug response (Roses, 2000 ...

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31-01-2019 дата публикации

MASSIVE PARALLEL METHOD FOR DECODING DNA AND RNA

Номер: US20190031706A1

This invention provides methods for attaching a nucleic acid to a solid surface and for sequencing nucleic acid by detecting the identity of each nucleotide analogue after the nucleotide analogue is incorporated into a growing strand of DNA in a polymerase reaction. The invention also provides nucleotide analogues which comprise unique labels attached to the nucleotide analogue through a cleavable linker, and a cleavable chemical group to cap the —OH group at the 3′-position of the deoxyribose. 2. A method for simultaneously sequencing a plurality of different nucleic acids which comprises simultaneously applying the method of to the plurality of different nucleic acids.4. A method for simultaneously sequencing a plurality of different nucleic acids which comprises simultaneously applying the method of to the plurality of different nucleic acids. This application claims the benefit of U.S. Provisional Application No. 60/300,894, filed Jun. 26, 2001, and is a continuation-in-part of U.S. Ser. No. 09/684,670, filed Oct. 6, 2000, the contents of both of which are hereby incorporated by reference in their entireties into this application.Throughout this application, various publications are referenced in parentheses by author and year. Full citations for these references may be found at the end of the specification immediately preceding the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains.The ability to sequence deoxyribonucleic acid (DNA) accurately and rapidly is revolutionizing biology and medicine. The confluence of the massive Human Genome Project is driving an exponential growth in the development of high throughput genetic analysis technologies. This rapid technological development involving chemistry, engineering, biology, and computer science makes it possible to move from studying single genes at a time to ...

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31-01-2019 дата публикации

SYNTHESIS OF DOUBLE-STRANDED NUCLEIC ACIDS

Номер: US20190032110A1
Принадлежит:

The present invention relates to a method for the synthesis of double-stranded nucleic acids from a wide variety of samples and comprises the use of these nucleic acids for deep sequence analysis. Also, the present invention relates to specific reagents used in the method of the present invention. Further, the invention relates to kits comprising reagents for the method of the invention and use of said kits. 1. A method for the synthesis of double stranded nucleic acid with a defined 3′ and 5′ terminal nucleotide sequence from a sample comprising single stranded nucleic acid comprising the steps of:a) providing a sample comprising single stranded or double stranded nucleic acid,b) adding at least 5 consecutive nucleotides to the 3-terminus of the single stranded or double stranded nucleic acid,c) hybridizing a priming oligonucleotide complementary to the added nucleotide sequence and synthesizing a cDNA or cRNA with a template dependent DNA or RNA polymerase to generate a double stranded nucleic acid,d) hybridizing a template switching oligonucleotide to said double stranded nucleic acid, ande) extending the 3′ end of the cDNA or cRNA strand to synthesize a double stranded nucleic acid, wherein one strand of the nucleic acid comprises the priming oligonucleotide, and a cDNA or a cRNA that is complementary to the single stranded nucleic acid and to the template switching oligonucleotide.2. The method according to claim 1 , wherein the sample is a liquid or solid biopsy or derived thereof.3. The method according to claim 1 , wherein the sample is obtained from fossils claim 1 , remnants of extinct organisms claim 1 , plants claim 1 , fruits and animals claim 1 , microbes claim 1 , bacteria claim 1 , or viruses.4. The method according to claim 1 , wherein the single stranded nucleic acid is DNA.5. The method according to claim 1 , wherein the consecutive nucleotides are identical claim 1 , or consist of identical dinucleotides claim 1 , trinucleotides claim 1 , ...

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31-01-2019 дата публикации

Methods and Systems for Processing Polynucleotides

Номер: US20190032129A1
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.

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31-01-2019 дата публикации

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

Номер: US20190032133A1
Принадлежит: Abbott Molecular, Inc.

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

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31-01-2019 дата публикации

HIGH PERFORMANCE MULTI-INPUT MICRORNA SENSORS AND USES THEREOF

Номер: US20190032141A1
Автор: Huh Jin, Weiss Ron
Принадлежит:

Provided herein are genetic circuits and cell state classifiers for detecting the microRNA profile of a cell. The cell state classifiers of the present disclosure are designed to incorporate multiple genetic circuits integrated together by transcriptional or translational control. Multiple inputs can be sensed simultaneously by coupling their detection to different portions of the genetic circuit such that the output molecule is produced only when the correct input profile of miRNAs is detected. The genetic circuits and cell state classifiers may be used in various applications (e.g., therapeutic or diagnostic applications). 1. A cell state classifier , the cell state classifier comprising:(i) a first sensor circuit comprising a promoter operably linked to a nucleotide sequence encoding a first activator and one or more target sites for a first set of microRNAs; (a) a promoter operably linked to a nucleotide sequence encoding a second activator and one or more target sites for a second microRNA;', '(b) a promoter that is activated by the second activator of (ii)(a), operably linked to a nucleotide sequence encoding a first repressor and one or more target sites for the second microRNA of (ii)(a); wherein the second activator is different from the first activator, and, '(ii) a second sensor circuit comprising(iii) a first signal circuit comprising a first activatable/repressible promoter that is activated by the first activator of (i) or repressed by the first repressor of (iii), operably linked to a nucleotide sequence encoding a first output molecule and one or more target sites for any one of the first set of microRNAs of (i).2. The cell state classifier of claim 1 , wherein the cell state classifier comprises a plurality of the second sensor circuit of (ii).3. The cell state classifier of claim 1 , wherein the first signal circuit of (iii) further comprises a nucleotide sequence encoding a third activator claim 1 , operably linked to the first activatable/ ...

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30-01-2020 дата публикации

METHODS AND COMPOSITIONS FOR CAPPING NUCLEIC ACIDS

Номер: US20200032322A1
Принадлежит: OMNIOME, INC.

A method for identifying a nucleic acid template that include (a) providing a plurality of primer-template hybrids, wherein a first hybrid of the plurality includes a first template hybridized to a first primer, and wherein a second hybrid of the plurality includes a second template hybridized to a second primer, the second primer having a ternary complex inhibitor moiety at the 3′ end; (b) delivering polymerases and nucleotides to the plurality, whereby the first hybrid binds a polymerase and nucleotide to form a stabilized ternary complex and whereby the second hybrid does not bind a polymerase and nucleotide to form a stabilized ternary complex; and (c) detecting the stabilized ternary complex to identify the first template. 1. A method for identifying a nucleic acid template , comprising(a) providing a plurality of primer-template hybrids, wherein a first hybrid of the plurality comprises a first template hybridized to a first primer, and wherein a second hybrid of the plurality comprises a second template hybridized to a second primer, the second primer comprising a ternary complex inhibitor moiety at the 3′ end;(b) delivering polymerases and nucleotides to the plurality, whereby the first hybrid binds a polymerase and nucleotide to form a stabilized ternary complex and whereby the second hybrid does not bind a polymerase and nucleotide to form a stabilized ternary complex; and(c) detecting the stabilized ternary complex to identify the first template.2. The method of claim 1 , further comprising (d) contacting the plurality with a primer extension reagent claim 1 , whereby the plurality of primer-template hybrids is modified by extending the first primer but not the second primer.3. The method of claim 2 , further comprising repeating (b) claim 2 , (c) and (d) for the modified plurality of primer-template hybrids claim 2 , thereby determining a sequence for at least a portion of the first template.4. The method of claim 3 , wherein (b) claim 3 , (c) and (d) ...

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30-01-2020 дата публикации

GENETIC MARKERS FOR DISCRIMINATION AND DETECTION OF VIRAL HEMORRHAGIC SEPTICEMIA VIRUS CAUSING INFECTIOUS AQUATIC ORGANISM DISEASES, AND METHOD OF DISCRIMINATING AND DETECTING THE VIRUS USING THE SAME

Номер: US20200032327A1
Принадлежит:

Genetic markers are described for discriminating or detecting viruses causing infectious aquatic organism diseases, and a method of discriminating and detecting the viruses using the same is disclosed, in which the method includes selecting and amplifying a DNA nucleotide sequence encoding a gene specific for viral hemorrhagic septicemia virus (VHSV), red sea bream iridovirus (RSIV) or infectious spleen and kidney necrosis virus (ISKNV), which is a virus causing red sea bream iridovirus disease, or Koi herpesvirus (KHV); hybridizing a peptide nucleic acid (PNA) that specifically recognizes the amplification product; controlling the temperature of the hybridization product to obtain a temperature-dependent melting curve; and discriminating the viral type or detecting whether or not fish are infected with the viral type by analyzing the obtained melting curve to determine a melting temperature. 1. A PNA probe consisting of SEQ ID NO: 2 , for discrimination or detection of a virus causing red sea bream iridovirus disease which is a virus causing infectious aquatic organism disease , wherein the virus causing red sea bream iridovirus disease is red sea bream iridovirus (RSIV) or infectious spleen and kidney necrosis virus (ISKNV).2. A PNA probe consisting of SEQ ID NO: 2 with one or more of a reporter and a quencher attached thereto , wherein the probe is for discrimination or detection of a virus causing red sea bream iridovirus disease which is a virus causing infectious aquatic organism disease.3. A composition for discrimination or detection of a virus causing red sea bream iridovirus disease , the composition comprising: a primer consisting of SEQ ID NO: 7 and a PNA probe consisting of SEQ ID NO: 2.4. A kit for discrimination or detection of a virus causing red sea bream iridovirus disease , the kit comprising: a primer consisting of SEQ ID NO: 7; and a PNA probe consisting of SEQ ID NO: 2.5. A method for detecting a virus causing red sea bream iridovirus disease , ...

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30-01-2020 дата публикации

GENETIC MARKERS FOR DISCRIMINATION AND DETECTION OF VIRAL HEMORRHAGIC SEPTICEMIA VIRUS CAUSING INFECTIOUS AQUATIC ORGANISM DISEASES, AND METHOD OF DISCRIMINATING AND DETECTING THE VIRUS USING THE SAME

Номер: US20200032328A1
Принадлежит:

Genetic markers are described for discriminating or detecting viruses causing infectious aquatic organism diseases, and a method of discriminating and detecting the viruses using the same is disclosed, in which the method includes selecting and amplifying a DNA nucleotide sequence encoding a gene specific for viral hemorrhagic septicemia virus (VHSV), red sea bream iridovirus (RSIV) or infectious spleen and kidney necrosis virus (ISKNV), which is a virus causing red sea bream iridovirus disease, or Koi herpesvirus (KHV); hybridizing a peptide nucleic acid (PNA) that specifically recognizes the amplification product; controlling the temperature of the hybridization product to obtain a temperature-dependent melting curve; and discriminating the viral type or detecting whether or not fish are infected with the viral type by analyzing the obtained melting curve to determine a melting temperature. 1. A PNA probe consisting of SEQ ID NO: 5 , for discrimination or detection of Koi herpesvirus (KHV) which is a virus causing infectious aquatic organism disease.2. A PNA probe consisting of SEQ ID NO: 5 with one or more of a reporter and a quencher attached thereto , wherein the probe is for discrimination or detection of Koi herpesvirus (KHV) which is a virus causing infectious aquatic organism disease.3. A composition for discrimination or detection of Koi herpesvirus (KHV) , the composition comprising: a primer consisting of SEQ ID NO: 10 and a PNA probe consisting of SEQ ID NO: 5.4. A kit for discrimination or detection of Koi herpesvirus (KHV) , the kit comprising: a primer consisting of SEQ ID NO: 10; and a PNA probe consisting of SEQ ID NO: 5.5. A method for detecting Koi herpesvirus (KHV) , the method comprising the steps of:(a) extracting a target nucleic acid from a fish sample;(b) amplifying a genetic marker nucleotide sequence for Koi herpesvirus (KHV) contained in the target nucleic acid by use of a primer pair capable of amplifying a fragment consisting of SEQ ID ...

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04-02-2021 дата публикации

DNA-TAGGED METHANOL RESPONSIVE POLYMER FOR SINGLE-STRANDED NUCLEIC ACID PRODUCTION

Номер: US20210032683A1
Принадлежит: Dana-Farber Cancer Institute, Inc.

Provided herein, in some embodiments, are methods and compositions for the production of long single-stranded DNA. 1. A method for producing single-stranded deoxyribonucleic acid (DNA) , the method comprising:(a) amplifying a DNA template in a polymerase chain reaction mixture that comprises the DNA template, a pair of primers, at least one of which is tagged with a MeRPy (a MeRPy-tagged primer) and comprises a cleavage site, a polymerase, and deoxynucleoside triphosphates to produce a double-stranded MeRPy-tagged amplicon; and(b) separating the double-stranded MeRPy-tagged amplicon into an untagged DNA strand and a MeRPy-tagged DNA strand comprising the cleavage site, and optionally isolating the untagged DNA strand.2. A method for producing single-stranded deoxyribonucleic acid (DNA) , the method comprising:(a) copolymerizing a 5′-acrylamide-tagged single-strand DNA (ssDNA) primer with acrylamide monomers to produce a methanol-responsive polymer (MeRPy)-tagged primer, wherein the ssDNA forward primer comprises a cleavage site;(b) optionally purifying the MeRPy-tagged forward primer;(c) amplifying a DNA template in a polymerase chain reaction mixture that comprises the DNA template, a pair of primers, at least one of which is tagged with a MeRPy (a MeRPy-tagged primer), a polymerase, and deoxynucleoside triphosphates to produce a double-stranded MeRPy-tagged amplicon; and(d) separating the double-stranded MeRPy-tagged amplicon into an untagged DNA strand and a MeRPy-tagged DNA strand comprising the cleavage site, and optionally isolating the untagged DNA strand.3. The method of or further comprising cleaving the MeRPy-tagged DNA strand at the cleavage site to produce MeRPy-tagged primer and single-stranded DNA.4. The method of further comprising isolating the MeRPy-tagged primer and/or the single-stranded DNA.5. The method of any one of - claim 4 , wherein the cleavage site is a 3′-deoxyuridine (dU).6. The method of any one of claim 4 , claim 4 , or claim 4 , ...

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11-02-2016 дата публикации

Methods of sequencing the immune repertoire

Номер: US20160040234A1
Принадлежит: Lineage Biosciences Inc USA

The invention provides a non-invasive technique for the detection and quantification of the immune repertoire, in a biological sample containing a plurality of distinct cell populations. Methods are conducted using sequencing technology to detect and enumerate immune repertoire within a heterogeneous biological sample.

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08-02-2018 дата публикации

CHEMICALLY-ENHANCED PRIMER COMPOSITIONS, METHODS AND KITS

Номер: US20180037931A1
Принадлежит:

A chemically-enhanced primer is provided comprising a negatively charged moiety (NCM), an oligonucleotide sequence having a) non-nuclease resistant inter-nucleotide linkages or b) at least one nuclease resistance inter-nucleotide linkage. The chemically-enhanced primer can be used for sequencing and fragment analysis. Methods for synthesizing the chemically-enhanced primer as well as a method of preparing DNA for sequencing, a method of sequencing DNA, and kits containing the chemically-enhanced primer are also provided. The method of sequencing DNA can comprise contacting amplification reaction products with the composition wherein excess amplification primer is degraded by the nuclease and the chemically-enhanced primer is essentially non-degraded. 115-. (canceled)16. A chemically-enhanced primer comprising:an oligonucleotide sequence having a) non-nuclease resistant inter-nucleotide linkages or b) at least one nuclease resistance inter-nucleotide linkage; anda negatively charged moiety (NCM).20. The chemically-enhanced primer of claim 19 , wherein the chemically-enhanced primer is fluorescently labeled.21. The chemically-enhanced primer of claim 19 , wherein the chemically-enhanced primer is not fluorescently labeled.22. The chemically-enhanced primer of claim 19 , wherein W is H or OH.23. The chemically-enhanced primer of claim 19 , wherein n is 3 or 6.24. The chemically-enhanced primer of claim 19 , wherein each instance of n is independently 3 or 6.25. The chemically-enhanced primer of claim 24 , wherein when x is greater than 5 claim 24 , a first plurality of n is selected to be 3 claim 24 , and a second plurality of n is selected to be 6.26. The chemically-enhanced primer of claim 19 , wherein x is 5 claim 19 , 8 claim 19 , 9 claim 19 , 10 claim 19 , or 15.27. The chemically-enhanced primer of claim 19 , wherein z is an integer of 5 to 30.28. The chemically-enhanced primer of claim 19 , having an OLIGO portion wherein OLIGO comprises a universal primer.29. ...

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08-02-2018 дата публикации

TRANSPOSON NUCLEIC ACIDS COMPRISING A CALIBRATION SEQUENCE FOR DNA SEQUENCING

Номер: US20180037935A1
Принадлежит:

Transposon nucleic acids comprising a transposon end sequence and a calibration sequence for DNA sequencing in the transposon end sequence. In one embodiment, the transposon end sequence is a Mu transposon end. A method for the generation of DNA fragmentation library based on a transposition reaction in the presence of a transposon end with the calibration sequence providing facilitated downstream handling of the produced DNA fragments, e.g., in the generation of sequencing templates. 1. A method for in vitro assembly of a plurality of transpososome complexes , comprising:a) contacting under a condition for forming a transpososome complex (i) a plurality of MuA transposase enzymes, (ii) a plurality of modified MuA transposon end sequences each including a calibration sequence and comprising the nucleotide sequence of SEQ ID NO:5, and (iii) a plurality of target DNA.2. The method of claim 1 , wherein the in vitro assembly of the plurality of transpososome complexes lack magnesium.3. The method of claim 1 , wherein the in vitro assembly of the plurality of transpososome complexes further comprises magnesium.4. The method of claim 3 , further comprising: incubating the plurality of transposome complexes under a condition for performing a transposition reaction claim 3 , wherein the transposition reaction results in fragmentation of the plurality of target DNA and incorporation of a MuA transposon end sequence from the plurality of MuA transposon end sequences into the ends of the fragmented target DNA claim 3 , thereby generating a plurality of target DNA fragments attached at both ends to a modified MuA transposon end sequence.5. The method of claim 4 , wherein the modified MuA transposon end sequences which are attached to both ends of the target DNA fragments further comprise a cleavage site.6. The method of claim 5 , further comprising: incubating the plurality of target DNA fragments with an enzyme that cleaves the cleavage site so that the modified MuA transposon ...

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08-02-2018 дата публикации

METHOD OF DNA SYNTHESIS

Номер: US20180037943A1
Принадлежит:

The present invention relates to an in vitro cell-free process for production of deoxyribonucleotides (DNAs) comprising at least one hairpin, corresponding DNA products and uses thereof, and oligonucleotides and kits useful in the process of the invention. 1. An in vitro cell-free process for production of a deoxyribonucleic acid (DNA) molecule which comprises a desired DNA sequence , the process comprising:(a) contacting an oligonucleotide immobilised on a solid support with a series of template oligonucleotides which overlap in sequence, in the presence of at least one DNA polymerase under conditions promoting template-dependent extension of the immobilised oligonucleotide to produce a first DNA strand which comprises the desired DNA sequence and a first portion of a protelomerase target sequence proximal to the solid support;(b) introducing a DNA sequence comprising a second portion of the protelomerase target sequence into a distal end of the first DNA strand, or into a second DNA strand, such that the first and second portions of the protelomerase target sequence thereby create the protelomerase target sequence proximal to the solid support; and(c) contacting the protelomerase target sequence proximal to the solid support with a protelomerase to thereby produce and release the DNA molecule from immobilisation.2. The process according to claim 1 , wherein the immobilised oligonucleotide of (a) comprises the first portion of the protelomerase target sequence.3. The process according to claim 1 , wherein the first portion of the protelomerase target sequence is introduced into the first DNA strand by template-dependent extension.4. The process according to claim 1 , wherein the DNA molecule is a single-stranded circular DNA molecule comprising at least one hairpin that comprises the first portion or the second portion of the protelomerase target sequence.5. The process according to claim 4 , wherein step (b) comprises template-dependent extension with one or more ...

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08-02-2018 дата публикации

NUCLEIC ACID AMPLIFICATION AND LIBRARY PREPARATION

Номер: US20180037946A1
Принадлежит: SIGMA-ALDRICH CO. LLC

Processes and kits for preparing a plurality of amplification products with reduced non-specific amplification artifacts. 1. A process for preparing a plurality of amplified nucleic acid products with reduced non-specific amplification artifacts , the process comprising:a) contacting one or more nucleic acids with at least one pair of amplification primers under amplification conditions to form a plurality of amplification products, each amplification primer comprising at least three nuclease-resistant groups in its 3′ region;b) contacting the plurality of amplification products with a 5′ to 3′ exonuclease and either a 3′ to 5′ single strand-specific exonuclease or a proofreading polymerase to form a plurality of digested amplification products,wherein the plurality of digested amplification products has a reduced level of non-specific amplification artifacts as compared to a standard nucleic acid amplification reaction performed with primers devoid of nuclease-resistant groups.2. The process of claim 1 , wherein each of the plurality of digested amplification products has a phosphate group at its 5′ end.3. The process of claim 1 , wherein each of the plurality of digested amplification products is blunt-ended or each of the plurality of digested amplification products has a 3′ overhang.4. The process of claim 1 , wherein the nuclease-resistant groups are chosen from phosphorothioate bonds claim 1 , 2′ O-methyl groups claim 1 , 2′ fluoro groups claim 1 , or a combination thereof.5. The process of claim 1 , wherein the 5′ to 3′ exonuclease is chosen from T7 exonuclease claim 1 , T5 exonuclease claim 1 , lambda exonuclease claim 1 , RecJ claim 1 , or a combination thereof.6. The process of claim 1 , wherein the 3′ to 5′ single strand-specific exonuclease is chosen from exonuclease I claim 1 , exonuclease T claim 1 , or a combination thereof.7. The process of claim 1 , wherein the proofreading polymerase is chosen from T4 DNA polymerase claim 1 , Klenow DNA polymerase ...

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08-02-2018 дата публикации

KITS AND METHODS FOR PATHOGEN DETECTION

Номер: US20180037947A1
Принадлежит:

Kits and methods for detecting pathogens without the need for laboratory equipment are disclosed. The kits and methods described herein allow for near-room temperature amplification of pathogen polynucleotides in a biological sample in a one-compartment reaction vessel. The kits and methods may be used to detect any target nucleic acid, such as DNA or RNA from a bacterial, fungal, or viral pathogen. 1. A kit for detection of a pathogen polynucleotide in a biological sample comprising:(a) a reaction vessel comprising: (1) a padlock probe comprising a 5′ end complementary to a first section of the pathogen polynucleotide and a 3′ end complementary to a second section of the pathogen polynucleotide, wherein the first section and second section of the pathogen polynucleotide sequence are located adjacent to each other; (2) a ligase that anneals the 5′ and 3′ ends of the padlock probe together to form a circular padlock probe; (3) a primer comprising a polynucleotide complementary to a portion of the padlock probe; (4) a polymerase; and (5) a reporter probe;(b) a reaction buffer that supports polynucleotide ligation and polymerization; and(c) a test strip.2Chlamydia tracomatisNeisseria gonorrhoeae.. The kit of claim 1 , wherein the pathogen is selected from the group consisting of human papillomavirus claim 1 , claim 1 , and3. The kit of or claim 1 , wherein the padlock probe comprises a polynucleotide sequence selected from the group consisting of SEQ ID NO:2 claim 1 , SEQ ID NO:3 claim 1 , SEQ ID NO:4 claim 1 , SEQ ID NO:8 claim 1 , SEQ ID NO:9 claim 1 , SEQ ID NO:10 claim 1 , SEQ ID NO:14 claim 1 , SEQ ID NO:15 claim 1 , SEQ ID NO:16 claim 1 , SEQ ID NO:20 claim 1 , and a variant having at least 90% sequence identity to any of the foregoing.4. The kit of any of - claim 1 , wherein the padlock probe comprises the polynucleotide sequences of SEQ ID NO:3 and SEQ ID NO:4 claim 1 , SEQ ID NO:9 and SEQ ID NO:10 claim 1 , SEQ ID NO:15 and SEQ ID NO:16 claim 1 , or variants ...

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24-02-2022 дата публикации

Analysis of Chromatin Using a Nicking Enzyme

Номер: US20220056537A1
Принадлежит: NEW ENGLAND BIOLABS, INC.

Provided herein, among other things, are various compositions and methods for analyzing chromatin. In some embodiments, the composition may comprise a mixture of a nicking enzyme, four dNTPs, at least one labeled dNTP and, optionally, a polymerase. In some embodiments, this method may comprise: obtaining a sample comprising chromatin, reacting the sample with the composition to selectively label the open chromatin in the sample, and analyzing the labeled sample. 142.-. (canceled)43. A method for detecting abnormal cells in or from a tissue section or biopsy wherein the abnormal cells have altered chromatin compared to normal cells , comprising:(a) reacting the cells with a composition comprising a mixture of a nicking enzyme, four dNTPs, and at least one labeled dNTP, and a polymerase to selectively label the chromatin;(b) incorporating the labeled dNTP into the chromatin of the cells to form labelled nucleic acid; and(c) analyzing the pattern of labelled dNMPs in the labeled cells.44. The method according to claim 43 , wherein (c) further comprises determining whether the pattern of labelled dNMPs correspond to a cancer diagnosis of the cells or tissues.45. The method according to claim 43 , wherein prior to (a) claim 43 , making the cells permeable.46. The method according to claim 43 , wherein the tissue section or biopsy is fixed.47. The method according to claim 45 , wherein the tissue section or biopsy is fixed.48. The method according to claim 43 , wherein after (b) reverse cross linking and isolating DNA from the cells.49. The method according to claim 43 , wherein following (b) claim 43 , labelling nuclei from the cells with a primary antibody.50. The method according to claim 49 , wherein after labelling with a primary antibody claim 49 , labelling with a secondary antibody.51. The method according to claim 49 , further comprising: visualizing the labelled nuclei using a fluorescent microscope. The mammalian genome is largely packaged into chromatin ...

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07-02-2019 дата публикации

IDENTIFICATION OF GENETIC MODIFICATIONS

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

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

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06-02-2020 дата публикации

METHODS OF IDENTIFYING AND CHARACTERIZING GENE EDITING VARIATIONS IN NUCLEIC ACIDS

Номер: US20200040378A1
Принадлежит: PIONEER HI-BRED INTERNATIONAL, INC.

Compositions and methods of identifying and characterizing potential gene editing on-target and off-target sites and/or edits in a nucleic acid are provided. 1. A method of identifying and characterizing variations of an edited polynucleotide , comprising:(a) creating at least one edit in a polynucleotide at an intended target site,(b) capturing the polynucleotide that comprises the intended target site, an off-target site, or a combination thereof,(c) amplifying the captured polynucleotide of (b) to create a pool of polynucleotides,(d) sequencing the pool of (c), and(e) assessing the pool of polynucleotides to identify sequences corresponding to the intended target site, sequences corresponding to an off-target site, or a combination thereof.2. The method of claim 1 , further comprising generating nucleic acid fragments of the captured polynucleotide and recovering said fragments to create an enriched DNA pool.3. The method of claim 2 , further comprising characterizing the sequence composition of the enriched DNA pool to determine the nature of the enriched pool.4. The method of claim 3 , wherein the nature of the enriched pool comprises the composition and abundance of each sequenced fragment species.513.-. (canceled)14. The method of claim 1 , wherein the polynucleotide in step (b) is captured by a molecule that binds to said polynucleotide but lacks substantial nuclease activity wherein the molecule further comprises a tag for protein purification or isolation.15. The method of claim 14 , wherein said molecule is a deactivated Cas9 (dCas9) claim 14 , wherein the dCas9 is complexed with a guide polynucleotide corresponding to a target sequence of interest on the polynucleotide of step (a).16. (canceled)17. The method of claim 14 , wherein said tag is selected from the group consisting of: His tag claim 14 , FLAG tag claim 14 , HA tag claim 14 , chitin binding protein (CBP) tag claim 14 , maltose binding protein (MBP) tag claim 14 , glutathione-S-transferase (GST ...

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06-02-2020 дата публикации

GENETIC MARKERS FOR DISCRIMINATION AND DETECTION OF VIRAL HEMORRHAGIC SEPTICEMIA VIRUS CAUSING INFECTIOUS AQUATIC ORGANISM DISEASES, AND METHOD OF DISCRIMINATING AND DETECTING THE VIRUS USING THE SAME

Номер: US20200040383A1
Принадлежит:

Genetic markers are described for discriminating or detecting viruses causing infectious aquatic organism diseases, and a method of discriminating and detecting the viruses using the same is disclosed, in which the method includes selecting and amplifying a DNA nucleotide sequence encoding a gene specific for viral hemorrhagic septicemia virus (VHSV), red sea bream iridovirus (RSIV) or infectious spleen and kidney necrosis virus (ISKNV), which is a virus causing red sea bream iridovirus disease, or Koi herpesvirus (KHV); hybridizing a peptide nucleic acid (PNA) that specifically recognizes the amplification product; controlling the temperature of the hybridization product to obtain a temperature-dependent melting curve; and discriminating the viral type or detecting whether or not fish are infected with the viral type by analyzing the obtained melting curve to determine a melting temperature. 1. A PNA probe consisting of SEQ ID NO: 1 , for discrimination or detection of viral hemorrhagic septicemia virus (VHSV) which is a virus causing infectious aquatic organism disease.2. A PNA probe consisting of SEQ ID NO: 1 with one or more of a reporter and a quencher attached thereto , wherein the probe is for discrimination or detection of viral hemorrhagic septicemia virus (VHSV) which is a virus causing infectious aquatic organism disease.3. A composition for discrimination or detection of viral hemorrhagic septicemia virus (VHSV) , the composition comprising: a primer consisting of SEQ ID NO: 6 and a PNA probe consisting of SEQ ID NO: 1.4. A kit for discrimination or detection of viral hemorrhagic septicemia virus (VHSV) , the kit comprising: a primer consisting of SEQ ID NO: 6; and a PNA probe consisting of SEQ ID NO: 1.5. A method for detecting viral hemorrhagic septicemia virus (VHSV) , the method comprising the steps of:(a) extracting a target nucleic acid from a fish sample;(b) amplifying a genetic marker nucleotide sequence for viral hemorrhagic septicemia virus ( ...

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06-02-2020 дата публикации

GENETIC MARKERS FOR DISCRIMINATION AND DETECTION OF VIRAL HEMORRHAGIC SEPTICEMIA VIRUS CAUSING INFECTIOUS AQUATIC ORGANISM DISEASES, AND METHOD OF DISCRIMINATING AND DETECTING THE VIRUS USING THE SAME

Номер: US20200040384A1
Принадлежит:

Genetic markers are described for discriminating or detecting viruses causing infectious aquatic organism diseases, and a method of discriminating and detecting the viruses using the same is disclosed, in which the method includes selecting and amplifying a DNA nucleotide sequence encoding a gene specific for viral hemorrhagic septicemia virus (VHSV), red sea bream iridovirus (RSIV) or infectious spleen and kidney necrosis virus (ISKNV), which is a virus causing red sea bream iridovirus disease, or Koi herpesvirus (KHV); hybridizing a peptide nucleic acid (PNA) that specifically recognizes the amplification product; controlling the temperature of the hybridization product to obtain a temperature-dependent melting curve; and discriminating the viral type or detecting whether or not fish are infected with the viral type by analyzing the obtained melting curve to determine a melting temperature. 1. A PNA probe consisting of SEQ ID NO: 3 , for discrimination or detection of red sea bream iridovirus (RSIV) which is a virus causing infectious aquatic organism disease.2. A PNA probe consisting of SEQ ID NO: 3 with one or more of a reporter and a quencher attached thereto , wherein the probe is for discrimination or detection of red sea bream iridovirus (RSIV) which is a virus causing infectious aquatic organism disease.3. A composition for discrimination or detection of red sea bream iridovirus (RSIV) , the composition comprising: a primer consisting of SEQ ID NO: 8 and a PNA probe consisting of SEQ ID NO: 3.4. A kit for discrimination or detection of Red sea bream iridovirus (RSIV) , the kit comprising: a primer consisting of SEQ ID NO: 8; and a PNA probe consisting of SEQ ID NO: 3.5. A method for detecting Red sea bream iridovirus (RSIV) , the method comprising the steps of:(a) extracting a target nucleic acid from a fish sample;(b) amplifying a genetic marker nucleotide sequence for Red sea bream iridovirus (RSIV) contained in the target nucleic acid by use of a primer ...

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06-02-2020 дата публикации

NOVEL SPIKE-IN OLIGONUCLEOTIDES FOR NORMALIZATION OF SEQUENCE DATA

Номер: US20200040393A1

The invention relates to novel spike-in oligonucleotides specifically for use in normalization of small RNA sequence data. The invention specifically provides sets each comprising at least two subsets of single stranded nucleic acid molecules, each nucleic acid molecule comprising a 5′ phosphate, a sequence of at least 3 randomized nucleotides, a core sequence of at least 8 core nucleotides containing at least one mismatch compared to a target sequence, a sequence of at least 3 randomized nucleotides, and a 3′ modification, wherein each subset comprises a plurality of nucleic acid molecules having an identical core nucleotide sequence and different randomized nucleotides, and wherein the nucleic acid molecules of each subset differ in at least one nucleotide of the core nucleotide sequence and the generation of a library containing the sets. The invention also relates to reference values in nucleotide sequencing and a method for determining the amount of target sequences in a sample. 1. A set comprising at least two subsets of single stranded nucleic acid molecules , each nucleic acid molecule comprising from the 5′ to 3′ direction:a) a 5′ phosphate,b) a sequence of at least 3 randomized nucleotides,c) a core sequence of at least 8 nucleotides which sequence contains two or more mismatches compared to a target sequence,d) a sequence of at least 3 randomized nucleotides, ande) a 3′ modification,wherein each of the at least two subsets comprises a plurality of nucleic acid molecules having an identical core nucleotide sequence and different randomized nucleotides, andwherein the nucleic acid molecules of each subset differ in at least one nucleotide of the core nucleotide sequence.2. The set according to claim 1 , wherein the plurality of nucleic acid molecules comprises randomized nucleotide sequences containing all four nucleotide combinations of A claim 1 , C claim 1 , G claim 1 , U or A claim 1 , C claim 1 , G claim 1 , T.3. The set according to claim 1 , wherein ...

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15-02-2018 дата публикации

Methods for Isolation, Identification, and Quantification of miRNAs

Номер: US20180044371A1
Принадлежит:

Method and compositions and kits for isolation, identification, and quantification of miRNAs and other small RNAs, including but not limited to, siRNAs, mRNAs, and snRNAs are disclosed. Methods of diagnosing a disease or its progression are also disclosed. 1. A method for isolating , identifying , or quantifying an miRNA from a sample of interest , comprising:a) contacting the sample of interest with an anti-miRNA probe covalently attached to a bead;b) incubating the sample of interest—bead mixture under suitable conditions to form hybridized complexes between the miRNA in the sample of interest and the anti-miRNA probes on the beads;c) washing the beads under suitable conditions to remove the unbound sample material; andd) isolating, identifying, or quantifying the miRNA bound to the anti-miRNA probes.2. The method according to claim 1 , wherein the miRNA is isolated from the anti-miRNA probe beads before identifying or quantifying the miRNA.3. The method according to claim 1 , wherein the identifying or quantifying the miRNA comprises using reverse transcription followed by real-time (RT) or quantitative polymerase chain reaction (q-PCR).4. The method according to claim 1 , wherein the beads are magnetic beads.5. The method according to claim 4 , wherein the beads are carboxylic acid beads.6. The method according to claim 4 , wherein the beads are DNA beads.7. The method according to claim 6 , wherein the beads are P1 DNA beads.8. The method according to claim 1 , wherein the anti-miRNA probe is attached to the beads using chemical synthesis.9. The method according to claim 1 , wherein the anti-miRNA probe is attached to the beads using enzymatic synthesis.10. The method according to claim 9 , wherein the enzymatic synthesis comprises:a) taking an oligonucleotide that has a sequence complementary to an miRNA of interest (anti-miRNA probe);b) adding a terminal didexoynucleotide to the 3′ end using a terminal transferase;c) adding a 5′-phosphate group to the ...

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15-02-2018 дата публикации

METHODS, COMPOSITIONS, AND KITS FOR GENERATING rRNA-DEPLETED SAMPLES OR ISOLATING rRNA FROM SAMPLES

Номер: US20180044660A1
Автор: Sooknanan Roy R.
Принадлежит:

The present invention provides methods, compositions, and kits for generating rRNA-depleted samples and for isolating rRNA from samples. In particular, the present invention provides compositions comprising affinity-tagged antisense rRNA molecules corresponding to substantially all of at least one rRNA molecule (e.g., 28S, 26S, 25S, 18S, 5.8S and 5S eukaryotic cytoplasmic rRNA molecules, 12S and 16S eukaryotic mitochondrial rRNA molecules, and 23S, 16S and 5S prokaryotic rRNA molecules) and methods for using such compositions to generate rRNA-depleted samples or to isolate rRNA molecules from samples. 1. A composition comprising antisense rRNA molecules complementary to at least 95% of a full-length sequence of at least one rRNA molecule selected from the group consisting of 28S , 26S , 25S , 18S , 5.8S , and 5S eukaryotic cytoplasmic rRNA molecules , 12S and 16S eukaryotic mitochondrial rRNA molecules , 23S , 16S , 4.5S , and 5S eukaryotic chloroplast rRNA molecules , and 23S , 16S , and 5S prokaryotic rRNA molecules , wherein the antisense rRNA molecules comprise affinity tags at a ratio of at least eight affinity tags per every 100 nucleobases of the antisense rRNA molecules.2. A method of generating a rRNA-depleted sample from an initial sample comprising: i) an initial sample comprising RNA molecules derived from at least one eukaryotic organism or species, wherein said RNA molecules comprise rRNA molecules and non-rRNA RNA molecules;', 'ii) a composition comprising antisense rRNA molecules complementary to at least 95% of at least one rRNA molecule selected from the group consisting of: 25S, 26S, 28S, 18S, 5.8S, and 5S eukaryotic cytoplasmic rRNA molecules and 12S and 16S eukaryotic mitochondrial rRNA molecules, wherein said antisense rRNA molecules comprise affinity-tags at a ratio of at least one affinity-tag per every 10 nucleobases of said antisense rRNA molecules; and', 'iii) a binding matrix comprising affinity-tag-binding molecules;, 'a) providing;'}b) ...

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03-03-2022 дата публикации

Resolving spatial arrays using deconvolution

Номер: US20220064630A1
Принадлежит: 10X Genomics Inc

Methods for determining a location of a feature on a spatial array include (a) providing an array of features on a substrate, where a feature of the array includes a barcoded oligonucleotide having, in a 5′ to 3′ direction, a spatial barcode, a cleavage domain, and a constant sequence; (b) hybridizing a priming oligonucleotide to the constant sequence; (c) extending the priming oligonucleotide using the barcoded oligonucleotide as a template; and (d) determining all or a portion of a sequence of the extended priming oligonucleotide corresponding to the spatial barcode, or a complement thereof, and a location of the extended priming oligonucleotide, and using the location of the extended priming oligonucleotide to determine the location of the feature on the spatial array.

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03-03-2022 дата публикации

Polymerase Chain Reaction Primers and Probes for Mycobacterium Tuberculosis

Номер: US20220064715A1

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

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08-05-2014 дата публикации

Mapping Cytosine Modifications

Номер: US20140127678A1
Принадлежит: New England Biolabs Inc

Methods, compositions and kits for selectively altering and detecting modified cytosine residues are provided.

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08-05-2014 дата публикации

Methods and compositions for nucleic acid amplification

Номер: US20140127700A1
Принадлежит: Gen Probe Inc

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

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14-02-2019 дата публикации

Methods for sample preparation

Номер: US20190048334A1
Автор: Joseph Dunham

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

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14-02-2019 дата публикации

IMPROVED AMPLIFICATION AND SEQUENCING METHODS

Номер: US20190048335A1
Принадлежит:

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

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14-02-2019 дата публикации

ELIMINATION OF PRIMER-PRIMER INTERACTIONS DURING PRIMER EXTENSION

Номер: US20190048410A1
Автор: Godwin Brian
Принадлежит:

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

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25-02-2021 дата публикации

Method for quickly homogenizing circular dna samples

Номер: US20210054443A1

The present application provides a method for quickly homogenizing circular DNA samples, comprising performing rolling circle amplification on circular DNAs in the samples, so that the concentration of the circular DNAs in the samples are homogenized.

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25-02-2021 дата публикации

METHODS OF IDENTIFYING COMBINATIONS OF TRANSCRIPTION FACTORS

Номер: US20210054448A1

Provided herein, in some embodiments, are methods and compositions for identifying combinations of transcription factors, for example, those involved in cell type conversion processes, such as cell differentiation. 1. A method of identifying at least one transcription factor , comprising: wherein each cargo element comprises', '(i) a first promoter operably linked to a first nucleotide sequence encoding a transcription factor and a barcode, and', '(ii) a terminator having a 5′ end,, '(a) contacting cells that comprise a transposase with a population of nucleic acids, wherein each nucleic acid comprises a cargo element flanked by terminal repeat sequences that are recognized by the transposase,'}wherein the barcode uniquely identifies the transcription factor and is located within 100 nucleotides of the 5′ end of the terminator;(b) detecting differences in gene expression in the cells compared to control cells and identifying differentiated cells; and(d) detecting one or more barcodes in the differentiated cells and identifying a transcription factor or a combination of transcription factors in the differentiated cells.2. The method of claim 1 , wherein the barcode is located within 50 nucleotides of the 5′ end of the terminator.3. The method of or claim 1 , wherein the transposase is a piggyBAC™ transposase.4. The method of or claim 1 , wherein the terminal repeat sequences are long terminal repeat sequences.5. The method of any one of - claim 1 , wherein the terminal repeat sequences are inverted terminal repeat sequences.6. The method of any one of - claim 1 , wherein each nucleic acid further comprises a second promoter operably linked to a second nucleotide sequence encoding a selection marker.7. The method of any one of - claim 1 , wherein each nucleic acid further comprises a second promoter operably linked to a second nucleotide sequence encoding an inducing agent.8. The method of any one of - claim 1 , wherein each nucleic acid further comprises a second ...

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23-02-2017 дата публикации

Safe Sequencing System

Номер: US20170051347A1
Принадлежит: JOHNS HOPKINS UNIVERSITY

The identification of mutations that are present in a small fraction of DNA templates is essential for progress in several areas of biomedical research. Though massively parallel sequencing instruments are in principle well-suited to this task, the error rates in such instruments are generally too high to allow confident identification of rare variants. We here describe an approach that can substantially increase the sensitivity of massively parallel sequencing instruments for this purpose. One example of this approach, called “Safe-SeqS” for (Safe-Sequencing System) includes (i) assignment of a unique identifier (UID) to each template molecule; (ii) amplification of each uniquely tagged template molecule to create UID-families; and (iii) redundant sequencing of the amplification products. PCR fragments with the same UID are truly mutant (“super-mutants”) if ≧95% of them contain the identical mutation. We illustrate the utility of this approach for determining the fidelity of a polymerase, the accuracy of oligonucleotides synthesized in vitro, and the prevalence of mutations in the nuclear and mitochondrial genomes of normal cells.

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21-02-2019 дата публикации

MIRNA PROFILING COMPOSITIONS AND METHODS OF USE

Номер: US20190054195A1
Принадлежит: The General Hospital Corporation

Disclosed herein is a nanosensor of miRNA activity in a target cell, and methods of use, for detection and diagnostic applications. The nanosensor comprises a delivery particle comprising an iron oxide crystal coated with a polymer, and a sensor oligonucleotide covalently attached to the polymer. The sensor oligonucleotide comprises a seed region, comprising a nucleic acid sequence that is completely complementary to the target miRNA and comprises a cleavage site which can be engaged and cleaved by the target miRNA. The sensor oligonucleotide also comprises two non-seed regions that each flank the seed region and are each comprised of a nucleic acid sequence that is complementary to the target miRNA to promote hybridization of the sensor oligonucleotide to the target miRNA, and members of a quencher-fluorophore pair. The quencher fluorophore pair members respectively flank the cleavage site and are separated by a distance that permits significant quenching of emitted fluorescent signal. 1. A sensor oligonucleotide for detection of activity of a target miRNA comprising:a) a seed region comprising a nucleic acid sequence that is completely complementary to a target miRNA and comprises a cleavage site which can be engaged by the target miRNA and cleaved by the target miRNA in complex with RNA induced silencing complex;b) two non-seed regions that each flank the seed region and are each comprised of nucleic acid sequences complementary to the target miRNA to promote hybridization of the sensor oligonucleotide to the target miRNA; andc) members of a quencher-fluorophore pair;wherein the quencher fluorophore pair members respectively flank the cleavage site and are separated by a distance that permits significant quenching of emitted fluorescent signal.2. The sensor oligonucleotide of claim 1 , that has a nucleic acid sequence that is completely complementary to the target miRNA sequence.3. The sensor oligonucleotide of claim 1 , that comprises RNA claim 1 , or a ...

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21-02-2019 дата публикации

MULTIPLEXED SINGLE MOLECULE RNA VISUALIZATION WITH A TWO-PROBE PROXIMITY LIGATION SYSTEM

Номер: US20190055594A1
Принадлежит:

SNAIL provides cost-efficient detection of specific nucleic acids in single cells, and may be combined with flow cytometry to simultaneously analyze large numbers of cells for a plurality of nucleic acids, e.g. at least one, to up to 5, up to 10, up to 15, up to 20 or more transcripts can be simultaneously analyzed, at a rate of up to about 50, 100, 250, 500 or more cells/second. The methods require only two primers for amplification, and may further include a detection primer. 1. A method for determining the abundance of a target nucleic acid in a single cell , the method comprising:contacting a fixed and permeabilized cell with at least one pair of SNAIL oligonucleotide primers under conditions permissive for specific hybridization, wherein the pair of primers comprises a Splint Primer Oligonucleotide (SPO) and a Padlock Oligonucleotide (PO), wherein each of SPO and PO comprise a first complementarity region (CR1 and CR1′, respectively) complementary to adjacent sequences on the target nucleic acid; and each of SPO and PO further comprise a second complementarity region (CR2 and CR2′) located adjacent to CR1 or CR1′; wherein CR2′ is a split region such that the 5′ and the 3′ ends of PO hybridize to CR2 such that after hybridization the 5′ and the 3′ ends of PO are positioned directly adjacent to one another;washing the cell free of unbound primer;contacting the cell with ligase wherein the PO is ligated to generate a closed circle;performing rolling circle amplification using the PO as a template and SPO as a primer for a polymerase;contacting the cell with a detection probe under conditions permissive for specific hybridization; anddetecting the level of bound detection probes to determine the abundance of the target nucleic acid.2. The method of claim 1 , wherein the SNAIL oligonucleotide primer pairs are denatured by heating before contacting the sample.3. The method of claim 1 , wherein the cell is present in a population of cells.4. The method of claim 3 , ...

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