ПРАЙМЕРЫ И ЗОНДЫ ДЛЯ ПОЛИМЕРАЗНОЙ ЦЕПНОЙ РЕАКЦИИ ДЛЯ ОБНАРУЖЕНИЯ MYCOBACTERIUM TUBERCULOSIS

Изобретение относится к области биотехнологии. Предложен зонд, состоящий из выделенной нуклеиновой кислоты, для обнаружения, обладающей лекарственной устойчивостью, содержащий последовательность зонда, которая по меньшей мере на 85% идентична последовательности, выбранной из перечисленных в Таблице 2, а также соответствующие набор и способ обнаружения, обладающей лекарственной устойчивостью. Изобретение может быть использовано в медицине. 3 н. и 18 з.п. ф-лы, 3 ил., 5 табл., 7 пр.

СУБСТРАТЫ НУКЛЕИНОВЫХ КИСЛОТ С ФЕРМЕНТАТИВНОЙ АКТИВНОСТЬЮ

... 1. Изолированный полинуклеотидный субстрат для каталитической нуклеиновой кислоты с ферментативной активностью, при этом указанный полинуклеотидный субстрат содержит последовательность N-N-N-N-N-N-N-N-rR-rY-N-N-N-N-N-N-N, гдеrR является пуриновым рибонуклеотидом;rY является пиримидиновым рибонуклеотидом;каждый из N-Nявляется нуклеотидом;шесть или более из N-Nявляются цитозиновыми нуклеотидами; именее трех из N-Nявляются гуаниновыми нуклеотидами.2. Изолированный полинуклеотидный субстрат по п. 1, где полинуклеотидный субстрат содержит или состоит из последовательности, определяемой любой из SEQ ID NO: 25-27, 29-30, 33, 72-90 или 172-175.3. Изолированный полинуклеотидный субстрат по п. 1, где семь или более или восемь или более из N-Nявляются цитозиновыми нуклеотидами.4. Изолированный полинуклеотидный субстрат по п. 3, где семь или более из N-Nявляются цитозиновыми нуклеотидами, а полинуклеотидный субстрат содержит или состоит из последовательности, определяемой любой из SEQ ID NO: 29, 73 ...

СПОСОБЫ ОПРЕДЕЛЕНИЯ ВОСПРИИМЧИВОСТИ ПАЦИЕНТА К ВНУТРИБОЛЬНИЧНОЙ ИНФЕКЦИИ И СОСТАВЛЕНИЯ ПРОГНОЗА РАЗВИТИЯ СЕПТИЧЕСКОГО СИНДРОМА

... 1. Способ определения восприимчивости пациента к внутрибольничной инфекции, согласно которому:a) получают биологический образец пациента и экстрагируют биологический материал из биологического образца;b) получают по меньшей мере один специфический реагент в отношении продукта экспрессии по меньшей мере одного гена-мишени, выбираемого среди генов-мишеней S100A9 и S100A8;c) определяют экспрессию по меньшей мере одного из генов-мишеней S100A9 и S100A8, причем сверхэкспрессия по отношению к определенной пороговой величине является указанием на восприимчивость к внутрибольничной инфекции.2. Способ по п.1, согласно которому на стадии с) определяют экспрессию гена-мишени S100A9 и гена-мишени S100A8, причем сверхэкспрессия гена-мишени S100A9 по отношению к определенной пороговой величине и сверхэкспрессия гена-мишени S100A8 по отношению к определенной пороговой величине являются указанием на восприимчивость к внутрибольничной инфекции.3. Способ по п.1, согласно которому биологический образец берут ...

Verfahren zur Vervielfältigung von Nukleinsäuresequenzen

FORTFÜHRENDE AMPLIFIZIERUNGSREAKTION

Method and apparatus to facilitate transmission of an encrypted rolling code

Detection of amplified products in nucleic acid assays

SINGLEPRIMER-NUCLEIC ACID-EXTENSION-PROCEED N

NUCLEIC ACID AMPLIFICATION ON BASIS OF BALL EMULSION

TWO-END SEQUENZIERUNG

HIV-1 DETECTION OF MEANS NUKLEINSÄUREAMPLIFIZIERUNG

NUCLEIC ACID AMPLIFICATION WITH DUPLEX INTERMEDIATE PRODUCTS

MULTIPLEXNUCLEIC ACID-OH-POINT

NUCLEIC ACID AMPLIFICATION ON BASIS OF BALL EMULSION

BY MEANS OF NUKLEINSÄUREANALOGS INDUCED TRANSKRIPTION OF DOPPELSTRÄNGIGER DNA

NUCLEIC ACID REINFORCEMENT

TRANSKRIPTIONSBASIERENDE DUPLICATION OF DOPPLESTRÄNGIGEN DNS ZIELMOLEKÜLEN

USE FROM RNA POLYMERASE TO THE IMPROVEMENT OF NUKEINSAEURE AMPLIFIKATIONSVERFAHREN

PROCEDURE FOR THE PROOF OF NUCLEIC ACID GOAL SEQUENCES OF MEANS IN VITRO TRANSKRIPTION OF A RNA PROMOTER

TRANSKRIPTIONBASIERTE NUCLEIC ACID OF REINFORCEMENT/CPROOF SYSTEMS

PROCEDURE FOR THE DUPLICATION OF NUCLEIC ACID SEQUENCES

PROCEDURE FOR THE RECOGNITION AND MEASUREMENT OF GESPLEISTEN NUCLEIC ACIDS

PROCEDURE FOR THE AMPLIFICATION OF NUCLEIC ACID SEQUENCES

Fed-batch in vitro transcription process

The present disclosure provides methods of ...

Detection of hepatitis b virus rna

Target-triggered amplification

Multiplexed quantitative detection of pathogens

Method for detecting and/or quantifying human DNA

The present invention relates to a method, kit and use of various nucleic acid sequences for deleting and/or quantifying one or more nucleic acids of a genome in a sample. Wherein the nucleic acid is amplified and the locus that is amplified is a multi copy locus within the genome, the multicopy locus has copies on at least two different chromosomes and the amplification product is detected and/or quantified.

Materials and methods for the generation of fully 2'-modified nucleic acid transcripts

Materials and methods for the generation of transcripts comprising modified nucleotides

Detection of HIV-1 by nucleic acid amplification

SINGLE PRIMER ISOTHERMAL NUCLEIC ACID AMPLIFICATION-ENHANCED ANALYTE DETECTION AND QUANTIFICATION

METHOD AND APPARATUS TO FACILITATE TRANSMISSION OF AN ENCRYPTION ROLLING CODE

METHOD AND APPARATUS TO FACILITATE TRANSMISSION OF AN ENCRYPTED ROLLING CODE AbsracuSJ gh Diou re An encrypted rolling code (11), a plurality of differing data bit order pattems (13),and a 5 pluraity of differing data inversion patterns 14) are provided. One then selects (15) a paidcuiar one of each of these pattems and uses those selected pattens as transmission characteristics when transmitting (16) at least part of the encrypted rolng code ...

Compositions and methods for detecting an RNA virus

The present invention provides methods for rapidly identifying an RNA viral infection using an isothermal nucleic acid amplification reaction that can be carried out extracted RNA in the context of a crude biological sample.

Methods of amplifying nucleic acid sequences

Methods are provided for nucleic acid amplification including contacting a double stranded nucleic acid with transposases bound to transposon DNA, wherein the transposon DNA includes a transposase binding site and an RNA polymerase promoter sequence, wherein the transposases/transposon DNA complex bind to target locations along the double stranded nucleic acid and cleave the double stranded nucleic acid into a plurality of double stranded fragments, with each double stranded fragment having the transposon DNA bound to each 5' end of the double stranded fragment, extending the double stranded fragments along the transposon DNA to make double stranded extension products having double stranded RNA polymerase promoter sequences at each end, contacting the double stranded extension products with an RNA polymerase to make a plurality of RNA transcripts of each double stranded extension product, reverse transcribing the RNA transcripts into single stranded copy DNA, fonning complementary strands ...

Internal controls for iosthermal nucleic acid amplification reactions

Methods for defining cell types

Rna polymerase chain reaction

Simple nucleic acid amplification process

SELF-SUSTAINED, SEQUENCE REPLICATION SYSTEM

METHOD FOR DETECTION OF NUCLEIC ACID TARGET SEQUENCES INVOLVING IN VITRO TRANSCRIPTION FROM AN RNA PROMOTER

Disclosed is a method of detecting the presence of a nucleic acid target sequence of interest, the method comprising the steps of: (a) adding first and second nucleic acid probes to a sample comprising the sequence of interest, so as to form a complex comprising three strands of nucleic acid, wherein the first probe comprises the full length sequence of a first strand of a double stranded promoter, the target sequence comprises an end part of a second strand of the double stranded promoter which is complementary to a part of the first strand, and the second probe comprises the rest of the second strand of the double stranded promoter which is complementary to a part of the first strand, such that a functional promoter is formed when the first probe is hybridised to both the target sequence and to the second probe; (b) adding a polymerase which recognises the promoter, so as to cause the de novo synthesis of nucleic acid from the promoter present in the complex; and (c) detecting directly ...

NUCLEIC ACID AMPLIFICATION PROCESS

This invention relates to a process for amplifying a specific nucleic acid sequence. The process involves synthesizing single-stranded RNA, single-stranded DNA and double-stranded DNA. The single-stranded RNA is a first template for a first primer, the single-stranded DNA is a second template for a second primer, and the double stranded DNA is a third template for synthesis of a plurality of copies of the first template. A sequence of the first primer or the second primer is complementary to a sequence of the specific nucleic acid and a sequence of the first primer or the second primer is homologous to a sequence of the specific nucleic acid. The amplification process may be used to increase the quantity of the specific nucleic acid sequence to allow detection, or to increase the purity of the specific nucleic acid sequence as a substitute for conventional cloning methodology.

METHOD FOR THE NON-SPECIFIC AMPLIFICATION OF NUCLEIC ACID

The present invention is concerned with a method for generating, in a non specific manner, multiple copies of RNA from a pool of mRNA's. Such a method is of particular importance in techniques for screening the differences in expression in given cell types or in cells under specific conditions. The present invention provides a non-selective poly A mRNA labeling and amplification method, i.e. a method not encompassing cDNA synthesis. The present invention is directed to a method for amplifying RNA by creating, in a non specific manner, multiple RNA copies starting from nucleic acid containing starting material comprising a pool of mRNA's each mRNA comprising a poly-A tail, wherein the material is contacted simultaneously with an oligonucleotide comprising an oligo-dT sequence, the sequence of a promoter recognized by an RNA polymerase and a transcription initiation region which is located between the oligo-dT sequence and the sequence of the promoter, and further with an enzyme having reverse ...

COMBINED EXPONENTIAL AND LINEAR AMPLIFICATION

Methods and compositions are provided for sensitive detection and quantitation of nucleic acids. Methods and compositions are further provided for genotyping. Probes of the invention allow target initiated amplification for single stranded, double stranded polynucleotides and pyrophosphate (PPi). DNA enzyme mediated detection method is also provided for detecting single stranded end products.

BIOREACTOR FOR RNA IN VITRO TRANSCRIPTION

The present invention relates to a bioreactor for RNA in vitro transcription, a method for RNA in vitro transcription, a module for transcribing DNA into RNA and an automated apparatus for RNA manufacturing. Further, the use of a bioreactor for RNA in vitro transcription as described herein is part of the present invention.The present invention relates to an RNA in vitro transcription reactor designed to be operable in an automated manner under GMP-compliant conditions. In particular, said RNA in vitro transcription reactor allows repetitive use of DNA template for various RNA in vitro transcription reactions. Further, the invention relates to an apparatus for RNA manufacturing comprising (a) a module for template DNA synthesis, (b) a module for transcribing DNA into RNA comprising said RNA in vitro transcription reactor, and, optionally, (c) a module for RNA formulation.

MUTANTS OF RNA POLYMERASES WITH INCREASED STABILITY

The present application relates to mutated RNA polymerases from bacteriophages that have increased stability, for example under high temperature conditions. One example of bacteriophage encoded RNA polymerase is the T7 RNA polymerase. 77 is a bacteriophage capable of infecting E. coli cells. Examples of other E. coli infecting T7- like bacteriophages are T3, .slzero.I, .slzero.II, W31, H, Y, A1, croC21, C22 and C23. An example of a Salmonella typhimurium infecting bacteriophage is SP6. The present invention is concerned with the RNA polymerases of T7-like bacteriophages that have been mutated. Due to these mutations the RNAP's have an increased stability. Preferred mutated RNA polymerases according to the invention are mutant RNA polymerases from 77 or SP3 bacteriophages. Due to the high homology between these enzymes, mutations in the T7 gene 1 sequence are likely to have the same effect in the corresponding gene sequence of the T3 bacteriophage. An especially preferred embodiment of the ...

TAGGED OLIGONUCLEOTIDES AND THEIR USE IN NUCLEIC ACID AMPLIFICATION METHODS

The present invention provides nucleic acid amplification methods that de sirably reduce or eliminate false positive amplification signals resulting f rom contaminating biological material, e.g., nucleic acid, that may be prese nt in one or more reagents used in an amplification reaction and/or that may be present in the environment in which an amplification reaction is perform ed. The invention offers the further advantage of requiring less stringent p urification and/or sterility efforts than conventionally needed in order to ensure that enzymes and other reagents used in amplification reactions, and the environment in which an amplification reaction is performed, are free of bacterial or other nucleic acid contamination that may yield false positive results.

METHOD AND APPARATUS TO FACILITATE TRANSMISSION OF AN ENCRYPTED ROLLING CODE

An encrypted rolling code (11), a plurality of differing data bit order patterns (13), and a plurality of differing data inversion patterns 14) are provided. One then selects (15) a particular one of each of these patterns and uses those selected patterns as transmission characteristics when transmitting (16) at least part of the encrypted rolling code.

SINGLE-PRIMER NUCLEIC ACID AMPLIFICATION METHODS

This specification is directed to novel methods of synthesizing multiple copies of a target nucleic acid sequence which are autocatalytic (i.e., able to cycle automatically without the need to modify reaction conditions such as temperature, pH, or ionic strength and using the product of one cycle in the next one). In particular, the present specification discloses a method of nucleic acid amplification which is robust and efficient, while reducing the appearance of side-products. The method uses only one primer, the "priming oligonucleotide," a promoter oligonucleotide modified to prevent polymerase extension from its 3'-terminus and, optionally, a means for terminating a primer extension reaction, to amplify RNA or DNA molecules in vitro, while reducing or substantially eliminating the formation of side-products.

METHODS AND COMPOSITIONS FOR DETECTING BACTERIAL NUCLEIC ACID AND DIAGNOSING BACTERIAL VAGINOSIS

Disclosed are methods for diagnosing Bacterial Vaginosis in a subject comprising performing an assay for the detection of any one or more of Lactobacillus sp., Gardneralla vaginalis, and Eggerthella sp. in a subject sample. Also disclosed are methods and compositions for detecting Lactobacillus sp., Gardneralla vaginalis, and/or Eggerthella nucleic acid in a sample.

POLYMERASE CHAIN REACTION PRIMERS AND PROBES FOR MYCOBACTERIUM TUBERCULOSIS

The present invention relates to novel primers and sloppy molecular beacon and molecular beacon probes for amplifying segments from different genes in Mycobacterium tuberculosis for identifying the presence of M.th DNA and/or resistance to antituberculosis drugs. This invention relates to primers, probes, and related uses in detecting M.tb and M.tb drug resistance. In one aspect, the invention provides an isolated oligonucleotide set or primer set for amplifying a portion of a M. tuberculosis region selected from the group consisting of rpoB gene, gyrA gene, gyrB gene, inhA promoter, rrs gene, eis promoter, embB gene, katG gene, dosR gene, IS61 10 gene, IS1081 gene.

PROCESS FOR THE SPECIFIC AMPLIFICATION OF NUCLEIC ACID SEQUENCES

A b s t r a c t Process for the specific production of nucleic acids based on the principle of transcription in which a promoter oligonucleotide and a template-specific oligonucleotide which can hybridize with it are used as a promoter reagent and a process for nucleic acid detection which is based on this process.

PROCESS, CONSTRUCT AND CONJUGATE FOR PRODUCING MULTIPLE NUCLEIC ACID COPIES

This invention provides inter alia an in vitro process for producing multiple specific nucleic acid copies in which the copies are produced under isostatic conditions, e.g., temperature, buffer and ionic strength, and independently of any requirement for introducing an intermediate structure for producing the copies. In other aspects, the invention provides in vitro processes for producing multiple specific nucleic acid copies in which the products are substantially free of any primer-coded sequences, such sequences having been substantially or all removed from the product to regenerate a primer binding site, thereby allowing new priming events to occur and multiple nucleic acid copies to be produced. This invention further provides a promoter--independent non-naturally occurring nucleic acid construct that produces a nucleic acid copy or copies without using or relying on any gene product that may be coded by the nucleic acid construct. Another aspect of this invention concerns a protein-nucleic ...

NUCLEIC ACID AMPLIFICATION BY TWO-ENZYME, SELF-SUSTAINED SEQUENCE REPLICATION

... 2096013 9208800 PCTABS00013 Novel methods are provided for nucleic acid amplification by continuous, substantially isothermal, self-sustained sequence replication ("3SR") utilizing RNA-dependent DNA polymerase activity, DNA-dependent DNA polymerase activity, RNAse H activity and DNA-dependent RNA polymerase activity. In one of the methods, before enzymatic activities can be provided by only two enzymes, a reverse transcriptase and a DNA-dependent RNA polymerase. The methods may employ two or three enzymes to provide the necessary enzymatic activities. Thus, in certain of the methods, an exogenous source of RNAse H, such as E. coli RNAse H, is employed in combination with a reverse transcriptase and a DNA-dependent RNA polymerase. In other of the methods of the present invention, reaction media are employed in which the inherent RNAse H activity of retroviral reverse transcriptases is effective to provide high levels of amplification so that only two enzymes, reverse transcriptase and DNA-dependent ...

METHOD FOR THE SYNTHESIS OF RIBONUCLEIC ACID (RNA)

METHOD FOR THE SENSITIVE DETECTION OF NUCLEIC ACIDS

Method for the sensitive detection of a target nucleic acid by hybridization with a probe nucleic acid. The latter contains a part which can hybridize with the target nucleic acid and a nucleic acid-specific part which does not hybridize with the target nucleic acid. The method further comprises cleavage of the probe nucleic acid, hybridization of a cleavage product of the probe nucleic acid containing the part that does not hybridize with the target nucleic acid with a matrix nucleic acid containing a part that can be hybridized with the cleavage product and a part that cannot be hybridized with the probe nucleic acid. The method also comprises the determination of the hybrid consisting of the cleavage product and the matrix nucleic acid and a reagent kit suitable for this purpose.

SELF-SUSTAINED SEQUENCE REPLICATION ELECTROCHEMILUMINESCENT NUCLEIC ACID ASSAY

This invention relates to an improved process for detecting and quantifying a desired nucleic acid sequence. This process involves synthesizing single stranded RNA, single stranded DNA, double-stranded DNA followed by detection using an electrochemiluminescent labeled binding species. The figure illustrates an electrochemiluminescent labeled nucleotide for polymerase incorporation in nucleic acid.

METHOD AND KIT FOR DEMONSTRATING MICROORGANISMS

... 2095843 9208805 PCTABS00013 Detection of microorganisms by first isolating them from the sample by means of antibodies and a solid support for the antibodies and then, optionally after isolation of the DNA from the bound microorganisms, carrying out a PCR. The antibodies to be used are specific for the microorganisms to be detected and most preferably generated against still living microorganisms. Especially magnetic particles are suitable as solid support. In PCR, primers are used that are specific for the microorganisms to be detected, most preferably based on an rRNA sequence or a DNA replication origin sequence.

DETECTION OF HUMAN RETROVIRUS INFECTION

A method for determining virus replication in human cells by human retrovirus using RNA amplification comprising detecting the hybridization of an RNA probe which specifically hybridizes with spliced RNA and not with genomic RNA. This method permits early detection of RNA replication resulting from primary infection without detecting non-replicating virus.

CONTINUOUS AMPLIFICATION REACTION

Continuous amplification reaction provides a method of amplifying a specific nucleic acid without the need to cycle a reaction. The method produces RNA transcripts which can be detected by a variety of methods. Amplification and detection kits are also provided.

DETECTION OF EXTRACELLULAR TUMOR-ASSOCIATED NUCLEIC ACID IN BLOOD PLASMA OR SERUM USING NUCLEIC ACID AMPLIFICATION ASSAYS

This invention relates to detection of specific extracellular nucleic acid in plasma or serum fractions of human or animal blood associated with neoplastic or proliferative disease. Specifically, the invention relates to detection of nucleic acid derived from mutant oncogenes or other tumor-associated DNA, and to those methods of detecting and monitoring extracellular mutant oncogenes or tumor-associated DNA found in the plasma or serum fraction of blood by using rapid DNA extraction followed by nucleic acid amplification with or without enrichment for mutant DNA. In particular, the invention relates to the detection, identification, or monitoring of the existence, progression or clinical status of benign, premalignant, or malignant neoplasms in humans or other animals that contain a mutation that is associated with the neoplasm through detection of the mutated nucleic acid of the neoplasm in plasma or serum fractions. The invention permits the detection of extracellular, tumorassociated ...

DNA DIAGNOSTICS BASED ON MASS SPECTROMETRY

Fast and highly accurate mass spectrometry-based processes for detecting a particular nucleic acid sequence in a biological sample are provided. Depending on the sequence to be detected, the processes can be used, for example, to diagnose a genetic disease or chromosomal abnormality; a predisposition to a disease or condition, infection by a pathogenic organism, or for determining identity or heredity.

Gene detection method

RNA AMPLIFICATION METHOD REQUIRING A SINGLE STEP OF HANDLING.

METHOD TO GENERATE TRANSCRIBED

La présente invention concerne une méthode pour générer des transcrits à partir • d'au moins une séquence ARN à amplifier comprenant une région «amorce » et une région d'intérêt et • d'une amorce d'amplification comprenant une région promotrice, et une région capable de s'hybrider sur ladite région « amorce » de la séquence ARN à amplifier, ladite méthode étant réalisée à température constante, et comprenant les étapes suivantes : a) on hybride ladite amorce avec l'ARN à amplifier, b) on élonge l'amorce par une activité enzymatique de transcriptase inverse afin de générer me séquence d'acide désoxyribonucléique complémentaire (ADNc) de l'ARN à amplifier, c) on clive l'ARN à amplifier, hybridé sur ledit ADNc, par une enzyme ayant une activité ribonucléase H, pour obtenir des fragments d'ARN à amplifier, hybridés sur ledit ADNc, d) on élonge les extrémités desdits fragments d'ARN à amplifier par une enzyme de transcritpase inverse et de déplacement de brin, pour obtenir des hybrides ARN-ADN ...

Promoter modified for ARN polymerase, its preparation and its applications

Oligonucléotide utilisable comme brin non-matrice de promoteur, dans la transcription d'une séquence d'une cible nucléotidique, en présence d'une ARN polymérase phagique choisie parmi celles des phages dont les ARN polymérases ont des promoteurs spécifiques comprenant une séquence consensus au moins depuis la position -17 jusqu'à la position -1, caractérisé par le fait: - qu'il comprend une séquence de coeur flanquée à l'une au moins de ses extrémités d'une séquence nucléotidique capable d'hybridation avec une séquence de la cible, - que ladite séquence de coeur est constituée par une séquence de 6 à 9 nucléotides consécutifs choisis dans la région -12 à -4 du brin non-matrice dudit promoteur spécifique, ou une séquence suffisamment homologue permettant de conserver la fonctionnalité de ladite ARN polymérase, - qu'une séquence flanquante est complémentaire d'une première région de la cible, et que l'autre séquence flanquante, lorsqu'elle est présente, est complémentaires d'une seconde région ...

METHOD FOR THE AMPLIFICATION AND DETECTION OF DNA USING A TRANSCRIPTION BASED AMPLIFICATION

The present invention is directed to a transcription based amplification method for the amplification of DNA targets starting from ds or ssDNA optionally present in a sample, comprising the steps of: - incubating the sample in an amplification buffer with one or more restriction enzymes capable of cleaving DNA at a selected restriction site, said restriction enzyme creating a defined 3' end on the said DNA strand(s), and a promoter- primer, said promoter-primer having a 5' region comprising the sequence of a promoter recognized by a DNA-dependent RNA polymerase and a 3' region complementary to the defined 3' end of the DNA strand, a second primer, having the opposite polarity of the promoter- primer and comprising the 5' end of the target sequence, and in case of ssDNA as the target DNA, a restriction primer; - maintaining the thus created reaction mixture under the appropriate conditions for a sufficient amount of time for a digestion by the restriction enzyme to take place; - subjecting ...

METHODS AND COMPOSITIONS FOR ISOTHERMAL WHOLE GENOME AMPLIFICATION

Disclosed are methods and compositions for amplification of genetic material, including isothermal WGA of single cells.

MULTIPLEXED QUANTITATIVE DETECTION OF PATHOGENS

The invention allows for the quantitative detection of a plurality of pathogens in a single sample. The method includes the amplification of a sample with a plurality of pathogen-specific primer pairs to generate amplicons of distinct sizes from each of the pathogen specific primer pairs. The method further includes the use of a plurality of competitor polynucleotide targets that correspond to each of the pathogen-specific primer pairs. The competitor polynucleotides are added to the reaction mixture at a known concentration to allow for the quantitation of the amount of pathogen in the sample. The method can be used for monitoring pathogen infection in an individual, preferably an immunocompromised individual.

COMPOSITIONS AND METHODS FOR DETECTING GROUP B STREPTOCOCCI

Compositions, methods and kits for detecting Group B streptococci. Particularly described are oligonucleotides that are useful as amplification primers and hybridization probes for detecting very low levels of Group B streptococci nucleic acids.

BEAD EMULSION NUCLEIC ACID AMPLIFICATION

Disclosed are methods for nucleic acid amplification wherein nucleic acid templates, beads, and amplification reaction solution are emulsified and the nucleic acid templates are amplified to provide clonal copies of the nucleic acid templates attached to the beads. Also disclosed are kits and apparatuses for performing the methods of the invention.

METHODS OF AMPLIFYING AND SEQUENCING NUCLEIC ACIDS

An apparatus and method for performing rapid DNA sequencing, such as genomic sequencing, is provided herein. The method includes the steps of preparing a sample DNA for genomic sequencing, amplifying the prepared DNA in a representative manner, and performing multiple sequencing reaction on the amplified DNA with only one primer hybridization step.

METHODS AND COMPOSITIONS FOR ANALYZING RIBONUCLEIC ACIDS

Methods and compositions for probe, amplification to detect, identify, quantitate, and/or analyze a targeted nucleic acid sequence. After hybridization between a probe and the targeted nucleic acid, the probe is modified to distinguish hybridized probe from unhybridized probe. Thereafter, the probe is amplified. Moreover, in specific embodiments, the present invention involves a chimeric probe that is particularly effective when the targeted nucleic acid sequence is short and/or has a relatively low concentration, such as with an miRNA molecule.

ISOTHERMAL TRANSCRIPTION BASED AMPLIFICATION ASSAY FOR THE DETECTION AND QUANTITATION OF MACROPHAGE DERIVED CHEMOKINE RNA

An isothermal transcription based amplification assay for MDC RNA uses primer combinations for sequences within the MDC gene. A quantitative control uses a mutant RNA for comparison.

Method for treating a solution in order to destroy any ribonucleic acid after amplification

The present invention provides a method for treating a solution containing among others, target nucleic acid to be amplified in order to destroy any ribonucleic acid that is present in the solution and that could possibly be amplified in another assay. The method is useful to avoid carry-over contamination between experiments. Thus, the present invention provides a method for treating a solution containing at least one target nucleic acid of interest to be amplified comprising the following steps: contacting suitable amplification reagents and at least one target nucleic acid in the presence of i. at least one ribonuclease (RNase), and ii. one ribonuclease inhibitor, the RNase being inhibited by the ribonuclease inhibitor; amplification reagents, RNase, ribonuclease inhibitor and target nucleic acid form the solution; performing a transcription-based amplification of the target; treating said solution in order to inactivate the ribonuclease inhibitor and activate the RNase; degrading any ...

Methods of small sample amplification

The present invention relates to the amplification of nucleic acids, preferably from mRNA. A primer and promoter are added to a target sequence to be amplified and then the target is amplified in an in vitro transcription reaction and the product of this reaction is used as template for subsequent rounds of amplification.

Selective amplification of target polynucleotide sequences

A method is provided for multiplying the number of copies of a target polynucleotide sequence comprising a series of primer hybridization, extending, and denaturing steps to provide an intermediate double-stranded DNA molecule containing a promoter sequence (through the use of a promoter-sequence-containing primer) incorporated upstream from the target sequence. The double-stranded DNA intermediate is then used to grow multiple RNA copies of the target sequence. The resulting RNA copies can be used as target sequences to produce further copies. Multiple cycles of this sort can thereby exponentially increase the number of target sequence copies.

COMPOSITIONS AND METHODS FOR DETECTION OF HEPATITIS A VIRUS NUCLEIC ACID

Nucleic acid oligomeric sequences and in vitro nucleic acid amplification and detection methods for detecting the presence of HAV RNA sequences in samples are disclosed. Kits comprising nucleic acid oligomers for amplifying and detecting HAV nucleic acid sequences are disclosed.

METHOD FOR MEASURING SURVIVIN mRNA

Disclosed is a method of amplifying and detecting mRNA of survivin gene in an RNA amplification process comprising: a step for forming a double-stranded DNA containing a promoter sequence by use of a combination of oligonucleotides consisting of a first primer having a sequence homologous with a portion of survivin mRNA and a second primer having a complementary sequence, wherein the promoter sequence is added to the 5'-end of either the first primer or the second primer with a reverse transcriptase, forming an RNA transcription product by use of an RNA polymerase with using the double-stranded DNA as template, and forming the double-stranded DNA by use of a reverse transcriptase by continuing to use the RNA transcription product as a template of DNA synthesis, measuring an amount of amplified RNA produced over time with an oligonucleotide probe designed so that signal properties change with the formation of a complementary double strand with the amplified RNA.

IMPROVED METHODS FOR GENERATING MULTIPLE RNA COPIES

The present invention is directed to a novel method of efficiently synthesizing, in a non-specific manner, multiple copies of a target RNA. The present invention also relates to kits relating to the same and the use of these copies for determining gene expression pattern. In particular, the present invention relates to a method for generating multiple RNA copies comprising the steps of (a) providing a sample comprising target RNA; wherein said sample is simultaneously contacted with an oligonucleotide comprising at its 5' side a promoter sequence recognized by an RNA polymerase, wherein each oligonucleotide further comprises a target hybridising sequence, which is a random sequence or a predetermined sequence and possibly a modification at its 3' terminal end in such a way that extension therefrom is prohibited; and, an enzyme having DNA polymerase activity; an enzyme having RNase H activity; an enzyme having RNA polymerase activity; and, sufficient amounts of dNTPs and rNTPs; and, (b) ...

METHOD FOR SCREENING CD14 AGONIST OR ANTAGONIST

An oligonucleotide for detection or amplification of a gene selected from the group consisting of Vibrio parahaemolyticus thermostable direct hemolysin-related hemolysin genes (trh1 and trh2) and Vibrio parahaemolyticus thermostable direct hemolysin gene (tdh2) or RNA derived therefrom is provided. Further, method for detecting trh1, trh2 or tdh2 using said oligonucleotide is provided.

Method for determining nucleic acids

A method for determining nucleic acids in a biological sample entails conversion of the nucleic acid sequences to be determined into single strands and reaction of these single strands with a polynucleotide probe. The polynucleotide probe consists of a region complementary to the nucleic acid to be determined and of an indicator region, and the change in conformation in the indicator region occurring on contact between the probe and the nucleic acid species to be detected under hybridisation conditions is detected.

Self-sustained, sequence replication system

The present disclosure provides details of an invention comprising an amplification system for the detection of target nucleic acids, most particularly target nucleic acid sequences, in an isothermal setting wherein all of the reagents necessary to conduct the amplification are present and the reactions are self-sustaining and continuous. Featured is a selective enzymatic digestion of a RNA/DNA duplex, formed by hybridization of a DNA primer with target nucleic acid, thus freeing the DNA strand for further hybridization followed by primer extension to provide a DNA duplex that can serve as a template for production of a plurality of transcripts that can be recycled and/or detected as such for deduced presence of target nucleic acid sequence.

METHOD OF AMPLIFYING AND DETECTING TARGET NUCLEIC ACID SEQUENCE BY USING THERMOSTABLE ENZYMES

OLIGONUCLEOTIDES FOR USE IN DETERMINING THE PRESENCE OF TRICHOMONAS VAGINALIS IN A SAMPLE

COMPOSITIONS AND METHODS FOR DETECTING AN RNA VIRUS

The present invention provides methods for rapidly identifying an RNA viral infection using an isothermal nucleic acid amplification reaction that can be carried out on extracted RNA in the context of a crude biological sample.

СУБСТРАТЫ НУКЛЕИНОВЫХ КИСЛОТ С ФЕРМЕНТАТИВНОЙ АКТИВНОСТЬЮ

Группа изобретений относится к области биотехнологии. Предложены изолированный полинуклеотидный субстрат для каталитической нуклеиновой кислоты с ферментативной активностью и его применение, способ детектирования присутствия мишени в образце, использующий указанный субстрат, а также набор для детекции молекулы-мишени и комплект для детекции молекулы-мишени, содержащие указанный субстрат, и набор для применения в сигнальном каскаде, содержащий указанный комплект. Предложенный изолированный полинуклеотидный субстрат способен эффективно каталитически модифицироваться в широком диапазоне температур. Предложенная группа изобретений может быть использована в биотехнологии для детектирования присутствия мишени в образце. 6 н. и 30 з.п. ф-лы, 13 ил., 25 табл., 10 пр.

СПОСОБ АМПЛИФИКАЦИИ

Изобретение относится к молекулярной биологии и технологии рекомбинантных ДНК. Сущность изобретения: проводят взаимодействие сегмента-мишени РКН с реакционной смесью, содержащей первый и второй праймеры и ферменты, обладающие активностью к РНК - и ДНК полимеразе с образованием матрицы, содержащей функциональный двунитевой промотор и последовательность сегмента-мишени РНК с последующим образованием РНК путем транскрипции с матрицей. 5 з. п. ф-лы, 1 ил.

SUBTRAHIERENDE AMPLIFIZIERUNG

TESTVERFAHREN, DAS AUF DER BILDUNG VON RINGFÖRMIGEN NUKLEINSÄUREN BASIERT

VERFAHREN ZUR BESTIMMUNG VON NUCLEINSAEUREN

METHODE FÜR DIE IN VITRO SYNTHESE VON KURZEN DOBBELSTRÄNGIGEN RNAS

Method and apparatus to facilitate transmission of an encrypted rolling code

Method for isothermal amplification of nucleic acids and method for detecting nucleic acids using simultaneous isothermal amplification of nucleic acids

IMPROVED PROCEDURE FOR THE QUANTIFICATION OF NUCLEIC ACIDS

ELIMINATION OF WRONG NEGATIVES IN NUKLEIN ACIDS

PROCEDURE FOR AMPLIFIZIERUNG AND/OR PROOF OF A NUCLEIC ACID SEQUENCE, PROOF REAGENT AND THEIR USES

NON--RADIOACTIVE HYBRIDISATIONS TEST PROCEDURE AND SENTENCE

PROCEDURE FOR THE DUPLICATION OF NUCLEIC ACID SEQUENCES

Synthesis of tagged nucleic acids

The present invention relates generally to methods, compositions and kits for synthesizing sense RNA molecules from one or more RNA molecules of interest in a sample. In exemplary embodiments, the methods use a terminal tagging oligoribonucleotide (rTTO) to join a DNA sequence tag to the 3′-termini of first-strand cDNA molecules. The use of an rTTO comprising ribonucleotides results in decreased oligonucleotide-derived background synthesis of RNA in the absence of sample RNA and, surprisingly and unexpectedly, also results in significantly increased yields of sense RNA molecules that exhibit sequences that are substantially identical to those of the RNA molecules of interest in the sample. The sense RNA molecules also have an RNA sequence tag on their 5′-termini that is useful for fixing the lengths of sense RNA molecules that are synthesized in a second or subsequent round.

Compositions and methods for detection of hepatitis a virus nucleic acid

Nucleic acid oligomeric sequences and in vitro nucleic acid amplification and detection methods for detecting the presence of HAV RNA sequences in samples are disclosed. Kits comprising nucleic acid oligomers for amplifying and detecting HAV nucleic acid sequences are disclosed.

Assay for detecting and quantifying hiv-1

Method of detecting HIV-1 nucleic acids using nucleic acid amplification and a molecular torch hybridization probe. The invented method is characterized by high levels of precision in the quantitation of HIV-1 targets at low copy numbers, and by accurate detection of different HIV-1 subtypes, including M group and O group variants.

Methods and compositions for detection of small rnas

Currently, the circularization of small RNAs is broadly regarded as an obstacle in ligation-related assays and explicitly avoided while short lengths of linear RNA targets is broadly recognized as a factor limiting use of conventional primers in PCR-related assays. In contrast, the disclosed invention capitalizes on circularization of small RNA targets or their conjugates with oligonucleotide adapters. The circular RNA templates provide amplification of the target sequences via synthesis of multimer nucleic acids that can be either labeled for direct detection or subjected to PCR amplification and detection. Structure of small circular RNAs and corresponding multimeric nucleic acids provide certain advantages over current methods including flexibility in design of conventional RT and PCR primers as well as use of 5′-overlapping dimer-primers for efficient and sequence-specific amplification of short target sequences. Our invention also reduces number of steps and reagents while increasing sensitivity and accuracy of detection of small RNAs with both 2′OH and 2′-OMe at their 3′ ends. Our invention increase sensitivity and specificity of detection of microRNAs and other small RNAs with both 2′OH and 2′-OMe at their 3′ ends while allowing us to distinguish these two forms from each other.

Sequence-specific methods for homogeneous, real-time detection of lamp products

Presented herein are methods and compositions for generating sequence-specific, secondary amplification products during Loop-mediated Isothermal Amplification (LAMP). Conventional LAMP produces a preponderance of high molecular weight DNA structures concatenated into self-complementary hairpins, which are not amenable to detection by routine probe-based hybridization methods, making multiplex detection of two or more targets or sequence variants in closed-tube formats extremely difficult. Provided herein, for example, are methods for generating secondary LAMP products bearing a fragment of the original target sequence embedded within low-molecular weight products that are devoid of competitive hairpin structures, the lack of which enhances probe-based detection of target sequences. These secondary products can, for example, be produced in real-time, during the LAMP process, and can provide the option of detecting multiple target sequences within a single tube using, e.g., a homogenous, real-time fluorescence format.

NUCLEIC ACID AMPLIFICATION

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 ...

Methods of Amplifying Nucleic Acid Sequences

Methods are provided for nucleic acid amplification including contacting a double stranded nucleic acid with transposases bound to transposon DNA, wherein the transposon DNA includes a transposase binding site and an RNA polymerase promoter sequence, wherein the transposases/transposon DNA complex bind to target locations along the double stranded nucleic acid and cleave the double stranded nucleic acid into a plurality of double stranded fragments, with each double stranded fragment having the transposon DNA bound to each 5′ end of the double stranded fragment, extending the double stranded fragments along the transposon DNA to make double stranded extension products having double stranded RNA polymerase promoter sequences at each end, contacting the double stranded extension products with an RNA polymerase to make a plurality of RNA transcripts of each double stranded extension product, reverse transcribing the RNA transcripts into single stranded copy DNA, forming complementary strands to the single stranded copy DNA to form a plurality of double stranded DNA amplicons corresponding to each double stranded fragment. 1. A method of nucleic acid amplification comprisingcontacting a double stranded nucleic acid with transposases bound to transposon DNA, wherein the transposon DNA includes a transposase binding site and an RNA polymerase promoter sequence, wherein the transposases/transposon DNA complex bind to target locations along the double stranded nucleic acid and cleave the double stranded nucleic acid into a plurality of double stranded fragments, with each double stranded fragment having the transposon DNA hound to each 5′ end of the double stranded fragment,extending the double stranded fragments along the transposon DNA to make double stranded extension products having double stranded RNA polymerase promoter sequences at each end,contacting the double stranded extension products with an RNA polymerase to make a plurality of RNA transcripts of each double ...

COMPOSITIONS AND METHODS FOR DETECTION OF HEPATITIS A VIRUS NUCLEIC ACID

Nucleic acid oligomeric sequences and in vitro nucleic acid amplification and detection methods for detecting the presence of HAV RNA sequences in samples are disclosed. Kits comprising nucleic acid oligomers for amplifying and detecting HAV nucleic acid sequences are disclosed. 1. A combination of at least two oligomers for amplifying a HAV target region comprising:for a first HAV target region, an oligomer of about 23 to 26 nt contained in the sequence of SEQ ID NO:138 that includes at least the sequence of SEQ ID NO:139 or SEQ ID NO:140, or an oligomer in a size range of about 19 to 25 nt contained in the sequence of SEQ ID NO: 141 and that contains at least one sequence of SEQ ID NOS:142 to 146, or a promoter primer oligomer in a size range of about 50 to 53 nt that includes a HAV target-specific portion of any one of SEQ ID NOS:21 to 27;for a second HAV target region, an oligomer of about 21 to 27 nt contained in the sequence of SEQ ID NO:60 or contained in the sequence of SEQ ID NO:86 that includes at least the sequence of SEQ ID NO:156, or a promoter primer oligomer in a size range of about 48 to 54 nt that includes a HAV target-specific portion of any one of SEQ ID NOS:29 to 32;for a third HAV target region, an oligomer of about 24 to 30 nt contained in the sequence of SEQ ID NO:147 that includes at least the sequence of SEQ ID NO:148, or is contained in the sequence of SEQ ID NO:157 and that includes at least the sequence of SEQ ID NO:158, or a promoter primer oligomer that includes a HAV target-specific portion of SEQ ID NO:31 or SEQ ID NO:32;for a fourth HAV target region, an oligomer of about 18 to 27 nt contained in the sequence of SEQ ID NO:93 or SEQ ID NO:95 and that contains at least the sequence of SEQ ID NO:97, SEQ ID NO:159, or SEQ ID NO:160, or a promoter primer oligomer that includes a HAV target-specific portion of SEQ ID NO:33;for a fifth HAV target region, an oligomer of about 19 to 31 nt contained in the sequence of SEQ ID NO:149 and that ...

METHODS AND COMPOSITIONS FOR ENRICHMENT OF AMPLIFICATION PRODUCTS

In some aspects, the present disclosure provides methods for enriching amplicons, or amplification products, comprising a concatemer of at least two or more copies of a target polynucleotide. In some embodiments, a method comprises sequencing the amplicons comprising at least two or more copies of a target polynucleotide. In some embodiments, the target polynucleotides comprise sequences resulting from chromosome rearrangement, including but not limited to point mutations, single nucleotide polymorphisms, insertions, deletions, and translocations including fusion genes. In some aspects, the present disclosure provides compositions and reaction mixtures useful in the described methods. 1. A method for enriching amplicons comprising a concatemer of at least two or more copies of a target polynucleotide , the method comprising:(a) generating a concatemer comprising a single-stranded polynucleotide from a circular target polynucleotide by extension of a first primer, the first primer comprising a first 3′ end that specifically hybridizes to the target polynucleotide via sequence complementarity and a first 5′ end comprising a first common sequence that does not specifically hybridize to the target polynucleotide via sequence complementarity;(b) generating a plurality of extension products containing one or more copies of the target polynucleotide by extension of a second primer comprising a second 3′ end that specifically hybridizes to the concatemer via sequence complementarity and a second 5′ end comprising a second common sequence that does not specifically hybridize to the concatemer via sequence complementarity, wherein the first common sequence and the second common sequence each comprise at least 10 contiguous nucleotides at a 5′ end and are at least 90% identical when optimally aligned; and(c) amplifying the plurality of extension products of step (b) under conditions to generate a plurality of amplicons, wherein amplicons comprising at least 2 or more copies of ...

Manufacturing methods for production of rna transcripts

Described are methods for production of RNA transcripts using a non-amplified, linearized DNA template in an in vitro transcription reaction. Enzymatic 5 ′ capping and oligo dT purification can also be included in the methods.

COMPOSITIONS TO DETECT ATOPOBIUM VAGINAE NUCLEIC ACID

The disclosed invention include nucleic acid oligomers that may be used as amplification oligomers, including primers, as capture probes for sample preparation, and detection probes for detection of 16S rRNA from in samples by using methods of specific nucleic acid amplification and detection. 1A. vaginaeA. vaginae,A. vaginaeA. vaginae. A composition comprising at least two amplification oligomers for amplifying a 16S rRNA of or a gene encoding 16S rRNA of wherein said amplification oligomers are configured to specifically hybridize to said 16S rRNA of or a gene encoding 16S rRNA of and generate an amplicon comprising a nucleotide sequence that is SEQ ID NO:43 , wherein at least one of the amplification oligomers comprises a target-specific sequence selected from the group consisting of SEQ ID NO:2 and SEQ ID NO:27 , and wherein at least one of the amplification oligomers comprises a 5′ promoter sequence.2. The composition of claim 1 , wherein a first oligomer member of the at least two amplification oligomers comprises the target-specific sequence of SEQ ID NO:2 and a second oligomer member of the at least two amplification oligomers comprises the target-specific sequence of SEQ ID NO:27.3. The composition of claim 2 , wherein the second oligomer member comprises the sequence of SEQ ID NO:3.4. The composition of claim 1 , wherein said promoter sequence is a T7 RNA polymerase promoter sequence.5. The composition of claim 4 , wherein said promoter sequence is SEQ ID NO:25.6. The composition of claim 5 , wherein at least one of the amplification oligomers comprises the sequence of SEQ ID NO:3.7A. vaginaeA. vaginae,A. vaginaeA. vaginae. A kit comprising at least two amplification oligomers for amplifying a 16S rRNA of or a gene encoding 16S rRNA of wherein said amplification oligomers are configured to specifically hybridize to said 16S rRNA of or a gene encoding 16S rRNA of and generate an amplicon comprising a nucleotide sequence that is SEQ ID NO:43 claim 5 , ...

COMPOSITIONS AND METHODS FOR DETECTING AN RNA VIRUS

The present invention provides methods for rapidly identifying an RNA viral infection using an isothermal nucleic acid amplification reaction that can be carried out extracted RNA in the context of a crude biological sample. 1. A method of amplifying a target polynucleotide in a reverse transcriptase and nicking amplification reaction , the method comprising:(a) contacting a target polynucleotide molecule with a reverse transcriptase primer in the presence of a reverse transcriptase and dNTPs under conditions permissive for cDNA synthesis, thereby generating a cDNA;(b) contacting the cDNA with forward and reverse primers each carrying at least one nicking enzyme recognition sequence within their respective 5′-terminal regions which specifically bind the cDNA with their respective 3′-terminal regions in the presence of a nicking enzyme, dNTPs, a and a strand-displacement polymerase under conditions permissive for the isothermal amplification of the cDNA, thereby generating amplicons.2. (canceled)3. The method of claim 1 , wherein the polynucleotide molecule is an Ebola virus (EBOV) claim 1 , human immunodeficiency virus (HIV) claim 1 , dengue virus claim 1 , influenza B virus claim 1 , or bovine diarrhea virus polynucleotide.48.-. (canceled)98. The method of any one of - claims 1 , wherein steps (a) and (b) are carried out in a single reaction.10. The method of claim 1 , wherein the reverse transcriptase enzyme and the strand-displacement DNA polymerase are the same or different enzymes.1115.-. (canceled)16. The method of claim 1 , wherein the method is carried out in about 5 claim 1 , 7 claim 1 , 10 claim 1 , 15 claim 1 , 20 claim 1 , 25 or thirty minutes.17. (canceled)18. The method of claim 21 , wherein the target polynucleotide molecule is not purified or isolated away from the biological sample.19. (canceled)20. The method of claim 1 , wherein no separate reverse transcriptase primer is required claim 1 , but the forward and/or reverse primers are used.21. The ...

METHOD OF PRODUCING RNA FROM CIRCULAR DNA AND CORRESPONDING TEMPLATE DNA

The present invention is concerned with a method of producing a target RNA using a circular DNA, wherein said method does not comprise a step of linearizing said circular DNA. The present invention further relates to a circular DNA comprising an RNA polymerase promoter sequence, followed by a sequence encoding a target RNA, followed by a sequence encoding a self-cleaving ribozyme, followed by an RNA polymerase terminator sequence element, wherein the latter element may comprise several RNA polymerase terminator sequences. Multimeric DNA obtained by rolling circle amplification of said circular DNA is also within the scope of the present invention. 2. The method according to claim 1 , wherein said circular DNA provided in step a) comprises an RNA polymerase terminator sequence element iv which comprises at least one Class II termination sequence and fails to comprise a Class I termination sequence.3. The method according to or claim 1 , wherein said circular DNA provided in step a) comprises an RNA polymerase terminator sequence element iv which comprises at least one VSV terminator sequence and/or at least one 1R11 variant rrnB T1 downstream terminator sequence.4. The method according to or claim 1 , wherein said circular DNA provided in step a) comprises an RNA polymerase terminator sequence element iv which comprises at least one VSV terminator sequence.5. The method according to any one of to claim 1 , wherein the in vitro transcription in step b) is carried out in the presence of naturally occurring nucleotides and at least one modified nucleotide claim 1 , wherein said at least one modified nucleotide at least partially replaces at least one naturally occurring nucleotide.6. The method according to any one of to claim 1 , wherein the in vitro transcription in step b) is carried out in the presence of a cap analog.7. The method according to any one of to claim 1 , wherein said target RNA is purified in step c) by at least one first and at least one second ...

HANDHELD NUCLEIC ACID-BASED ASSAY FOR RAPID IDENTIFICATION

A method for identifying a predefined target organism includes extracting a nucleic acid from a sample to form an extracted nucleic acid, amplifying the extracted nucleic acid to form a nucleic acid amplicon, tagging the nucleic acid amplicon with a capture probe and a detector partner to form a detector partner-nucleic acid amplicon-capture probe complex, and performing a detection assay on the detector partner-nucleic acid amplicon-capture probe complex to identify whether the predefined target organism is present in the sample. 1. A method for identifying a predefined target organism , the method comprising:extracting a nucleic acid from a sample to form an extracted nucleic acid;amplifying the extracted nucleic acid to form a nucleic acid amplicon;tagging the nucleic acid amplicon with a capture probe and a detector partner to form a detector partner-nucleic acid amplicon-capture probe complex, the detector partner comprising an antibody labeled with a detector moiety; andperforming a detection assay on the detector partner-nucleic acid amplicon-capture probe complex to identify whether the predefined target organism is present in the sample.2. The method of claim 1 , further comprising performing size exclusion chromatography or selective binding between extracting the nucleic acid and amplifying the extracted nucleic acid claim 1 , and amplifying the extracted nucleic acid and tagging the nucleic acid amplicon.3. The method of claim 1 , further comprising adding a flow buffer to the detector partner-nucleic acid amplicon-capture probe complex prior to performing the detection assay on the detector partner-nucleic acid amplicon-capture probe complex.4. The method of claim 1 , wherein amplifying the extracted nucleic acid to form the nucleic acid amplicon comprises isothermally amplifying the extracted nucleic acid.5. The method of claim 4 , wherein isothermally amplifying the extracted nucleic acid comprises performing nucleic acid sequence-based amplification ...

IMIDAZOLYL PYRIMIDINYLAMINE COMPOUNDS AS CDK2 INHIBITORS

The present application provides imidazolyl pyrimidinylamine inhibitors of cyclin-dependent kinase 2 (CDK2), as well as pharmaceutical compositions thereof, and methods of treating cancer using the same. 2. The compound of claim 1 , or a pharmaceutically acceptable salt thereof claim 1 , wherein Ris halo claim 1 , CN claim 1 , or Chaloalkyl.3. The compound of claim 1 , or a pharmaceutically acceptable salt thereof claim 1 , wherein Ris H.4. The compound of claim 1 , or a pharmaceutically acceptable salt thereof claim 1 , wherein n is 0 or 1.5. The compound of claim 1 , or a pharmaceutically acceptable salt thereof claim 1 , wherein Ris selected from Calkyl claim 1 , Chaloalkyl claim 1 , Ccycloalkyl claim 1 , phenyl claim 1 , 4-7 membered heterocycloalkyl claim 1 , 5-6 membered heteroaryl claim 1 , Ccycloalkyl-Calkyl claim 1 , phenyl-Calkyl claim 1 , 4-7 membered heterocycloalkyl-Calkyl claim 1 , and 5-6 membered heteroaryl-Calkyl; wherein said Calkyl claim 1 , Chaloalkyl claim 1 , Ccycloalkyl claim 1 , phenyl claim 1 , 4-7 membered heterocycloalkyl claim 1 , 5-6 membered heteroaryl claim 1 , Ccycloalkyl-Calkyl claim 1 , phenyl-Calkyl claim 1 , 4-7 membered heterocycloalkyl-Calkyl claim 1 , and 5-6 membered heteroaryl-Calkyl are each optionally substituted by 1 claim 1 , 2 claim 1 , or 3 independently selected Rsubstituents.6. The compound of claim 1 , or a pharmaceutically acceptable salt thereof claim 1 , wherein:{'sup': 5A', 'a51', 'c51', 'd51', '5B, 'sub': 1-6', '1-6', '3-4', '1-6', '1-6', '3-4, 'each Ris independently selected from H, halo, CN, Calkyl, Chaloalkyl, Ccycloalkyl, OR, and NRR, wherein said Calkyl, Chaloalkyl, and Ccycloalkyl are each optionally substituted with 1 or 2 independently selected Rsubstituents;'}{'sup': a51', 'c51', 'd51, 'sub': 1-6', '1-6, 'each R, R, and Ris independently selected from H, Calkyl, and Chaloalkyl; and'}{'sup': '5B', 'sub': 1-3', '1-3', '1-3', '1-3', '1-3', '1-3, 'each Ris independently selected from H, halo, CN, OH, ...

Nucleic Acid Enzyme Substrates

The invention relates to nucleic acid substrates for catalytic nucleic acid enzymes and methods utilising the substrates.

Polymerase Chain Reaction Primers and Probes for Mycobacterium Tuberculosis

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 ...

Methods and compositions for nucleic acid amplification

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.

COMPOSITIONS, KITS AND RELATED METHODS FOR THE DETECTION AND/OR MONITORING OF LISTERIA

Provided are compositions, kits, and methods for the identification of . In certain aspects and embodiments, the compositions, kits, and methods may provide improvements in relation to specificity, sensitivity, and speed of detection. 1ListeriaL. monocytogenes, L. innocua, L. grayi, L. ivanovii, L. welshimeri, L. murrayiL. seeligeriBrochothrix thermosphactaErysipelothrix rhusiopathiae. A set of oligonucleotides configured to amplify in an amplification reaction a nucleic acid sequence from , and without substantial amplification of a nucleic acid or an nucleic acid comprising a first T7 provider oligonucleotide and a first primer oligonucleotide ,{'i': 'Listeria', 'wherein the first T7 provider oligonucleotide targets a sequence in a first nucleic acid region corresponding to nucleotide positions of about 15-91 of SEQ ID NO: 103; and'}{'i': 'Listeria', 'wherein the first primer oligonucleotide targets a sequence in a second nucleic acid region corresponding to nucleotide positions of about 90-156 of SEQ ID NO: 103.'}2. The set of oligonucleotides of claim 1 , wherein the first T7 provider oligonucleotide comprises a hybridizing sequence joined at its 5′ end to a T7 promoter sequence claim 1 , wherein the hybridizing sequence is 19-24 nucleobases in length and comprises the hybridizing sequence of SEQ ID NO: 8 claim 1 , SEQ ID NO: 9 claim 1 , SEQ ID NO: 10 claim 1 , SEQ ID NO: 11 claim 1 , SEQ ID NO: 12 claim 1 , SEQ ID NO: 13 claim 1 , SEQ ID NO: 14 claim 1 , or SEQ ID NO: 15.3. The set of oligonucleotides of claim 2 , wherein the first T7 provider oligonucleotide has a nucleobase sequence comprising the nucleobase sequence of SEQ ID NO: 8 claim 2 , SEQ ID NO: 9 claim 2 , SEQ ID NO: 10 claim 2 , SEQ ID NO: 11 claim 2 , SEQ ID NO: 12 claim 2 , SEQ ID NO: 13 claim 2 , SEQ ID NO: 14 claim 2 , SEQ ID NO: 15 claim 2 , or SEQ ID NO: 12 having a guanine at a position corresponding to nucleotide position 58 of SEQ ID NO: 103.4. The set of oligonucleotides of claim 2 , ...

NUCLEIC ACID AMPLIFICATION

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. 1. A method of synthesizing a double stranded cDNA of an RNA molecule , said method comprisinga) annealing an RNA molecule with a first oligonucleotide comprising a primer operably linked to a promoter region to form a first complex,b) synthesizing a first strand cDNA by reverse transcription of said first complex to form hybrid(s) of RNA and first strand cDNA,c) degrading first oligonucleotides not used in a) orb) above with exonuclease activity, andd) annealing said first strand cDNA, after denaturing the hybrid(s) or degrading the RNA from said hybrid(s), with a plurality of second oligonucleotides comprising a random primer region to form a population of second complexes, ande) forming double stranded cDNA from said population of second complexes with DNA polymerase activity.2. The method of wherein said RNA is mRNA.3. The method of wherein said RNA is part of a cellular mRNA preparation.4. The method of wherein said first oligonucleotide comprises a primer containing an oligo or poly dT sequence.5. The method of wherein said oligo or poly dT sequence is at least about eight dT in length.6. The method of wherein said random primer region comprises at least about six random nucleotides.7. The method of wherein said random primer region comprises at least about nine random nucleotides.8. The method of wherein said DNA polymerase activity is DNA dependent.9. The method of wherein said DNA dependent polymerase activity is selected from exonuclease deficient Klenow claim 8 , Taq polymerase activities claim 8 , and combinations of exonuclease deficient Klenow and/or Taq polymerase activities.10. A method of amplifying RNA ...

Universal or Broad Range Assays and Multi-Tag Sample Specific Diagnostic Process Using Non-Optical Sequencing

and amplifying the DNA, or the RNA following reverse transcription with a reverse transcriptase; and contacting the amplification product with one or more species-, organism- or virus-specific detectable marker.

Automated cell processing methods, modules, instruments, and systems

In an illustrative embodiment, automated multi-module cell editing instruments are provided to automate multiple edits into nucleic acid sequences inside one or more cells.

CANOLA VARIETY D3156M

Provided is a canola variety designated D3156M and seed, plants and plant parts thereof produced from a cross of inbred varieties. Methods for producing a canola variety comprise crossing canola variety D3156M with another canola plant. Methods for producing a canola plant containing in its genetic material one or more traits introgressed into D3156M through backcross conversion and/or transformation, and to the canola seed, plant and plant part produced thereby are described. Canola variety D3156M, the seed, the plant produced from the seed, plant parts and variants, mutants, and minor modifications of canola variety D3156M are disclosed. 1. A canola variety D3156M , representative seed of the variety having been deposited under ATCC Accession Number PTA-124073.2. A seed of the canola variety of .3. The seed of claim 2 , further comprising a seed treatment on the surface of the seed.4. The seed of claim 3 , wherein the treatment comprises a fungicide claim 3 , insecticide or combination thereof.5. A plant or plant part of the canola variety of .6. A method comprising cleaning seed harvested from the plant of .7. A method for producing canola oil comprising processing seed harvested from the plant of .8. A method of producing a canola seed claim 2 , the method comprising planting the seed of to produce a subsequent generation of seed; harvesting the subsequent generation of seed; and planting the subsequent generation of seed.9. The seed of claim 2 , further comprising a trait introduced by backcrossing or genetic transformation.10. The seed of claim 9 , wherein the trait is selected from the group consisting of male sterility claim 9 , a site for site-specific recombination claim 9 , abiotic stress tolerance claim 9 , altered phosphate claim 9 , altered antioxidants claim 9 , altered fatty acids claim 9 , altered essential amino acids claim 9 , altered carbohydrates claim 9 , herbicide resistance claim 9 , insect resistance and disease resistance.11. The seed of ...

CANOLA VARIETY 45M38

Provided is a canola variety designated 45M38 and seed, plants and plant parts thereof produced from a cross of inbred varieties. Methods for producing a canola variety comprise crossing canola variety 45M38 with another canola plant. Methods for producing a canola plant containing in its genetic material one or more traits introgressed into 45M38 through backcross conversion and/or transformation, and to the canola seed, plant and plant part produced thereby are described. Canola variety 45M38, the seed, the plant produced from the seed, plant parts and variants, mutants, and minor modifications of canola variety 45M38 are disclosed. 1. A canola variety 45M38 , representative seed of the variety having been deposited under ATCC Accession Number PTA-124074.2. A seed of the canola variety of .3. The seed of claim 2 , further comprising a seed treatment on the surface of the seed.4. The seed of claim 3 , wherein the treatment comprises a fungicide claim 3 , insecticide or combination thereof.5. A plant or plant part of the canola variety of .6. A method comprising cleaning seed harvested from the plant of .7. A method for producing canola oil comprising processing seed harvested from the plant of .8. A method of producing a canola seed claim 2 , the method comprising planting the seed of to produce a subsequent generation of seed; harvesting the subsequent generation of seed; and planting the subsequent generation of seed.9. The seed of claim 2 , further comprising a trait introduced by backcrossing or genetic transformation.10. The seed of claim 9 , wherein the trait is selected from the group consisting of male sterility claim 9 , a site for site-specific recombination claim 9 , abiotic stress tolerance claim 9 , altered phosphate claim 9 , altered antioxidants claim 9 , altered fatty acids claim 9 , altered essential amino acids claim 9 , altered carbohydrates claim 9 , herbicide resistance claim 9 , insect resistance and disease resistance.11. The seed of claim 9 ...

CANOLA VARIETY 45H37

Provided is a canola variety designated 45H37 and seed, plants and plant parts thereof produced from a cross of inbred varieties. Methods for producing a canola variety comprise crossing canola variety 45H37 with another canola plant. Methods for producing a canola plant containing in its genetic material one or more traits introgressed into 45H37 through backcross conversion and/or transformation, and to the canola seed, plant and plant part produced thereby are described. Canola variety 45H37, the seed, the plant produced from the seed, plant parts and variants, mutants, and minor modifications of canola variety 45H37 are disclosed. 1. A canola variety 45H37 , representative seed of the variety having been deposited under ATCC Accession Number PTA-124075.2. A seed of the canola variety of .3. The seed of claim 2 , further comprising a seed treatment on the surface of the seed.4. The seed of claim 3 , wherein the treatment comprises a fungicide claim 3 , insecticide or combination thereof.5. A plant or plant part of the canola variety of .6. A method comprising cleaning seed harvested from the plant of .7. A method for producing canola oil comprising processing seed harvested from the plant of .8. A method of producing a canola seed claim 2 , the method comprising planting the seed of to produce a subsequent generation of seed; harvesting the subsequent generation of seed; and planting the subsequent generation of seed.9. The seed of claim 2 , further comprising a trait introduced by backcrossing or genetic transformation.10. The seed of claim 9 , wherein the trait is selected from the group consisting of male sterility claim 9 , a site for site-specific recombination claim 9 , abiotic stress tolerance claim 9 , altered phosphate claim 9 , altered antioxidants claim 9 , altered fatty acids claim 9 , altered essential amino acids claim 9 , altered carbohydrates claim 9 , herbicide resistance claim 9 , insect resistance and disease resistance.11. The seed of claim 9 ...

CANOLA VARIETY 45CM36

Provided is a canola variety designated 45CM36 and seed, plants and plant parts thereof produced from a cross of inbred varieties. Methods for producing a canola variety comprise crossing canola variety 45CM36 with another canola plant. Methods for producing a canola plant containing in its genetic material one or more traits introgressed into 45CM36 through backcross conversion and/or transformation, and to the canola seed, plant and plant part produced thereby are described. Canola variety 45CM36, the seed, the plant produced from the seed, plant parts and variants, mutants, and minor modifications of canola variety 45CM36 are disclosed. 1. A canola variety 45CM36 , representative seed of the variety having been deposited under ATCC Accession Number PTA-124076.2. A seed of the canola variety of .3. The seed of claim 2 , further comprising a seed treatment on the surface of the seed.4. The seed of claim 3 , wherein the treatment comprises a fungicide claim 3 , insecticide or combination thereof.5. A plant or plant part of the canola variety of .6. A method comprising cleaning seed harvested from the plant of .7. A method for producing canola oil comprising processing seed harvested from the plant of .8. A method of producing a canola seed claim 2 , the method comprising planting the seed of to produce a subsequent generation of seed; harvesting the subsequent generation of seed; and planting the subsequent generation of seed.9. The seed of claim 2 , further comprising a trait introduced by backcrossing or genetic transformation.10. The seed of claim 9 , wherein the trait is selected from the group consisting of male sterility claim 9 , a site for site-specific recombination claim 9 , abiotic stress tolerance claim 9 , altered phosphate claim 9 , altered antioxidants claim 9 , altered fatty acids claim 9 , altered essential amino acids claim 9 , altered carbohydrates claim 9 , herbicide resistance claim 9 , insect resistance and disease resistance.11. The seed of ...

METHODS AND COMPOSITIONS FOR ENRICHMENT OF AMPLIFICATION PRODUCTS

In some aspects, the present disclosure provides methods for enriching amplicons, or amplification products, comprising a concatemer of at least two or more copies of a target polynucleotide. In some embodiments, a method comprises sequencing the amplicons comprising at least two or more copies of a target polynucleotide. In some embodiments, the target polynucleotides comprise sequences resulting from chromosome rearrangement, including but not limited to point mutations, single nucleotide polymorphisms, insertions, deletions, and translocations including fusion genes. In some aspects, the present disclosure provides compositions and reaction mixtures useful in the described methods. 1. A method for processing or analyzing a cell-free biological sample of a subject , comprising:(a) obtaining said cell-free biological sample of said subject;(b) using a nucleic acid molecule derived from said cell-free biological sample to generate a circular nucleic acid molecule; and(c) subjecting said circular nucleic acid molecule or derivative thereof to sequencing to identify a sequence of said nucleic acid molecule derived from said cell-free biological sample.2. The method of claim 1 , further comprising processing said sequence to identify a cancer in said subject.3. The method of claim 2 , wherein said sequence is computer processed against a reference to identify one or more genetic variants claim 2 , and wherein said one or more genetic variants identify said cancer.4. The method of claim 1 , wherein said sequence of said circular nucleic acid molecule or derivative thereof comprises a junction sequence in which a 5′ end and a 3′ end of said nucleic acid molecule are joined.5. The method of claim 4 , wherein said sequence of said nucleic acid molecule identifies said 5′ end or said 3′ end of said nucleic acid molecule.6. The method of claim 1 , wherein (a) comprises ligating ends of said nucleic acid molecule to one another.7. The method of claim 1 , wherein (a) comprises ...

MANUFACTURING METHODS FOR PRODUCTION OF RNA TRANSCRIPTS

Described are methods for production of RNA transcripts using a non-amplified, linearized DNA tempate in an in vitro transcription reaction. Enzymatic 5′ capping and oligo dT purification can also be included in the methods. 1. A method for producing a purified composition comprising a capped RNA transcript for a gene of interest , the method comprising:(a) providing a sample comprising a linear, non-amplified DNA template, the DNA template comprising an RNA polymerase promoter sequence operatively linked to a target sequence coding for the gene of interest, a poly A tail sequence, an endonuclease recognition site sequence immediately downstream of the poly A tail sequence, and optionally a 5′ untranslated region (UTR) and/or a 3′ UTR;(b) contacting the sample with a RNA polymerase and ribonucleotidesunder conditions sufficient for vitro transcription to produce a first composition comprising an uncapped RNA transcript, wherein at least 80% of the RNA transcript is full-length uncapped RNA transcript;(c) purifying the uncapped RNA transcript using oligo dT affinity purification;(d) capping the uncapped RNA transcript to produce a second composition comprising a capped RNA transcript; and(e) purifying the capped RNA transcript from the second composition by anion exchange chromatography,thereby producing the purified composition comprising a capped RNA transcript.2. The method of claim 1 , wherein at least 95% of the uncapped RNA transcript in the first composition is full-length uncapped RNA transcript.3. The method of claim 1 , wherein the percent full length uncapped RNA transcript is determined using reverse phase HPLC and measured by peak area of full length relative to total peak area.4. The method of claim 1 , wherein the method does not comprising treating the composition with DNase.5. (canceled)6. The method of claim 1 , wherein the method does not comprise a polymerase chain reaction (PCR) amplification step.7. The method of claim 1 , wherein the DNA ...

METHODS FOR QUANTITATIVE AMPLIFICATION AND DETECTION OVER A WIDE DYNAMIC RANGE

Disclosed are compositions and methods for making differentiable amplicon species at unequal ratios using a single amplification system in a single vessel. The number of differentiable amplicons and their ratios to one another are chosen to span the required linear dynamic range for the amplification reaction and to accommodate limitations of the measuring system used to determine the amount of amplicon generated. Unequal amounts of distinguishable amplicon species are generated by providing unequal amounts of one or more amplification reaction components (e.g., distinguishable amplification oligomers, natural and unnatural NTP in an NTP mix, or the like). The amount of target nucleic acid present in a test sample is determined using the linear detection range generated from detection of one or more amplicon species having an amount within the dynamic range of detection. 1. A method for detecting a target nucleic acid sequence in a sample comprising:(a) providing a sample suspected of containing a target nucleic acid; wherein the at least two differentiable amplicon species are generated using a single amplification oligomer that hybridizes to one strand of the target nucleic acid and at least two amplification oligomers that hybridize to the complementary strand of the target nucleic acid,', 'wherein the at least two amplification oligomers hybridizing to the complementary strand of the target nucleic acid are provided in different amounts and have distinct nucleic acid sequences, such that the defined ratio of the at least two differentiable amplicon species is generated and whereby the at least two differentiable amplicon species differ in nucleic acid composition; and, '(b) generating from the target nucleic acid a defined ratio of at least two differentiable amplicon species,'} wherein a first amplicon species is detectable in a first linear range representing a first concentration of the target nucleic acid in the sample to a second concentration of the target ...

FED-BATCH IN VITRO TRANSCRIPTION PROCESS

The present disclosure provides methods of in vitro transcribing a ribonucleic acid (RNA) of interest. In some embodiments, such methods include determining consumption rates of nucleoside triphosphates (NTPs). 1. A method of determining percent (%) nucleoside triphosphates (NTPs) consumption of an in vitro transcription (IVT) reaction comprising:(a) conducting an IVT reaction with a reaction mixture that comprises known initial NTP concentrations, a deoxyribonucleic acid (DNA) encoding a ribonucleic acid (RNA) of interest, and an RNA polymerase;(b) measuring individual NTP concentrations at discrete intervals over a period of time; and(c) calculating a percent (%) consumption value for each NTP of the reaction mixture.2. The method of claim 1 , wherein the NTPs comprise adenosine triphosphate (ATP) claim 1 , cytidine triphosphate (CTP) claim 1 , uridine triphosphate (UTP) claim 1 , and guanosine triphosphate (GTP).3. The method of claim 1 , wherein step (b) comprises (i) dividing individual NTP consumption rate over total NTP consumption rate.4. The method of claim 3 , wherein the individual NTP consumption rate is calculated by measuring individual NTP concentrations at discrete intervals over a period of time; and the total NTP consumption rate is calculated by measuring total NTP concentration at discrete intervals over a period of time.5. The method of claim 4 , wherein the individual NTP concentration and total NTP concentration measurements are collected until the concentration of at least one of the NTPs drops below a threshold concentration.6. The method of claim 5 , wherein the threshold concentration is above zero (0) millimolar (mM) claim 5 , optionally wherein the threshold concentration is 5 mM to 20 mM claim 5 , and optionally wherein the threshold concentration is within 5%-75% of the initial NTP concentration.7. The method of claim 1 , wherein the known initial NTP concentrations comprise equimolar NTP concentrations of each of [ATP] claim 1 , [CTP] ...

SYSTEM

The present disclosure describes technologies that permit sensitive detection of nucleic acids of interest (i.e., nucleic acids whose nucleotide sequence is or includes a target sequence). 1. A system comprising:a plurality of nucleic acid molecules having different nucleotide sequences; a first ligation oligonucleotide whose nucleotide sequence includes a templating element and a first target hybridization element; and', 'a second ligation oligonucleotide whose nucleotide sequence includes a second target hybridization element and a Cas recognition element; and', 'optionally one or more bridging oligonucleotides whose nucleotide sequence is or comprises one or more additional target hybridization elements,, 'a set of ligation oligonucleotides, comprisingwherein the target hybridization elements bind to different portions of a common target site, so that, when the plurality of nucleic acid molecules includes at least one nucleic acid molecule whose nucleotide sequence includes the target site, then the set of ligation oligonucleotides hybridizes to the target site and forms a gapped nucleic acid strand susceptible to ligation with a ligase to generate a ligated strand.2. The system of claim 1 , further comprising a ligase.3. The system of claim 1 , wherein one or more of the oligonucleotides is associated with a solid support.4. The system of claim 1 , wherein nucleic acids of the plurality are associated with a solid support.5. The system of claim 1 , further comprising a second set of ligation oligonucleotides directed to a second target site claim 1 , different from that of the first set of ligation oligonucleotides.6. The system of claim 5 , wherein the templating elements of the first and second sets of ligation oligonucleotides are the same.7. The system of claim 5 , wherein the templating elements of the first and second sets of ligation oligonucleotides are different.8. The system of any one of - claim 5 , wherein the Cas recognition elements of the first ...

Nucleic acid amplification

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.

gRNA STABILIZATION IN NUCLEIC ACID-GUIDED NICKASE EDITING

The present disclosure provides compositions of matter, methods and instruments for nucleic acid-guided nickase/reverse transcriptase fusion editing in live cells. Editing efficiency is improved using fusion proteins (e.g., the nickase-RT fusion) that retain certain characteristics of nucleic acid-directed nucleases (e.g., the binding specificity and ability to cleave one or more DNA strands in a targeted manner) combined with reverse transcriptase activity. Editing cassettes are employed, comprising a gRNA and a repair template where the 3′ end of the repair template is protected from degradation. 1. A CREATE fusion editing cassette for performing nucleic acid-guided nickase/reverse transcriptase fusion editing comprising from 3′ to 5′: an RNA stabilization moiety;', 'a linker region;', 'a primer binding region capable of binding to a nicked target DNA;', 'a nick-to-edit region; and', 'a region of post-edit homology;, 'an RNA repair template comprising a guide sequence; and', 'a scaffold region., 'a gRNA comprising2. The CREATE fusion editing cassette of claim 1 , wherein the RNA stabilization moiety is a G quadraplex claim 1 , an RNA hairpin claim 1 , an RNA pseudoknot or an exoribonuclease resistant RNA.3. The CREATE fusion editing cassette of claim 2 , wherein the RNA stabilization moiety is a G quadraplex.4. The CREATE fusion editing cassette of claim 3 , wherein the RNA stabilization moiety is a G quadraplex selected from SEQ ID No: 1; SEQ ID No: 2; SEQ ID No: 3; SEQ ID No: 4; SEQ ID No: 5; SEQ ID No: 6; SEQ ID No: 7; SEQ ID No: 8; SEQ ID No: 9; and SEQ ID No: 10.5. The CREATE fusion editing cassette of claim 3 , wherein the RNA stabilization moiety is a G quadraplex selected from SEQ ID No: 11; SEQ ID No: 12; SEQ ID No: 13; SEQ ID No: 14; SEQ ID No: 15; SEQ ID No: 16; SEQ ID No: 17; SEQ ID No: 18; SEQ ID No: 19; and SEQ ID No: 20.6. The CREATE fusion editing cassette of claim 3 , wherein the RNA stabilization moiety is a G quadraplex selected from SEQ ID No: ...

NUCLEIC ACID ENZYME SUBSTRATES

The invention relates to nucleic acid substrates for catalytic nucleic acid enzymes and methods utilising the substrates. 152-. (canceled)53. A method for modifying a polynucleotide substrate to provide a detectable effect , the method comprising contacting the substrate with a catalytic nucleic acid enzyme wherein:{'sub': 1', '2', '3', '4', '5', '6', '7', '8', '9', '10', '11', '12', '13', '14', '15', '1', '15', '5', '13', '9', '9', '15, 'the polynucleotide substrate comprises a sequence N-N-N-N-N-N-N-N-rR-rY-N-N-N-N-N-N-Nwherein rR is a purine ribonucleotide, rY is a uracil ribonucleotide, each of N-Nare deoxyribonucleotides, six or more of N-Nare cytosine nucleotides, Nis a cytosine nucleotide, and less than three of N-Nare guanine nucleotides;'}the catalytic nucleic acid enzyme is an MNAzyme or a DNAzyme; andcatalytic modification of the polynucleotide substrate by the catalytic nucleic acid enzyme during said contacting provides the detectable effect.54. The method according to claim 53 , wherein the catalytic nucleic acid is a catalytically active multi-component nucleic acid enzyme (MNAzyme) claim 53 , and the method comprises:(a) providing first and second oligonucleotide partzyme components of the MNAzyme, wherein the first oligonucleotide partzyme and the second oligonucleotide partzyme are capable of self-assembling upon binding to a target to form the MNAzyme;(b) providing the isolated polynucleotide substrate, wherein upon hybridization to the MNAzyme the polynucleotide substrate is capable of being catalytically modified by the MNAzyme to thereby provide the detectable effect; (1) self-assembly of the MNAzyme upon binding to the target, and', '(2) catalytic activity of the MNAzyme upon hybridizing to the polynucleotide substrate to thereby provide the detectable effect; and, '(c) contacting the two or more oligonucleotide partzyme components with a sample putatively containing the target under conditions permitting(d) detecting the detectable effect ...

USING RANDOM PRIMING TO OBTAIN FULL-LENGTH V(D)J INFORMATION FOR IMMUNE REPERTOIRE SEQUENCING

Disclosed herein include systems, methods, compositions, and kits for labeling nucleic acid targets in a sample. In some embodiments, nucleic acid targets (e.g., mRNAs) are initially barcoded on the 3′ end and are subsequently barcoded on the 5′ end following a template switching reaction and intermolecular and/or intramolecular hybridization and extension. 5′- and/or 3′-barcoded nucleic acid targets can serve as templates for amplification reactions and/or random priming and extension reactions to generate a sequencing library. The method can comprise generating a full-length sequence of the nucleic acid target by aligning a plurality of sequencing reads. Immune repertoire profiling methods are also provided in some embodiments. 1. A method for labeling nucleic acid targets in a sample , comprising:contacting copies of a nucleic acid target with a plurality of oligonucleotide barcodes, wherein each oligonucleotide barcode comprises a first universal sequence, a molecular label, and a target-binding region capable of hybridizing to the nucleic acid target;generating a plurality of barcoded nucleic acid molecules each comprising the target-binding region and a complement of the target-binding region; (i) an oligonucleotide barcode of the plurality of oligonucleotide barcodes,', '(ii) the barcoded nucleic acid molecule itself, and', '(iii) a different barcoded nucleic acid molecule of the plurality of barcoded nucleic acid molecules;, 'hybridizing the complement of the target-binding region of each barcoded nucleic acid molecule with the target-binding region of one or more ofextending 3′-ends of the plurality of barcoded nucleic acid molecules to generate a plurality of extended barcoded nucleic acid molecules each comprising the first molecular label and a second molecular label;amplifying the plurality of extended barcoded nucleic acid molecules using a target-specific primer capable of hybridizing to a sequence of the nucleic acid target and a primer comprising ...

Compositions Including A Double Stranded Nucleic Acid Molecule And A Stem-Loop Oligonucleotide

The present invention concerns preparation of DNA molecules, such as a library, using a stem-loop oligonucleotide. In particular embodiments, the invention employs a single reaction mixture and conditions. In particular, at least part of the inverted palindrome is removed during the preparation of the molecules to facilitate amplification of the molecules. Thus, in specific embodiments, the DNA molecules are suitable for amplification and are not hindered by the presence of the palindrome. 154-. (canceled)55. A kit comprising:a stem-loop oligonucleotide comprising an inverted repeat and a loop;a polymerase; anda ligase.56. The kit according to claim 55 , wherein the stem loop oligonucleotide further comprises:(a) a non-replicable base or sequence; or(b) at least one deoxy-uridine.57. The kit according to claim 56 , wherein the stem loop oliogucleotide comprises a non-replicable base or sequence.58. The kit according to claim 57 , wherein at least part of the non-replicable base or sequence is present in the loop of the oligonucleotide or in a sequence of the stem adjacent to the loop.59. The kit according to claim 58 , wherein the non-replicable base or sequence comprises an abasic site or sequence claim 58 , hexaethylene glycol claim 58 , or a bulky chemical moiety attached to the sugar-phosphate backbone or the base.60. The kit according to claim 56 , wherein the stem loop oligonucleotide further comprises at least one deoxy-uridine.61. The kit according to claim 60 , wherein loop of the stem-loop oligonucleotide comprises at least one deoxy-uridine.62. The kit according to claim 61 , wherein the kit further comprises deoxy-uridine glycosylase.63. The kit according to claim 55 , wherein the ligase is a DNA ligase.64. The kit according to claim 55 , wherein the polymerase is a DNA polymerase.65. The kit according to claim 55 , wherein the stem-loop oligonucleotide comprises a 5′ OH.66. The kit according to claim 55 , wherein the kit further comprises an ...

Compartmentalized Central Dogma Activities Within Artificial Cell

The present invention provides cell-free systems comprising: (i) a transcription compartment; (ii) a barrier; and (iii) a translation compartment, methods for producing proteins and performing post-translational modifications thereon using such systems, and kits comprising such systems.

RAPID NUCLEIC ACIDS SEPARATION AND SAMPLE PREPARATION VIA HOLLOW-CENTERED SILICA MICROSPHERE

Disclosed herein are method for separating, amplifying, or detecting a nucleic acid from a sample may comprise contacting a sample lysate with a plurality of buoyant, inorganic, nucleic-acid-capture microspheres. The nucleic-acid-capture microspheres may comprise unicellular hollow microspheres having a diameter between 5 and 300 μm and/or a true particle density between 0.05 and 0.60 grams/cm. The microspheres may comprise hollow soda-lime-borosilicate microspheres. In some embodiments, the microspheres comprises hollow soda-lime-borosilicate microspheres surrounded by an amorphous silica shell. Also disclosed are kits for performing the methods. 143.-. (canceled)44. A method for separating a nucleic acid from a sample , the method comprising:(a) contacting a sample lysate with a plurality of nucleic-acid-capture microspheres to form a lysate dispersion; wherein the lysate dispersion comprises a lysate continuous phase and a particulate phase and wherein the particulate phase comprises the plurality of nucleic-acid-capture microspheres and an adsorbed nucleic acid obtained from the sample;(b) separating the lysate continuous phase from the particulate phase comprising the plurality of inorganic, nucleic-acid-capture microspheres and the adsorbed nucleic acid obtained from the sample; and(c) contacting the particulate phase with an eluent to form an eluate comprising the nucleic acid obtained from the sample.45. The method of claim 44 , wherein the plurality of nucleic-acid-capture microspheres comprise unicellular hollow microspheres having a diameter between 5 and 300 μm and a true particle density between 0.05 and 0.60 grams/cm.46. The method of claim 44 , wherein the plurality of nucleic acid capture microsphere comprise soda-lime-borosilicate microspheres.47. The method of claim 44 , wherein the separating step (b) comprises mechanically separating the lysate continuous phase from the particulate phase.48. The method of claim 47 , wherein the nucleic-acid- ...

ENHANCED NUCLEIC ACID IDENTIFICATION AND DETECTION

The present invention relates to assays, including amplification assays, conducted in the presence of modulators. These assays can be used to detect the presence of particular nucleic acid sequences. In particular, these assays can allow for genotyping or other genetic analysis. 156.-. (canceled)57. A method , comprising:providing a volume suspected of containing target nucleic acids;dispersing said volume among a plurality of areas, such that said plurality of areas comprises a distribution of nucleic acids said distribution capable of generating digital nucleic acid amplification signals; andconducting an isothermal nucleic acid amplification reaction in said plurality of areas in the presence of a modulator, wherein said modulator modulates said isothermal nucleic acid amplification in the presence of said target nucleic acids and wherein said modulator acts on said target nucleic acids within a region amplified by said isothermal nucleic acid amplification reaction.58. The method of claim 57 , wherein within said distribution claim 57 , said plurality of areas each comprise at most one of said target nucleic acids.59. The method of claim 57 , wherein said modulation comprises producing a difference in amplification efficiency.60. The method of claim 59 , wherein said difference in amplification efficiency produces a positive amplification signal in a subset of said plurality of areas.61. The method of claim 60 , wherein said positive amplification signal in said subset of said plurality of areas is diagnostic of the presence of one or more distinct target nucleic acids within said volume.62. The method of claim 57 , wherein said modulator comprises an agent selected from the group consisting of a restriction enzyme claim 57 , an oligonucleotide claim 57 , a ligase enzyme claim 57 , an engineered or non-natural nuclease claim 57 , a nucleic acid modifying enzyme claim 57 , an artificial nucleic acid or nucleic acid analog claim 57 , a modified nucleotide acid ...

METHODS FOR ACCURATE PARALLEL QUANTIFICATION OF NUCLEIC ACIDS IN DILUTE OR NON-PURIFIED SAMPLES

The present invention disclosure relates to a next generation DNA sequencing method and use for accurate and massively parallel quantification of one or more nucleic acid targets, for example in large volumes of unpurified sample material. More particularly, the invention is related to a method and a kit comprising probes for detecting and quantifying genetic targets in complex samples. The invention includes one or more target-specific nucleic acid probes per genetic target and a bridge oligo or bridge oligo complex. 1. A method for the high-throughput detection of one or more target nucleotide sequence in a plurality of samples , the method comprising the steps of:(i) providing for each target nucleotide sequence in each of the samples:a first probe, a second probe and a bridge oligo or a plurality of oligonucleotides capable of annealing to each other to form a bridge oligo complex,wherein the first probe comprises, starting from the 5′ end of the molecule, a first bridge oligo-specific sequence, optionally a first sequence barcode, and a first target specific portion at the 3′ end of first probe;and wherein the second probe comprises, starting from the 5′ end of the molecule, a second target specific portion, optionally a second sequence barcode, and a second bridge oligo-specific sequence at the 3′ end of second probe;and wherein the bridge oligo or bridge oligo complex contains sequences complementary to the first bridge oligo-specific sequence and the second bridge oligo-specific sequence in the first probe and the second probe, respectively, and optionally a third barcode;and wherein at least one of the first sequence barcode or the second sequence barcode or the third barcode is present in the first probe or the second probe or the bridge oligo or bridge oligo complex, respectively;and wherein at least one of the first probe or the second probe or the bridge oligo or bridge oligo complex comprises a first capture moiety,(ii) contacting, for each of the one ...

METHODS AND COMPOSITIONS FOR DETECTION OF SMALL RNAS

Currently, the circularization of small RNAs is broadly regarded as an obstacle in ligation-related assays and explicitly avoided while short lengths of linear RNA targets is broadly recognized as a factor limiting use of conventional primers in PCR-related assays. In contrast, the disclosed invention capitalizes on circularization of small RNA targets or their conjugates with oligonucleotide adapters. The circular RNA templates provide amplification of the target sequences via synthesis of multimer nucleic acids that can be either labeled for direct detection or subjected to PCR amplification and detection. Structure of small circular RNAs and corresponding multimeric nucleic acids provide certain advantages over current methods including flexibility in design of conventional RT and PCR primers as well as use of 5′-overlapping dimer-primers for efficient and sequence-specific amplification of short target sequences. Our invention also reduces number of steps and reagents while increasing sensitivity and accuracy of detection of small RNAs with both 2′OH and 2′-OMe at their 3′ ends. Our invention increase sensitivity and specificity of detection of microRNAs and other small RNAs with both 2′OH and 2′-OMe at their 3′ ends while allowing us to distinguish these two forms from each other. 146-. (canceled)47. A method of detecting the presence of at least one target RNA in a sample , said method comprising:a) ligating an adapter or linker oligonucleotide to the 3′ or 5′ end of the at least one target RNA to produce at least one extended target polynucleotide;b) circularizing the at least one extended target polynucleotide by ligating the 5′-end of the at least one extended target polynucleotide to its 3′-end to produce at least one circularized extended target polynucleotide; i) hybridizing said at least one circularized extended target polynucleotide with an oligonucleotide reverse transcriptase (RT) primer that is complementary to a part of the extended target ...

OLIGONUCLEOTIDES FOR USE IN DETERMINING THE PRESENCE OF TRICHOMONAS VAGINALIS IN A SAMPLE

Methods for use in multiplex amplification and or detection of . The multiphase amplification provides fast, quantitative, sensitive detection with lower variability at low analyte concentrations. Described are detection probes, capture probes, amplification oligonucleotides, nucleic acid compositions, probe mixes, methods, and kits useful for amplifying and determining the presence of in a test sample. 1T. vaginalis. An amplification oligonucleotide for use in amplifying a target nucleic acid sequence in a sample comprising: a promoter primer comprising a nucleic acid sequence having target specific sequence with at least 90% identity to SEQ ID NO: 42 , 43 , 44 , 45 , 46 , 47 , 48 , or a complement thereof , and having a promoter sequence for a T7 RNA polymerase joined at its 5′ end.2. The amplification oligonucleotide of claim 1 , wherein the promoter sequence for the T7 RNA polymerase comprises SEQ ID NO: 65 or 66.3. The amplification oligonucleotide of claim 1 , wherein the promoter primer comprises a nucleic acid sequence having at least 90% identity to SEQ ID NO: 4 claim 1 , 5 claim 1 , 6 claim 1 , 7 claim 1 , 8 claim 1 , 9 claim 1 , 10 claim 1 , 11 claim 1 , or 12.4. A set of amplification oligonucleotides comprising the amplification oligonucleotide of any one of - and one or more additional amplification oligonucleotides suitable for use in amplification of one or more additional target nucleic acids.5T. vaginalis. An amplification oligonucleotide or use in amplifying a target nucleic acid sequence in a sample comprising: a non-promoter primer claim 1 , wherein the non-promoter primer comprises a nucleic acid sequence having at least 90% identity to SEQ ID NO: 13 claim 1 , 14 claim 1 , 15 claim 1 , 16 claim 1 , 17 claim 1 , 18 claim 1 , 19 claim 1 , or a complement thereof.6. A set of amplification oligonucleotides comprising the non-promoter primer of and one or more additional non-promoter primers suitable for use in amplification of one or more ...

NUCLEIC ACID AMPLIFICATION AND TESTING

Methods for amplifying a target nucleic acid by self-sustained amplification methods are described. The methods are designed, in particular, to be carried out without use of specialised lab facilities or instruments. Compositions, lyophilised formulations, and kits for carrying out the methods are also described. 146-. (canceled)47. A method of nucleic acid amplification , comprising amplifying a target nucleic acid by a self-sustained transcription-based amplification reaction which is carried out at a temperature less than 50° C. , and in the presence of a reconstituted lyophilised formulation , wherein the lyophilised formulation comprises a polysaccharide , a low molecular weight saccharide , and enzyme activities required for carrying out the self-sustained amplification reaction , wherein the enzyme activities are provided by one or more enzymes , and wherein the lyophilised formulation does not include cofactors or substrates required for amplification of the target nucleic acid using the enzyme activities.48. A method of nucleic acid amplification , comprising amplifying a target nucleic acid by a self-sustained transcription-based amplification reaction which is carried out at a temperature less than 50° C. , and in the presence of a reconstituted lyophilised formulation , wherein the lyophilised formulation comprises a polysaccharide , a low molecular weight saccharide , and enzyme activities required for carrying out the self-sustained amplification reaction , wherein the enzyme activities are provided by one or more enzymes , and wherein the lyophilised formulation does not include a buffering agent.49. The method of claim 47 , wherein the lyophilised formulation does not include a buffering agent claim 47 , or the buffering agent is present at a concentration of less than 1 mM.50. The method of claim 47 , further comprising reconstituting the lyophilised formulation prior to amplifying the target nucleic acid.51. The method of claim 47 , wherein the ...

Automated cell processing methods, modules, instruments, and systems

In an illustrative embodiment, automated multi-module cell editing instruments are provided to automate multiple edits into nucleic acid sequences inside one or more cells.

SPLIT-CYCLE AND TAPE AMPLIFICATION

Methods and compositions are provided for improved nucleic acid amplification assays. In some embodiments, the nucleic acid amplification assay is a tagged amplicon primer extension (TAPE) nucleic acid amplification reaction. 1. A plurality of mixture partitions , the individual mixture partitions comprising: a) a 3′ hybridization region that specifically hybridizes to the mutant target sequence; and', 'b) a mutation-specific 5′ tail region that does not hybridize to any nucleic acid fragments in the nucleic acid sample;, 'i) a mutation-specific 5′-tailed primer pair, wherein the mutation-specific 5′-tailed primer pair hybridizes to and specifically amplifies target DNA template molecules from a nucleic acid sample that comprise a mutant target sequence, if present, wherein the primers of the mutation specific 5′-tailed primer pair comprise a) a 3′ hybridization region that specifically hybridizes to the wild-type target sequence; and', 'b) a wild-type specific 5′ tail region that does not hybridize to any nucleic acid fragments in the nucleic acid sample, wherein the wild-type specific 5′ tail region is a different sequence than the mutation-specific 5′ tail region; and, 'ii) a wild-type specific 5′-tailed primer pair, wherein the wild-type specific 5′-tailed primer pair hybridizes to and specifically amplifies target DNA template molecules comprising a wild-type target sequence, if present, wherein the primers of the wild-type specific 5′-tailed primer pair compriseiii) a mutation specific flanking primer pair, wherein the mutation specific flanking primer pair hybridizes to and specifically amplifies amplicons comprising the 5′ tail regions of the mutation specific 5′-tailed primer pair, if present;iv) a wild-type specific flanking primer pair, wherein the wild-type specific flanking primer pair hybridizes to and specifically amplifies amplicons comprising the 5′ tail regions of the wild-type specific 5′-tailed primer pair, if present; andv) a thermostable ...

RANDOM-PRIMED TRANSCRIPTASE IN-VITRO TRANSCRIPTION METHOD FOR RNA AMPLIFICATION

A random-primed reverse transcriptase-in vitro transcription method of linearly amplifying RNA is provided. According to the methods of the invention, source RNA (or other single-stranded nucleic acid), preferably, mRNA, is converted to double-stranded cDNA using two random primers, one of which comprises a RNA polymerase promoter sequence (“promoter-primer”), to yield a double-stranded cDNA that comprises a RNA polymerase promoter that is recognized by a RNA polymerase. Preferably, the primer for first-strand cDNA synthesis is a promoter-primer and the primer for second-strand cDNA synthesis is not a promoter-primer. The double-stranded cDNA is then transcribed into RNA by the RNA polymerase, optimally in the presence of a reverse transcriptase that is rendered incapable of RNA-dependent DNA polymerase activity during this transcription step. The subject methods produce linearly amplified RNA with little or no 3′ bias in the sequences of the nucleic acid population amplified. 1. A method for amplifying one or more single stranded nucleic acids , said method comprising:(a) contacting said one or more single stranded nucleic acids with a first set of oligonucleotides, each of said oligonucleotides in said first set comprising a promoter sequence and a sequence from a set of random sequences of at least 4 nucleotides, a second set of oligonucleotides, each of said oligonucleotides in said second set comprising one of a set of random sequences of at least four nucleotides and one or more enzymes that alone or in combination catalyze the synthesis of double-stranded cDNA, under-conditions suitable for the production of double-stranded cDNA; and(b) contacting the double-stranded cDNA produced in, step (a) with a RNA polymerase that recognizes said promoter sequence and ribonucleotides under conditions suitable to effect transcription, thereby producing sense or antisense RNA copies corresponding to said one or more single stranded nucleic acids.2. The method of claim 1 , ...

SAMPLE INDEXING FOR SINGLE CELLS

Disclosed herein include systems, methods, compositions, and kits for sample identification. A sample indexing composition can comprise, for example, a protein binding reagent associated with a sample indexing oligonucleotide. Different sample indexing compositions can include sample indexing oligonucleotides with different sequences. Sample origin of cells can be identified based on the sequences of the sample indexing oligonucleotides. Sample indexing oligonucleotides can be barcoded using barcoded and lengthened using daisy-chaining primers. 1. A composition , comprising a first oligonucleotide-associated antibody and a second oligonucleotide-associated antibody , whereinthe first oligonucleotide-associated antibody comprises a first antibody capable of specifically binding to a first protein target and is associated with a first sample indexing oligonucleotide, wherein the first sample indexing oligonucleotide comprises a binding site for a first target sequence, a binding site for a first universal primer, and a first sample indexing sequence, andthe second oligonucleotide-associated antibody comprises a second antibody capable of specifically binding to a second protein target and is associated with a second sample indexing oligonucleotide, wherein the second sample indexing oligonucleotide comprises a binding site for a second target sequence, a binding site for a second universal primer, and a second sample indexing sequence, andwherein the first sample indexing sequence and the second sample indexing sequence comprise different sequences, and the first protein target and the second protein target are the same.2. The composition of claim 1 , wherein the first antibody and the second antibody are the same.3. The composition of claim 1 , wherein (1) in the first sample indexing oligonucleotide claim 1 , the first sample indexing sequence is located between the binding site for the first target sequence and the binding site for the first universal primer claim ...

SPLIT-CYCLE AND TAPE AMPLIFICATION

Methods and compositions are provided for improved nucleic acid amplification assays. 2. The plurality of mixture partitions of claim 1 , wherein the mixture partitions comprise emulsion droplets.34-. (canceled)5. The plurality of mixture partitions of claim 1 , wherein at least about 10% and no more than about 90% of the plurality of mixture partitions comprise multiple target DNA template molecules.6. (canceled)7. The plurality of mixture partitions of wherein the target DNA template in the mixture partitions is at an average concentration of about 1-5 copies per partition.8. The plurality of mixture partitions of claim 1 , wherein at least one of the target DNA templates in the mixture partitions comprises a mutant target sequence.910-. (canceled)11. The plurality of mixture partitions of claim 1 , wherein the plurality comprises from about 500 to about 10 claim 1 ,000 claim 1 ,000 mixture partitions.12. (canceled)13. The plurality of mixture partitions of claim 1 , wherein the 3′ hybridization regions of the wild-type and mutant 5′-tailed primer pairs comprise annealing temperatures to the wild-type and mutant target DNA template molecules respectively that are at least about 5° C. less than the annealing temperatures of the mutation specific and wild-type specific flanking primers.1419-. (canceled)20. The plurality of mixture partitions of claim 1 , wherein the 3′ hybridization regions of the wild-type and mutant 5′-tailed primer pairs comprise annealing temperatures to the wild-type and mutant target DNA template molecules respectively that are at least about 5° C. more than the annealing temperatures of the mutation specific and wild-type specific flanking primers.2126-. (canceled)27. A method for quantitating a frequency of wild-type and mutant target nucleic acid fragments in a nucleic acid sample claim 1 , the method comprising:(a) forming a plurality of mixture partitions of any one of the preceding claims;(b) incubating the mixture partitions under ...

MULTIPHASE NUCLEIC ACID AMPLIFICATION

Improved methods for use in nucleic acid amplification, including multiplex amplification, where the amplification is carried out in two or more distinct phases are disclosed. The first phase amplification reaction preferably lacks one or more components required for exponential amplification. The lacking component is subsequently provided in a second, third or further phase(s) of amplification, resulting in a rapid exponential amplification reaction. The multiphase protocol results in faster and more sensitive detection and lower variability at low analyte concentrations. Compositions for carrying out the claimed methods are also disclosed. 1. A method of amplifying a target nucleic acid sequence present in a sample , the method comprising the steps of: 'wherein the first phase linear amplification reaction generates a first amplification product; and', '(a) performing a first phase linear amplification reaction that amplifies the target nucleic acid sequence under conditions that do not support exponential amplification thereof,'}(b) performing a second phase amplification reaction under conditions allowing exponential amplification of the first amplification product, thereby generating a second amplification product that comprises the target nucleic acid sequence or the complement thereof.2. The method of claim 1 ,wherein the first phase linear amplification reaction takes place in a first phase amplification reaction mixture,wherein the second phase amplification reaction takes place in a second phase amplification reaction mixture, andwherein the first phase amplification reaction mixture comprises an agent that inhibits exponential amplification of the target nucleic acid sequence.3. The method of claim 1 ,wherein the first phase linear amplification reaction takes place in a first phase amplification reaction mixture,wherein the second phase amplification reaction takes place in a second phase amplification reaction mixture, andwherein the second phase ...

METHODS AND COMPOSITIONS FOR DETECTION OF SMALL RNAS

Currently, the circularization of small RNAs is broadly regarded as an obstacle in ligation-related assays and explicitly avoided while short lengths of linear RNA targets is broadly recognized as a factor limiting use of conventional primers in PCR-related assays. In contrast, the disclosed invention capitalizes on circularization of small RNA targets or their conjugates with oligonucleotide adapters. The circular RNA templates provide amplification of the target sequences via synthesis of multimer nucleic acids that can be either labeled for direct detection or subjected to PCR amplification and detection. Structure of small circular RNAs and corresponding multimeric nucleic acids provide certain advantages over current methods including flexibility in design of conventional RT and PCR primers as well as use of 5′-overlapping dimer-primers for efficient and sequence-specific amplification of short target sequences. Our invention also reduces number of steps and reagents while increasing sensitivity and accuracy of detection of small RNAs with both 2′OH and 2′-OMe at their 3′ ends. Our invention increase sensitivity and specificity of detection of microRNAs and other small RNAs with both 2′OH and 2′-OMe at their 3′ ends while allowing us to distinguish these two forms from each other. 146-. (canceled)47. A method of detecting the presence of one or more target RNAs in a sample , said method comprising:a) circularizing said one or more target RNAs in said sample by ligating the 5′-end of each of said one or more target RNAs to its 3′-end to produce one or more circularized target RNAs, wherein the one or more circularized target RNAs is from 10-100 nucleotides; i) hybridizing said one or more circularized target RNAs with a set of oligonucleotide RT primers comprising at least one primer specific for each of said one or more target RNAs;', 'ii) enzymatically extending said oligonucleotide RT primers bound to said one or more circularized target RNAs by a polymerase ...

METHODS AND COMPOSITIONS FOR NUCLEIC ACID AMPLIFICATION

Compositions, reaction mixtures, and methods for performing an amplification reaction, including multiplex amplification reaction, wherein the method comprises using one or more amplification oligomer complexes comprising linked first and second amplification oligomer members. In one aspect, the amplification oligomer complex is hybridized to a target nucleic acid, the target nucleic acid with hybridized amplification oligomer complex is then captured, and other components are washed away. Target sequences of the target nucleic acids are pre-amplified to generate a first amplification product. The first amplification product is amplified in one or more secondary amplification reactions to generate second amplification products. 197-. (canceled)98. A method for simultaneously amplifying at least two different target nucleic acid sequences contained in a sample comprising the steps of: (i) wherein each of said target capture oligomers and each of said amplification oligomer complexes hybridize to different target nucleic acid sequences,', '(ii) wherein each of said amplification oligomer complexes comprises a non-promoter primer oligonucleotide having a first 3′ target specific sequence and a promoter oligonucleotide having a second 3′ target specific sequence and a 5′ promoter sequence, wherein the promoter oligonucleotide optionally comprises a blocked 3′ terminus, and wherein the non-promoter primer oligonucleotide is directly or indirectly joined to the promoter oligonucleotide;, '(a) contacting a sample with at least two different target capture oligomers and with at least two different amplification oligomer complexes;'}(b) contacting said at least two target capture oligomers in step (a) with a solid support and immobilized probe for performing a target capture;(c) washing said sample after target capture;(d) pre-amplifying said target nucleic acid sequences using said amplification oligomer complexes, under conditions configured to maintain the amplification ...

BEAD EMULSION NUCLEIC ACID AMPLIFICATION

Disclosed are methods for nucleic acid amplification wherein nucleic acid templates, beads, and amplification reaction solution are emulsified and the nucleic acid templates are amplified to provide clonal copies of the nucleic acid templates attached to the beads. Also disclosed are kits and apparatuses for performing the methods of the invention. 144.-. (canceled)45. A composition comprising a population of nucleic acid template-carrying beads each comprising a plurality of species of template nucleic acid molecules , wherein each bead comprises more than 10 ,000 amplified copies of the species of template nucleic acid molecules attached thereto , wherein the population of beads are suspended in a microreactor comprising a water-in-oil emulsion , and wherein a plurality of the microreactors includes only one bead.46. The composition of claim 45 , wherein the attachment of each of the amplified copies of the species of template nucleic acid molecules to the bead is mediated by chemical groups or oligonucleotides that are bound to the surface of the bead.47. The composition of claim 46 , wherein the attachment of each of the amplified copies of the species of template nucleic acid molecules is a covalent chemical attachment.48. The composition of claim 46 , wherein the attachment of each of the amplified copies of the species of template nucleic acid molecules is a non-covalent attachment.49. The composition of claim 45 , wherein each bead comprises a single nucleic acid species.50. The composition of claim 45 , wherein the beads are made from a material selected from the group consisting of cellulose claim 45 , cellulose derivatives claim 45 , acrylic resins claim 45 , glass claim 45 , silica gels claim 45 , polystyrene claim 45 , gelatin claim 45 , polyvinyl pyrrolidone claim 45 , co-polymers of vinyl and acrylamide claim 45 , polystyrene cross-linked with divinylbenzene claim 45 , polyacrylamide latex gels claim 45 , polystyrene claim 45 , dextran claim 45 , ...

METHODS FOR QUANTITATIVE AMPLIFICATION AND DETECTION OVER A WIDE DYNAMIC RANGE

Disclosed are compositions and methods for making differentiable amplicon species at unequal ratios using a single amplification system in a single vessel. The number of differentiable amplicons and their ratios to one another are chosen to span the required linear dynamic range for the amplification reaction and to accommodate limitations of the measuring system used to determine the amount of amplicon generated. Unequal amounts of distinguishable amplicon species are generated by providing unequal amounts of one or more amplification reaction components (e.g., distinguishable amplification oligomers, natural and unnatural NTP in an NTP mix, or the like). The amount of target nucleic acid present in a test sample is determined using the linear detection range generated from detection of one or more amplicon species having an amount within the dynamic range of detection. 2. The method of claim 1 , wherein the single amplification oligomer that hybridizes to one strand of the target nucleic acid is a promoter-based amplification oligomer.3. The method of claim 1 , wherein the single amplification oligomer that hybridizes to one strand of the target nucleic acid is a primer.4. The method of claim 1 , wherein the at least two amplification oligomers that hybridize to the complementary strand of the target nucleic acid are promoter-based amplification oligomers.5. The method of claim 1 , wherein the at least two amplification oligomers that hybridize to the complementary strand of the target nucleic acid are primers.6. The method of claim 1 , wherein the number of copies of each amplicon species differs by at least three orders of magnitude.7. The method of claim 1 , wherein the number of copies of each amplicon species differs by at least four orders of magnitude.8. The method of claim 1 , wherein the amounts of the at least two amplification oligomers hybridizing to the complementary strand of the target nucleic acid differ by at least two orders of magnitude.9. The ...

Compositions and methods for detecting human papillomavirus nucleic acid

Disclosed are nucleic acid oligomers, including amplification oligomers, capture probes, and detection probes, for detection of a human papillomavirus (HPV) nucleic acid. Also disclosed are methods of specific nucleic acid amplification and detection using the disclosed oligomers, as well as corresponding reaction mixtures and kits.

SPLIT-CYCLE AND TAPE AMPLIFICATION

Methods and compositions are provided for improved nucleic acid amplification assays. In some embodiments, the nucleic acid amplification assay is a tagged amplicon primer extension (TAPE) nucleic acid amplification reaction. 1. A reaction mixture for performing a tagged amplicon primer extension (TAPE) nucleic acid amplification reaction , the mixture comprising:(i) a target DNA template molecule from a nucleic acid sample, wherein the target DNA template molecule comprises a target sequence; (a) a 3′ hybridization region of at least 10 nucleotides in length and no more than 30 nucleotides in length that is configured to specifically hybridize to the target sequence of the target DNA template molecule and generate a first primer extension product in the nucleic acid amplification reaction; and', '(b) a 5′ tail region of at least 10 nucleotides in length that is not complementary to the target sequence of the target DNA template molecule;, '(ii) a forward primer comprising (a) a 3′ hybridization region of at least 10 nucleotides in length and no more than 30 nucleotides in length that is configured to specifically hybridize to the first primer extension product and generate a second primer extension product in in the nucleic acid amplification reaction; and', '(b) a 5′ tail region of at least 10 nucleotides in length that is not complementary to the target sequence of the target DNA template molecule, wherein the 5′ tail region of the reverse primer is a reverse complement of the 5′ tail region of the forward primer; and, '(iii) a reverse primer comprising(iv) a thermostable polymerase.2. The reaction mixture of claim 1 , wherein the thermostable polymerase comprises 3′ to 5′ exonuclease activity.3. The reaction mixture of claim 1 , wherein the 3′ hybridization region of the forward primer and/or reverse primer comprises a discriminatory nucleotide that is complementary to a mutant target DNA template molecule comprising a mutant target sequence but not ...

Polymerase Chain Reaction Primers and Probes for Mycobacterium Tuberculosis

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

METHODS FOR QUANTITATIVE AMPLIFICATION AND DETECTION OVER A WIDE DYNAMIC RANGE

Disclosed are compositions and methods for making differentiable amplicon species at unequal ratios using a single amplification system in a single vessel. The number of differentiable amplicons and their ratios to one another are chosen to span the required linear dynamic range for the amplification reaction and to accommodate limitations of the measuring system used to determine the amount of amplicon generated. Unequal amounts of distinguishable amplicon species are generated by providing unequal amounts of one or more amplification reaction components (e.g., distinguishable amplification oligomers, natural and unnatural NTP in an NTP mix, or the like). The amount of target nucleic acid present in a test sample is determined using the linear detection range generated from detection of one or more amplicon species having an amount within the dynamic range of detection. 2. The method of claim 1 , wherein the single amplification oligomer that hybridizes to one strand of the target nucleic acid is a promoter-based amplification oligomer.3. The method of claim 1 , wherein the single amplification oligomer that hybridizes to one strand of the target nucleic acid is a primer.4. The method of claim 1 , wherein the at least two amplification oligomers that hybridize to the complementary strand of the target nucleic acid are promoter-based amplification oligomers.5. The method of claim 1 , wherein the at least two amplification oligomers that hybridize to the complementary strand of the target nucleic acid are primers.6. The method of claim 1 , wherein the number of copies of each amplicon species differs by at least three orders of magnitude.7. The method of claim 1 , wherein the number of copies of each amplicon species differs by at least four orders of magnitude.8. The method of claim 1 , wherein the amounts of the at least two amplification oligomers hybridizing to the complementary strand of the target nucleic acid differ by at least two orders of magnitude.9. The ...

FAR-RED DYE PROBE FORMULATIONS

Disclosed are formulations, including both liquid and lyophilized formulations, comprising a far-red dye probe and a non-linear surfactant or foamban. Also disclosed are related methods for preparing a lyophilized far-red dye probe formulation as well as related kits and diagnostic products. 1. A stabilized far-red dye probe formulation comprising:a far-red dye probe comprising a far-red dye conjugated to a carrier molecule;a non-linear surfactant at a concentration of greater than about 0.05% (v/v); andat least one buffering agent;wherein the formulation is an aqueous solution.2. The formulation of claim 1 , wherein the far-red dye is a far-red cyanine dye.3. The formulation of claim 2 , wherein the far-red cyanine dye is selected from the group consisting of Cyanine5 and Cyanine5.5.4. The formulation of claim 1 , wherein the non-linear surfactant is selected from the group consisting of a polyoxyethylene sorbitan fatty acid ester and digitonin.5. The formulation of claim 4 , wherein the non-linear surfactant is a polyoxyethylene sorbitan fatty acid ester selected from the group consisting of polysorbate 20 claim 4 , polysorbate 40 claim 4 , and polysorbate 60.6. The formulation of claim 1 , wherein the non-linear surfactant concentration is from about 0.06% (v/v) to about 20% (v/v) claim 1 , from about 0.06% (v/v) to about 10% (v/v) claim 1 , from about 0.1% (v/v) to about 20% (v/v) claim 1 , or from about 0.1% (v/v) to about 10% (v/v).7. The formulation of claim 1 , wherein the non-linear surfactant concentration is from about 0.5% (v/v) to about 20% (v/v) claim 1 , from about 0.5% (v/v) to about 10% (v/v) claim 1 , from about 1% (v/v) to about 20% (v/v) claim 1 , or from about 1% (v/v) to about 10% (v/v).8. The formulation of claim 1 , wherein the at least one buffering agent is Tris.9. The formulation of claim 8 , wherein the Tris buffering agent is present at a concentration of from about 5 mM to about 50 mM.10. The formulation of claim 1 , wherein the carrier ...

NOVEL MIRNA BIOMARKERS AND USE THEREOF

The present invention relates to novel isolated nucleic acid molecules (novel miRNAs and novel miRNA precursor molecules) as well as vectors, host cells, primers, cDNA-transcripts, polynucleotides derived from said isolated nucleic acid molecules and their use in diagnosis and therapy. Furthermore the present invention relates to methods and kits for diagnosing a disease, such as Multiple Sclerosis (MS) or Alzheimer's Disease (AD) employing said novel isolated nucleic acid molecules (novel miRNAs molecules). 1. An isolated nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 2 , SEQ ID NO: 1 , SEQ ID NO: 3 to SEQ ID NO: 37 , a fragment thereof , and a nucleotide sequence with at least 90% , 94% , 96% or greater sequence identity thereto.2. An isolated nucleic molecule that is a complement to nucleic acid molecules of .3. A vector comprising an isolated nucleic acid molecule according to .4. A host cell transformed with an isolated nucleic acid molecule according .5. (canceled)6. A primer for reverse transcribing an isolated nucleic acid molecule of .7. A cDNA-transcript of an isolated nucleic acid molecule of .8. A set of primer pairs for amplifying a cDNA-transcript of .9. A polynucleotide for detecting an isolated nucleic acid molecule of .10. A cDNA-transcript of claim 1 , hybridized to an isolated nucleic acid molecule of .11. (canceled)12. A method for treating a disease comprising the step of:administering an effective amount of an isolated nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 1, SEQ ID NO: 3 to SEQ ID NO: 37, a fragment thereof, and a nucleotide sequence with at least 90%, 94%, 96% or greater sequence identity thereto to a subject in need thereof.13. A method for diagnosing and/or prognosing of a disease comprising the steps of:determining an expression profile of at least one nucleic acid molecule, which is differentially ...

ISOTHERMAL AMPLIFICATION USING OLIGOCATION-CONJUGATED PRIMER SEQUENCES

Provided herein are methods and kits for isothermal nucleic acid amplifications that use an oligocation-oligonucleotide conjugate primer for amplifying a target nucleic acid to generate amplicons. Isothermal DNA amplification methods that employ a strand displacing DNA polymerase and polyamine-oligonucleotide conjugate primer are also provided. 1. A kit for isothermal DNA amplification comprising:a DNA polymerase having strand displacement activity; andan oligocation-oligonucleotide conjugate primer, wherein the oligocation-oligonucleotide conjugate primer comprises an oligocation moiety covalently conjugated to the 5′ end of an oligonucleotide sequence.2. The kit of claim 1 , wherein the nucleotide sequence is a random oligonucleotide sequence.3. The kit of claim 1 , wherein the oligocation moiety comprises at least one spermine moiety.5. The kit of claim 1 , wherein the DNA polymerase is a Phi29 DNA polymerase.6. A kit for isothermal DNA amplification comprising:an oligocation-oligonucleotide conjugate primer;a DNA polymerase having a strand displacement activity;a dNTP mixture; andan oligocation-oligonucleotide conjugate primer, wherein the oligocation-oligonucleotide conjugate primer comprises an oligocation moiety covalently conjugated to the 5′ end of an oligonucleotide sequence and wherein the oligocation moiety comprises at least one lysine moiety.7. The kit of claim 6 , wherein the oligonucleotide sequence is a random oligonucleotide sequence or a partially constrained oligonucleotide sequence.8. The kit of claim 6 , wherein the oligonucleotide sequence comprises a nucleotide analogue.9. The kit of claim 6 , wherein the oligonucleotide sequence comprises a phosphorothioate linkage between a 3′ terminal nucleotide and a nucleotide that is adjacent to the 3′ terminal nucleotide.10. The kit of claim 9 , wherein the oligonucleotide sequence is NNNNN*N.11. The kit of claim 6 , wherein the oligocation-oligonucleotide conjugate primer comprises a random pentamer ...

Sample Preparation Vessels, Microfluidic Circuits, and Systems and Methods for Sample Preparation, Extraction, and Analysis

The invention generally provides a sample preparation vessel including a flexible substrate defining at least one sealable opening adapted and configured to receive a solid sample; at least one fitting; and at least one filter adjacent to the at least one fitting, the filter adapted and configured to permit extracted fluids to exit the vessel while retaining solid particles, as well as vessels, circuits, systems, and related methods for sample preparation, extraction, and analysis. 1. A sample preparation vessel comprising:a flexible substrate defining at least one sealable opening adapted and configured to receive a solid sample;at least one fitting; andat least one filter adjacent to the at least one fitting, the filter adapted and configured to permit extracted fluids to exit the vessel while retaining solid particles.2. A microfluidic circuit comprising:a fluidic path;a first row of windows, each window including a chamber and an optical lens dome on a first surface of the microfluidic circuit; andan outlet adapted and configured for coupling with additional rows of windows.3. A system comprising:a first port in fluid communication with at least one fluid reservoir and adapted and configured for removable coupling with a sample preparation vessel, the one or more ports collectively;a second port adapted and configured to receive a sample from a sample mixing circuit;a first receptacle adapted and configured to receive the sample preparation vessel; anda second receptacle adjacent to the first receptacle, the second receptacle adapted and configured to receive the sample mixing circuit and hold the sample mixing circuit in fluid communication with the sample preparation vessel.4. The system of claim 3 , further comprising a homogenizer adapted and configured to press against the sample preparation vessel and substantially homogenize the contents thereof.59-. (canceled)10. A method for extracting an analyte from a sample claim 3 , the method comprising:{'claim-ref ...

Extracellular mRNA Markers of Muscular Dystrophies in Human Urine

Described herein are methods for diagnosing and monitoring subjects with diseases associated with aberrant splicing, based upon detecting properly spliced isoforms and mis-spliced isoforms in a urine sample from the subject. 1. A method , comprising:obtaining a sample comprising urine from a subject who has, or is suspected to have, a disease associated with aberrant mRNA splicing;isolating extracellular mRNA in the sample;determining one or more selected mRNA in the sample, wherein the one or more selected mRNA is aberrantly spliced in the subject, and is suspected to be present in a plurality of spliced isoforms in the sample, wherein the spliced isoforms comprise properly spliced isoforms and mis-spliced isoforms;quantitating levels of the properly spliced isoforms and mis-spliced isoforms of the selected mRNA in the sample; anddetermining a ratio of the properly spliced isoforms to the mis-spliced isoforms in the sample.2. A method of diagnosing a disease associated with aberrant mRNA splicing , the method comprising obtaining a sample comprising urine from a subject who has , or is suspected to have , a disease associated with aberrant mRNA splicing;isolating extracellular mRNA in the sample;determining one or more selected mRNA in the sample, wherein the one or more selected mRNA is aberrantly spliced in the subject, and is suspected to be present in a plurality of spliced isoforms in the sample, wherein the spliced isoforms comprise properly spliced isoforms and mis-spliced isoforms;quantitating levels of the properly spliced isoforms and mis-spliced isoforms of the one or more selected mRNAs in the sample;determining a ratio of the properly spliced isoforms to the mis-spliced isoforms of the one or more selected mRNAs in the sample; andcomparing the ratio of properly spliced to mis-spliced in a subject to a reference ration, wherein a ratio in the subject that is less than the reference ratio indicates the presence of a disease associated with aberrant mRNA ...

DEVICE FOR PARTICLE MANIPULATION

An operation pipe and a device equipped with the operation pipe, which use a gel to perform operations such as separation, extraction, purification, elution, recovery, analysis and the like of target components that are biological components such as nucleic acids. More specifically, an operation pipe and a device, with which it is possible to perform operations such as separation, extraction, purification, elution, recovery, analysis and the like of target components in a sealable pipe by operating magnetic particles in the pipe under a magnetic field from outside of the pipe. 1. An operation pipe for operating target components , comprising:a hollow pipe, having a closable open end for supplying a sample containing the target components on one side and a closed end on the other side, and having an operation pipe portion on the open end side and a recovery pipe portion on the closed end side;an operation medium, which is filled in the operation pipe portion so that gel layers and aqueous liquid layers are alternately multi-layered in the longitudinal direction of the hollow pipe, wherein a layer length of the gel layers and a layer length of the aqueous liquid layers are determined by the length in the longitudinal direction of the hollow pipe;a recovery medium, which is filled in the recovery pipe portion so that a gel layer and an aqueous liquid layer which is in contact with the closed end are multi-layered, wherein the aqueous liquid layer in contact with the closed end has a predetermined volume, and the layer length of the gel layer is determined by the length in the longitudinal direction of the hollow pipe; andmagnetic particles for capturing and transporting the target components;wherein the magnetic particles pass through the gel layer in a gel state and move in the longitudinal direction of the operation pipe due to application of a magnetic field.2. The operation pipe according to claim 1 , wherein an inner diameter of the hollow pipe is 0.1 mm-5 mm.3. ...

Automated cell processing methods, modules, instruments, and systems

In an illustrative embodiment, automated multi-module cell editing instruments are provided to automate multiple edits into nucleic acid sequences inside one or more cells.

COMPOSITIONS AND METHODS FOR PROCESSING AND AMPLIFICATION OF DNA, INCLUDING USING MULTIPLE ENZYMES IN A SINGLE REACTION

The present invention concerns preparation of DNA molecules, such as a library, using a stem-loop oligonucleotide. In particular embodiments, the invention employs a single reaction mixture and conditions. In particular, at least part of the inverted palindrome is removed during the preparation of the molecules to facilitate amplification of the molecules. Thus, in specific embodiments, the DNA molecules are suitable for amplification and are not hindered by the presence of the palindrome. 154-. (canceled)55. A kit comprising:a stem-loop oligonucleotide comprising an inverted repeat and a loop, wherein the stem loop oligonucleotide further comprises:(a) a non-replicable base or sequence; or(b) at least one deoxy-uridine.56. The kit according to claim 55 , wherein the stem loop oligonucleotide comprises a non-replicable base or sequence.57. The kit according to claim 56 , wherein at least part of the non-replicable base or sequence is present in the loop of the oligonucleotide or in a sequence of the stem adjacent to the loop.58. The kit according to claim 55 , wherein the non-replicable base or sequence comprises an abasic site or sequence claim 55 , hexaethylene glycol claim 55 , or a bulky chemical moiety attached to the sugar-phosphate backbone or the base.59. The kit according to claim 55 , wherein the stem loop oligonucleotide further comprises at least one deoxy-uridine.60. The kit according to claim 59 , wherein loop of the stem-loop oligonucleotide comprises at least one deoxy-uridine.61. The kit according to claim 60 , wherein the kit further comprises deoxy-uridine glycosylase.62. The kit according to claim 55 , wherein the kit further comprises a ligase.63. The kit according to claim 55 , wherein the kit further comprises a DNA polymerase.64. The kit according to claim 55 , wherein the stem-loop oligonucleotide comprises a 5′ OH.65. The kit according to claim 55 , wherein the kit further comprises an endonuclease.66. The kit according to claim 55 , ...

DISEASE RESISTANT PLANT METHODS AND COMPOSITIONS

The present invention provides methods and compositions for producing elite lines of corn exhibiting anthracnose stalk rot (ASR) resistance. Also provided in the present invention are corn plants exhibiting ASR resistance resulting from such methods, and methods for breeding corn such that the ASR resistance traits may be transferred to a desired genetic background. 1. A method of obtaining a corn plant with enhanced anthracnose stalk rot resistance , said method comprising:a) obtaining a DNA sample from at least one corn plant or part thereof from a population of corn plants;b) detecting in the DNA sample the presence of an anthracnose stalk rot resistance allele, wherein said allele is within 5 cM of a “C” corresponding to position 101 of SEQ ID NO:96, and wherein the “C” at said position is associated with enhanced anthracnose stalk rot resistance;c) selecting at least a first plant comprising said allele and enhanced anthracnose stalk rot resistance compared to a plant lacking said allele;d) crossing the plant selected in step c) with a second corn plant, wherein said second corn plant lacks or is heterozygous for said anthracnose stalk rot resistance allele;e) collecting seeds from the cross of step d); andf) growing at least one progeny corn plant from the seeds of step e); wherein said progeny corn plant comprises said allele and has enhanced anthracnose stalk rot resistance compared to a corn plant lacking said allele.2. The method of claim 1 , wherein said selecting comprises detecting a polymorphism located in a chromosomal segment flanked by marker loci TIDP3136 and pzb00414.3. The method of claim 2 , wherein said polymorphism is located in a chromosomal segment flanked by marker loci SEQ ID NOs:4 and 54.4. The method of claim 2 , wherein said polymorphism is located in a chromosomal segment flanked by marker loci SEQ ID NO: 52 and SEQ ID NO: 8.5. The method of claim 4 , wherein said chromosomal segment is flanked by marker loci SEQ ID NO: 52 and SEQ ID ...

METHODS AND COMPOSITIONS FOR NUCLEIC ACID AMPLIFICATION

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

LIGATION-BASED RNA AMPLIFICATION

Methods of amplification, purification and detection of nucleic acid sequences especially RNA are described. One aspect of the method involves the hybridisation and subsequent ligation of a nucleic acid structure to the nucleic acid sequence desired to be manipulated. The methods require that the nucleic acid structure comprises a double stranded region and a single stranded region. The single stranded region is complementary to the RNA sequence of interest. The double stranded region may also contain additional functionalities which are then used subsequently in the method. 1. A method of producing a ligated nucleic acid molecule comprising:a) supplying RNA other than poly A;b) supplying one or more nucleic acids having a double stranded region and a single stranded 3′ terminal region; andc) hybridizing the single stranded 3′ terminal region of the nucleic acid sequence to the RNA and ligating one 5′ end of the double stranded region of the nucleic acid to the 3′ end of the RNA by enzymatic means.2. The method of claim 1 , wherein the nucleic acids of step b) comprise DNA.3. The method of claim 1 , wherein the nucleic acids incorporates one or more features selected from the group consisting of:a) a nucleotide sequence that can subsequently be used as a promoter sequence for RNA synthesis; andb) a Tag which can be used to label the nucleic acid or manipulate the nucleic acid.4. The method of claim 3 , further comprising transcribing the product obtained using the nucleotide sequence of 1) and of tag 2) with RNA polymerase to produce a 5′ sequence tagged cRNA.5. The method of claim 4 , wherein the 5′ tagged cRNA molecule is ligated with a second double stranded DNA sequence comprising a double stranded region and a single stranded region.6. The method of claim 5 , wherein the ligated RNA-DNA molecule product is further transcribed by RNA polymerase to produce multiple copies of RNA containing tags at the 5′ and 3′ end of the RNA molecule.7. The method of claim 6 , ...

Compositions and methods for detecting an rna virus

The present invention provides methods for rapidly identifying an RNA viral infection using an isothermal nucleic acid amplification reaction that can be carried out extracted RNA in the context of a crude biological sample.

METHODS AND COMPOSITIONS FOR NUCLEIC ACID AMPLIFICATION

Compositions, reaction mixtures, and methods for performing an amplification reaction, including multiplex amplification reaction, wherein the method comprises using one or more amplification oligomer complexes comprising linked first and second amplification oligomer members. In one aspect, the amplification oligomer complex is hybridized to a target nucleic acid, the target nucleic acid with hybridized amplification oligomer complex is then captured, and other components are washed away. Target sequences of the target nucleic acids are pre-amplified to generate a first amplification product. The first amplification product is amplified in one or more secondary amplification reactions to generate second amplification products. 188.-. (canceled)89. A target capture reaction mixture , wherein the reaction mixture comprises at least one target nucleic acid , at least one target capture oligonucleotide and at least one amplification oligomer complex , wherein each of said amplification oligomer complexes comprises a first amplification oligomer member having a first target specific sequence that is joined to a second amplification oligomer member having a second target specific sequence , wherein the first amplification oligomer member is a non-promoter primer and the second amplification oligomer member is a promoter primer , and wherein each of said target capture oligomers and each of said amplification oligomer complexes specifically hybridizes to a different target nucleic acid sequence.90. The target capture reaction mixture of claim 89 , further comprising at least one solid support.91. The target capture reaction mixture of claim 90 , wherein said solid support is a magbead.92. The target capture reaction mixture of claim 89 , wherein said at least one target capture oligomer is a wobble probe.93. The target capture reaction mixture of claim 89 , further comprising from about 0.05M to about 4.2M of an imidazolium compound.9497.-. (canceled)98. The target ...

COMPOSITIONS AND METHODS FOR PROCESSING AND AMPLIFICATION OF DNA, INCLUDING USING MULTIPLE ENZYMES IN A SINGLE REACTION

The present invention concerns preparation of DNA molecules, such as a library, using a stem-loop oligonucleotide. In particular embodiments, the invention employs a single reaction mixture and conditions. In particular, at least part of the inverted palindrome is removed during the preparation of the molecules to facilitate amplification of the molecules. Thus, in specific embodiments, the DNA molecules are suitable for amplification and are not hindered by the presence of the palindrome. 154-. (canceled)55. An oligonucleotide-attached nucleic acid molecule comprising:a double stranded nucleic acid molecule; anda stem-loop oligonucleotide comprising an inverted repeat and a loop;wherein one strand of the stem-loop oligonucleotide is attached to one strand of the double stranded nucleic acid molecule.56. The oligonucleotide-attached nucleic acid molecule according to claim 55 , wherein the double stranded nucleic acid molecule is a double stranded DNA molecule.57. The oligonucleotide-attached nucleic acid molecule according to claim 55 , wherein the oligonucleotide-attached nucleic acid molecule further comprises a non-covalent junction separating one strand of the stem-loop oligonucleotide from one strand of the double stranded nucleic acid molecule.58. The oligonucleotide-attached nucleic acid molecule according to claim 57 , wherein the non-covalent junction comprises a nick claim 57 , a gap claim 57 , or a 5′ flap structure.59. The oligonucleotide-attached nucleic acid molecule according to claim 55 , wherein the stem-loop oligonucleotide comprises a 5′ OH.60. The oligonucleotide-attached nucleic acid molecule according to claim 55 , wherein the stem-loop oligonucleotide further comprises:(a) a non-replicable base or sequence; or(b) at least one deoxy-uridine.61. The oligonucleotide-attached nucleic acid molecule according to claim 55 , wherein the stem loop oligonucleotide comprises a non-replicable base or sequence.62. The oligonucleotide-attached nucleic acid ...

METHODS AND COMPOSITIONS FOR NUCLEIC ACID AMPLIFICATION

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.

Automated cell processing methods, modules, instruments, and systems

In an illustrative embodiment, automated multi-module cell editing instruments are provided to automate multiple edits into nucleic acid sequences inside one or more cells.

Sample indexing for single cells

Disclosed herein include systems, methods, compositions, and kits for sample identification. A sample indexing composition can comprise, for example, a protein binding reagent associated with a sample indexing oligonucleotide. Different sample indexing compositions can include sample indexing oligonucleotides with different sequences. Sample origin of cells can be identified based on the sequences of the sample indexing oligonucleotides. Sample indexing oligonucleotides can be barcoded using barcoded and lengthened using daisy-chaining primers.

COMPOSITIONS AND METHODS FOR DETECTING HUMAN PAPILLOMAVIRUS NUCLEIC ACID

Disclosed are nucleic acid oligomers, including amplification oligomers, capture probes, and detection probes, for detection of a human papillomavirus (HPV) nucleic acid. Also disclosed are methods of specific nucleic acid amplification and detection using the disclosed oligomers, as well as corresponding reaction mixtures and kits. 1. A capture probe oligomer for selectively separating a human papillomavirus type 33 (HPV33) or type 31 (HPV31) target nucleic acid from other components in a sample , wherein the capture probe oligomer is selected from the group consisting of(a) an HPV33-specific capture probe oligomer comprising a target-hybridizing sequence consisting of SEQ ID NO:50; and(b) an HPV31-specific capture probe oligomer comprising a target-hybridizing sequence consisting of SEQ ID NO:52;wherein the capture probe oligomer further comprises an immobilized probe-binding region that is not complementary to the HPV33 or HPV31 target nucleic acid.2. The capture probe oligomer of claim 1 , wherein said capture probe oligomer is the HPV33-specific capture probe oligomer.3. The capture probe oligomer of claim 1 , wherein said capture probe oligomer is the HPV31-specific capture probe oligomer.4. The capture probe oligomer of claim 1 , wherein the immobilized probe-binding region comprises a homopolymeric tail of about 10 to 40 nucleotides.5. The capture probe oligomer of claim 4 , wherein the immobilized probe-binding region is selected from the group consisting of Ato Aand TAto TA.6. The capture probe oligomer of claim 5 , whereinthe nucleotide sequence of the HPV33-specific capture probe oligomer consists of SEQ ID NO:51, and/orthe nucleotide sequence of the HPV31-specific capture probe oligomer consists of SEQ ID NO:53.7. The capture probe oligomer of claim 1 , where the capture probe oligomer is contained within a target capture reaction mixture.8. The capture probe oligomer of claim 6 , wherein the target capture reaction mixture comprises both the HPV33- ...

COMPOSITIONS, METHODS AND KITS TO DETECT HERPES SIMPLEX VIRUS NUCLEIC ACID

The disclosed invention is related to methods, compositions, kits and isolated nucleic acid sequences for targeting Herpes Simplex Virus (HSV) nucleic acid (eg. HSV-1 and/or HSV-2 nucleic acid). Compositions include amplification oligomers, detection probe oligomers and/or target capture oligomers. Kits and methods comprise at least one of these oligomers. 183-. (canceled)84. The isolated nucleic acid of substantially corresponding to the sequence set forth in any of SEQ ID Nos: 13 claim 88 , 14 claim 88 , 19 claim 88 , and 20.85. The isolated nucleic acid of claim 84 , comprising claim 84 , consisting or consisting essentially of the sequence set forth in SEQ ID Nos: 3 claim 84 , 4 claim 84 , 5 claim 84 , 6 claim 84 , 7 claim 84 , 8 claim 84 , 9 claim 84 , 10 claim 84 , 11 claim 84 , 12 claim 84 , 13 claim 84 , 14 claim 84 , 15 claim 84 , 16 claim 84 , 17 claim 84 , 18 claim 84 , 19 claim 84 , 20 claim 84 , 21 or 22 or the corresponding isolated RNA sequence.86. The isolated DNA or RNA sequence according to claim 84 , wherein said sequence is the complement or the reverse complement thereof.87. An isolated nucleic acid of comprising claim 88 , consisting or consisting essentially of a nucleic acid sequence corresponding to nucleotides 124 to 230 of SEQ ID NO:1 or corresponding to nucleotides 113 to 200 of SEQ ID NO:2.88. An isolated nucleic acid substantially corresponding to a nucleic acid sequence corresponding to nucleotides 124 to 230 of SEQ ID NO:1 or corresponding to nucleotides 113 to 200 of SEQ ID NO:2 claim 88 , wherein the isolated nucleic acid is linked to a label which leads to a detectable signal.89. The isolated nucleic acid sequence according to claim 88 , wherein the sequence is the complement or the reverse complement thereof.90. The isolated nucleic acid sequence according to claim 88 , wherein the sequence is RNA.91. The isolated nucleic acid sequence according to claim 88 , wherein the sequence is double stranded DNA.92. The isolated nucleic ...

ENHANCED NUCLEIC ACID IDENTIFICATION AND DETECTION

The present invention relates to assays, including amplification assays, conducted in the presence of modulators. These assays can be used to detect the presence of particular nucleic acid sequences. In particular, these assays can allow for genotyping or other genetic analysis. 156.-. (canceled)58. The method of claim 57 , further comprisingafter the providing and prior to the conducting, dispersing said volume among a plurality of areas, such that said plurality of areas comprises a distribution of nucleic acids said distribution capable of generating digital nucleic acid amplification signals,wherein the conducting is performed in the dispersed volume in said plurality of areas.59. The method of claim 58 , wherein said plurality of areas each comprises at most one copy of said target nucleic acid.60. The method of claim 58 , further comprising prior to the conducting claim 58 , providing a modulator to said plurality of areas.61. The method of claim 57 , wherein the providing comprises providing a first volume comprising a first nucleic acid and a second volume comprising a second nucleic acid.62. The method of claim 61 , further comprisingafter the providing and prior to the conducting, dispersing said first volume among a plurality of first areas and dispersing said second volume among a plurality of second areas,wherein the conducting is performed in the dispersed first volume in said plurality of first areas and in the dispersed second volume in said plurality of second areas.63. The method of claim 62 , wherein said plurality of first areas each comprise at most one copy of said first nucleic acid and said plurality of second areas each comprise at most one copy of said second nucleic acid.64. The method of claim 62 , further comprising prior to the conducting claim 62 , providing a modulator to said plurality of first areas or to said plurality of second areas.65. The method of claim 61 , further comprising detecting a difference in said isothermal nucleic ...

COMPOSITIONS AND METHODS FOR DETECTION OF HEPATITIS A VIRUS NUCLEIC ACID

Nucleic acid oligomeric sequences and in vitro nucleic acid amplification and detection methods for detecting the presence of HAV RNA sequences in samples are disclosed. Kits comprising nucleic acid oligomers for amplifying and detecting HAV nucleic acid sequences are disclosed. 1. A combination of at least two oligomers for amplifying a HAV target region comprising:a first oligomer comprising a target-specific sequence of about 24 to 28 nt contained in the sequence of SEQ ID NO:147 that includes at least the sequence of SEQ ID NO:148, and optionally joined at its 5′ end to a promoter sequence, and a second oligomer comprising a target-specific sequence of about 24 to 30 nt, contained in the sequence of SEQ ID NO:157 and that includes at least the sequence of SEQ ID NO:158.23.-. (canceled)4. The combination of at least two oligomers of claim 1 , wherein the first oligomer comprises a sequence consisting of: SEQ ID NO:31 claim 1 , or SEQ ID NO:32 claim 1 , or comprises a target specific sequence consisting of SEQ ID NO:61 further comprising a promoter sequence attached to its 5′ end claim 1 , SEQ ID NO:62 further comprising a promoter sequence attached to its 5′ end claim 1 , or SEQ ID NO:148 further comprising a promoter sequence attached to its 5′ end; and the second oligomer comprises a target specific sequence of SEQ ID NO:89 claim 1 , SEQ ID NO:90 claim 1 , or SEQ ID NO:91.58.-. (canceled)9. The combination of at least two oligomers of claim 1 , further comprising at least one capture probe oligomer selected from the group consisting of SEQ ID NOS:1 to 7 and SEQ ID NOS:8-14 linked to a moiety that allows it to bind a solid support.10. The combination of at least two oligomers of claim 1 , further comprising at least one detection probe oligomer comprising a target-specific sequence selected from the group consisting of SEQ ID NO:115; optionally wherein said nucleic acid detection oligomer further comprises a 2′-O-methyl linkages claim 1 , a chemiluminescent ...

SAMPLE INDEXING FOR SINGLE CELLS

Disclosed herein include systems, methods, compositions, and kits for sample identification. A sample indexing composition can comprise, for example, a protein binding reagent associated with a sample indexing oligonucleotide. Different sample indexing compositions can include sample indexing oligonucleotides with different sequences. Sample origin of cells can be identified based on the sequences of the sample indexing oligonucleotides. Sample indexing oligonucleotides can be barcoded using barcoded and lengthened using daisy-chaining primers. 130.-. (canceled)31. A method for cell identification , comprising: wherein each of the first plurality of cells and each of the second plurality of cells comprises one or more cellular component targets,', 'wherein each of the two sample indexing compositions comprises a cellular component binding reagent associated with a sample indexing oligonucleotide,', 'wherein the cellular component binding reagent is capable of specifically binding to at least one of the one or more cellular component targets, and', 'wherein the sample indexing oligonucleotide comprises a sample indexing sequence, and wherein sample indexing sequences of the two sample indexing compositions comprise different sequences;, '(a) contacting a first plurality of cells and a second plurality of cells with two sample indexing compositions, respectively,'} 'wherein the plurality of barcodes each comprises a cell label sequence, and wherein at least two of the plurality of barcodes comprise an identical cell label sequence;', '(b) barcoding the sample indexing oligonucleotides using a plurality of barcodes to create a plurality of barcoded sample indexing oligonucleotides,'}(c) obtaining sequencing data of the plurality of barcoded sample indexing oligonucleotides;(d) identifying a cell label sequence associated with two or more sample indexing sequences in the sequencing data obtained; and(e) removing sequencing data associated with the cell label sequence ...

LINEAR DOUBLE STRANDED DNA COUPLED TO A SINGLE SUPPORT OR A TAG AND METHODS FOR PRODUCING SAID LINEAR DOUBLE STRANDED DNA

The present invention is concerned with linear double stranded DNA, which is coupled to a single support or a tag at the 3′ end of its non-coding strand and methods for producing said linear double stranded DNA. The present invention further relates to the use of said linear double stranded DNA in an RNA in vitro transcription reaction and also to a method for producing RNA in vitro. The present invention also relates to a bioreactor for RNA in vitro transcription. 1. A linear double stranded DNA comprising a coding strand and a non-coding strand , wherein said DNA comprises (i) a coding sequence element encoded by the coding strand in the direction of from 5′ to 3′ of the coding strand and (ii) an RNA polymerase promotor sequence element upstream of the coding sequence element , wherein said non-coding strand is coupled at its 3′ end to a support or a tag , and wherein said support or tag is the only support or tag coupled to said DNA.2. The linear double stranded DNA according to claim 1 , wherein said non-coding strand is coupled at its 3′ end to a support or a tag via a triazole.3. The linear double stranded DNA according to or claim 1 , wherein said tag is biotin.4. The linear double stranded DNA according to claim 3 , wherein said biotin is associated with streptavidin claim 3 , preferably a streptavidin coated bead claim 3 , most preferably a streptavidin coated magnetic bead.5. The linear double stranded DNA according to any one of to claim 3 , wherein the coding sequence element is flanked by a 5′ UTR and/or a 3′ UTR element.6. A method for producing linear double stranded DNA comprising a coding strand and a non-coding strand claim 3 , wherein said non-coding strand is coupled at its 3′ end to a support or a tag claim 3 , comprising the steps of:(a) providing linear double stranded DNA comprising a coding sequence element encoded by the coding strand, followed at the 3′ end by a restriction site element;(b) incubating said DNA with (i) a modified ...

SYSTEMS AND METHODS FOR PRE-ANALYTICAL SUBSTRATE PROCESSING

Some embodiments presented in this disclosure concern an Automated Tissue Dissection (ATD) System. An ATD system is a one stop, and potentially low-cost, system to perform dissections on a substrate from pathologist digital mark or pen mark on the substrate using non-contact and/or mechanical method to extract a Formalin-Fixed Paraffin-Embedded (FFPE) tissue sample with: (a) only the ROI or ROIs as area to be saved; and (b) remove or decompose nucleic acid content in the region of no interest (RONI) and collect all tissue sample from a standard microscope substrate into a specific container. 1. A method of processing a biological sample for a biological assay , comprising (a) contacting the biological sample with a contact medium comprising a particulate substance and pressurized air under conditions sufficient to effectuate at least partial transfer of a component in the biological sample to the contact medium; and (b) removing the contact medium from the biological sample.2. The method of claim 1 , wherein the biological sample is processed for analysis of one or more analytes of diagnostic interest.3. The method of claim 1 , wherein the biological sample comprises punch biopsy specimens claim 1 , needle biopsy specimens claim 1 , fresh tissues claim 1 , tissue cultures claim 1 , frozen tissue specimen claim 1 , neutral formalin-treated tissues claim 1 , organs claim 1 , organelles claim 1 , formalin fixed paraffin embedded (FFPE) tissues claim 1 , ethanol-fixed paraffin-embedded (EFPE) tissues claim 1 , hematoxylin and eosin (H&E) stained tissues claim 1 , or glutaraldehyde fixed tissues.4. The method of claim 1 , comprising contacting a region of interest (ROI) claim 1 , a region of non-interest (RONI) claim 1 , or all regions in the biological sample with the contact medium; preferably contacting a region of interest (ROI) with the contact medium.5. The method of claim 1 , wherein the biological sample comprises at least one analyte of diagnostic interest ...

Improved method for nucleic acid amplification by NASBA

Selective terminal tagging of nucleic acids

A method is provided for adding a terminal sequence tag to nucleic acid molecules for use in RNA or DNA amplification. The method involves contacting with a mixture of oligonucleotides, each having a sequence tag template, a random sequence and a blocked 3′ terminus, under conditions such that, the random sequence anneals with the nucleic acid molecules and the nucleic acid molecules are extended using the sequence tag template as template. For synthesis of RNA from DNA molecules having terminal sequence tags, the method includes forming DNA templates having a double stranded promoter sequence and synthesizing RNA from the DNA templates. For amplification of sequences from DNA molecules having terminal sequence tags, the method includes forming DNA templates by extension of one primer having a sequence that is complementary to the terminal sequence tag and another primer having a sequence that is derived form one of the DNA molecules.

Array-based methods for producing ribonucleic acids

Methods and compositions for generating pluralities of ribonucleic acids are provided. In the subject methods, an array is employed as a template in an in vitro transcription reaction. Also provided are the arrays employed in the subject methods and kits for practicing the subject methods. The ribonucleic acid pluralities produced by the subject methods find use in a variety of different applications, including differential gene expression analysis and gene-silencing applications.

Single-primer nucleic acid amplification methods

The present invention is directed to novel methods of synthesizing multiple copies of a target nucleic acid sequence which are autocatalytic (i.e., able to cycle automatically without the need to modify reaction conditions such as temperature, pH, or ionic strength and using the product of one cycle in the next one). In particular, the present invention discloses a method of nucleic acid amplification which is robust and efficient, while reducing the appearance of side-products. The method uses only one primer, the “priming oligonucleotide,” a promoter oligonucleotide modified to prevent polymerase extension from its 3′-terminus and, optionally, a means for terminating a primer extension reaction, to amplify RNA or DNA molecules in vitro, while reducing or substantially eliminating the formation of side-products. The method of the present invention minimizes or substantially eliminates the emergence of side-products, thus providing a high level of specificity. Furthermore, the appearance of side-products can complicate the analysis of the amplification reaction by various molecular detection techniques. The present invention minimizes or substantially eliminates this problem, thus providing an enhanced level of sensitivity.

Transcription-based nucleic acid amplification / detection system

Assay for detection of human parvovirus B19 nucleic acid

Nucleic acid oligomers specific for human parvovirus B19 genomic DNA are disclosed. An assay for amplifying and detecting human parvovirus B19 nucleic acid in biological specimens is disclosed. Compositions for detecting the presence of parvovirus B19 genomic DNA in human biological specimens are disclosed.

Modified oligonucleotides and methods for determining the presence of a nucleic acid analyte in a sample

The present invention concerns oligonucleotides containing one or more modified nucleotides which increase the binding affinity of the oligonucleotides to target nucleic acids having a complementary nucleotide base sequence. These modified oligonucleotides hybridize to the target sequence at a faster rate than unmodified oligonucleotides having an identical nucleotide base sequence. Such modified oligonucleotides include oligonucleotides containing at least one 2′-O-methylribofuranosyl moiety joined to a nitrogenous base. Oligonucleotides can be modified in accordance with the present invention to preferentially bind RNA targets. The present invention also concerns methods of using these modified oligonucleotides and kits containing the same.

Compositions, methods and kits for determining the presence of Trichomonas vaginalis in a test sample

The present invention relates to oligonucleotides useful for determining the presence of Trichomonas vaginalis in a test sample. The oligonucleotides of the present invention may be incorporated into detection probes, helper probes, capture probes and amplification oligonucleotides, and used in various combinations thereof.

Method for the sensitive detection of nucleic acids

Methods of amplifying and sequencing nucleic acids

An apparatus and method for performing rapid DNA sequencing, such as genomic sequencing, is provided herein. The method includes the steps of preparing a sample DNA for genomic sequencing, amplifying the prepared DNA in a representative manner, and performing multiple sequencing reaction on the amplified DNA with only one primer hybridization step.

Methods of amplifying and sequencing nucleic acids

An apparatus and method for performing rapid DNA sequencing, such as genomic sequencing, is provided herein. The method includes the steps of preparing a sample DNA for genomic sequencing, amplifying the prepared DNA in a representative manner, and performing multiple sequencing reaction on the amplified DNA with only one primer hybridization step.

Methods of amplifying and sequencing nucleic acids

An apparatus and method for performing rapid DNA sequencing, such as genomic sequencing, is provided herein. The method includes the steps of preparing a sample DNA for genomic sequencing, amplifying the prepared DNA in a representative manner, and performing multiple sequencing reaction on the amplified DNA with only one primer hybridization step.

Methods of amplifying and sequencing nucleic acids

An apparatus and method for performing rapid DNA sequencing, such as genomic sequencing, is provided herein. The method includes the steps of preparing a sample DNA for genomic sequencing, amplifying the prepared DNA in a representative manner, and performing multiple sequencing reaction on the amplified DNA with only one primer hybridization step.

Methods of amplifying and sequencing nucleic acids

An apparatus and method for performing rapid DNA sequencing, such as genomic sequencing, is provided herein. The method includes the steps of preparing a sample DNA for genomic sequencing, amplifying the prepared DNA in a representative manner, and performing multiple sequencing reaction on the amplified DNA with only one primer hybridization step.

Methods of amplifying and sequencing nucleic acids

An apparatus and method for performing rapid DNA sequencing, such as genomic sequencing, is provided herein. The method includes the steps of preparing a sample DNA for genomic sequencing, amplifying the prepared DNA in a representative manner, and performing multiple sequencing reaction on the amplified DNA with only one primer hybridization step.

Stabilization of antimicrobial coatings

Described herein is a coated building panel comprising: a substrate comprising a first major surface opposite a second major surface; a coating atop at least one of the first major surface or the second major surface, the coating comprising: a binder composition; an antimicrobial composition comprising a cationic compound; a stabilization composition comprising: a silicate compound; and a surfactant having an HLB value between about 10 and about 14.

Method of assay of target nucleic acid

A simple and accurate method for assay of a single-stranded RNA containing a specific nucleic acids sequence in a sample at almost constant temperature by using at least the following reagents (A) to (I), which comprises a step of adding the reagents (A) to (I) one by one (in any order), in combinations of at least two or all at once and a step of measuring a fluorescent signal in the presence of the reagent (I) at least once after addition of at least the reagents (A) to (H); (A) a first single-stranded oligonucleic acid complementary to a sequence neighboring the 5' end of the specific nucleic acids sequence in the single-stranded RNA, (B) a second single-stranded oligo DNA complementary to a 3'-end sequence within the specific nucleic acids sequence, (C) an RNA-dependent DNA polymerase, (D) deoxyribonucleoside triphosphates, (E) a third single-stranded oligo DNA having (1) a promoter sequence for a DNA-dependent RNA polymerase, (2) an enhancer sequence for the promoter and (3) a 5'-end sequence within the specific nucleic acids sequence, in this order from the 5' end, (F) a DNA-dependent DNA polymerase, (G) a DNA-dependent RNA polymerase, (H) ribonucleoside triphosphates, and (I) a fourth single-stranded oligo DNA complementary to the specific nucleic acids sequence which is labeled so that it gives off a measurable fluorescent signal on hybridization with a nucleic acid containing the specific nucleic acids sequence.

Quantification method of target nucleic acid

In vitro rna transcription bioreactor

FIELD: biotechnology.SUBSTANCE: invention relates to biotechnology. Described is a bioreactor for RNA transcriptionin vitro. Reactor for RNA transcriptionin vitro,designed to operate in an automated manner in conditions corresponding to GMP principles. In particular, said reactor for RNA transcriptionin vitroallows multiple use of DNA matrix for various reactions of RNA transcriptionin vitro. Disclosed is the use of the described bioreactor for RNA transcriptionin vitro. Disclosed is a method of RNA transcriptionin vitro.Described is a module for transcription of DNA into RNA, as well as an automated device for producing RNA, containing the described bioreactor. Apparatus for producing RNA comprises (a) a module for synthesis of a DNA matrix, (b) a module for transcribing DNA into RNA, comprising said reactor for transcribing RNAin vitro, and optionally (c) a module for generating RNA.EFFECT: invention extends the range of means for RNA transcription.75 cl, 20 dwg, 1 ex РОССИЙСКАЯ ФЕДЕРАЦИЯ (19) RU (11) (13) 2 765 877 C1 (51) МПК C12N 15/10 (2006.01) C12Q 1/6865 (2018.01) ФЕДЕРАЛЬНАЯ СЛУЖБА ПО ИНТЕЛЛЕКТУАЛЬНОЙ СОБСТВЕННОСТИ (12) ОПИСАНИЕ ИЗОБРЕТЕНИЯ К ПАТЕНТУ (52) СПК B01F 13/0809 (2021.08); B01F 13/0818 (2021.08); B01F 15/065 (2021.08); B01F 15/00896 (2021.08) (21)(22) Заявка: 2020141236, 28.06.2019 (24) Дата начала отсчета срока действия патента: Дата регистрации: 04.02.2022 28.06.2018 EP PCT/EP2018/067504 (73) Патентообладатель(и): КЬЮРВАК АГ (DE), ТЕСЛА АУТОМАЦИОН ГМБХ (DE) (45) Опубликовано: 04.02.2022 Бюл. № 4 (85) Дата начала рассмотрения заявки PCT на национальной фазе: 28.01.2021 (86) Заявка PCT: 2 7 6 5 8 7 7 R U (87) Публикация заявки PCT: WO 2020/002598 (02.01.2020) Адрес для переписки: 129090, Москва, ул. Б. Спасская, 25, стр. 3, ООО "Юридическая фирма Городисский и Партнеры" (54) БИОРЕАКТОР ДЛЯ ТРАНСКРИПЦИИ РНК IN VITRO (57) Реферат: Изобретение относится к биотехнологии. для транскрипции ДНК в РНК, а также Описан биореактор для транскрипции РНК in ...