The present disclosure relates, according to some embodiments, to compositions, methods, and/or kits for producing vaccinia capping enzyme. For example, active, heterodimers of vaccinia capping enzyme may be produced as fusions comprising D1 and D12 subunits. Vaccinia capping enzyme fusion proteins may further comprise a linker.
The present disclosure relates to compositions, kits, and methods of making RNA vaccines having an appropriate cap structure. Systems, apparatus, compositions, and/or methods may include and/or use, in some embodiments, non-naturally occurring single-chain RNA capping enzymes. In some embodiments, an RNA capping enzyme may include an FCE variant having (a) an amino acid sequence at least 90% identical to positions 1 to 878 of SEQ ID NO: 1, and/or (b) one or more substitutions relative to SEQ ID NO: 1 at a position selected from positions corresponding to positions 215, 337, 572, 648, and 833 (e.g., a position selected from positions corresponding to position 215, 337, and 572) of SEQ ID NO: 1.
Compositions and methods are described that are directed to specific and sensitive methods of target nucleic acid detection and more specifically detecting target nucleic acids directly from biological samples. The compositions and methods were developed to be easy to use involving a minimum number of steps and giving rapid and consistent results either at point of care or in high throughput situations. The compositions and methods are directed to labelled probes and their uses in Loop-Mediated Isothermal Amplification (LAMP) diagnostic tests to detect target DNA from the environment or from an individual and also to detect specific variants of the target DNA, both with similar sensitivity. The compositions and methods may use any single improvement or combination of improvements selected from thermolabile enzyme variants, poloxamers, various salts, indicators and one or more LAMP primer sets for detecting single and/or multiple targets, probes for detecting variants of the targets including SARS-CoV-2 variants and lateral flow devices.
C12Q 1/70 - Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
G01N 21/78 - Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
Compositions, methods and kits are provided that include an inhibitory oligonucleotide RNase inhibitor capable of inhibiting one or more types of RNase that coexist with biological samples or are introduced in the laboratory, thereby protecting RNA in the sample from degradation. More than one type of oligonucleotide RNase inhibitor may be combined in a mixture to inhibit a plurality of different RNases. Single oligonucleotides were identified to have inhibitory activity for a plurality of different RNases. The RNase oligonucleotide inhibitor may be immobilized on beads or other surface. It may be stored in a lyophilized form or in solution.
C12Q 1/6848 - Nucleic acid amplification reactions characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
Provided herein, among other things, is a method for deaminating a double-stranded nucleic acid. In some embodiments, the method may comprise contacting a double-stranded DNA substrate that comprises cytosines and a double-stranded DNA deaminase having an amino acid sequence that is at least 80% identical to any of SEQ ID NOS: 21, 40, 47, 49, 50, 55, 58, 59, 62, 63, 65, 67, 70, 71, 76, 106, 107, 110, 112, 114, 117, 163 and/or 164 to produce a deamination product that comprises deaminated cytosines. Enzymes and kits for performing the method are also provided.
The present disclosure relates, according to some embodiments, to compositions and analysis of RNA (e.g., dephosphorylated oligoribonucleotides) including, for example, natural and/or synthetic RNAs. A composition may comprise, for example, an endoribonuclease having an amino acid sequence that (i) corresponds to an amino acid sequence of a first species (e.g., Homo sapiens, Escherichia coli, Aspergillus oryzae, Momordica charantia, Pyrococcus furiosus, Cucumis sativus, and Sus scrofa) or (ii) is a non-naturally occurring sequence; and/or an RNA end repair enzyme having an amino acid sequence that (i) corresponds to an amino acid sequence of a species other than the first species (e.g., a bacterial species or a bacteriophage species) or (ii) is a non-naturally occurring sequence.
The present disclosure relates, according to some embodiments, to immobilized enzyme compositions and methods for cleaving polynucleotide molecules including, for example, double-stranded DNA. Immobilized enzymes may comprise, for example, an enzyme (e.g., a type IIS restriction endonuclease, an RNAP, a capping enzyme), a support (e.g., a magnetic bead), and optionally, a linker disposed between the enzyme and the support. In some embodiments, methods may include contacting an immobilized enzyme with a polynucleotide substrate to form reaction products, separating the immobilized enzyme from the reaction products, and optionally reusing the immobilized enzymes in one or more subsequent reactions. preparing a library for sequencing. For example, a method may comprise (a) in a coupled reaction, (i) contacting a population of nucleic acid fragments with a tailing enzyme to produce tailed fragments, and (ii) ligating to the tailed fragments a sequencing adapter with a ligase to produce adapter-tagged fragments; and/or separating adapter-tagged fragments from the tailing enzyme and the ligase to produce separated adapter-tagged fragments and, optionally, separated tailing enzyme and/or separated ligase.
Provided herein, among other things, is a method for deaminating a double-stranded nucleic acid. In some embodiments, the method may comprise contacting a double-stranded DNA substrate that comprises cytosines and a double-stranded DNA deaminase having an amino acid sequence that is at least 80% identical to any of SEQ ID NOS: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 19, 24, 26, 27, 28, 33, 40, 49, 50, 63, 95, 96, 97, and/or 99 to produce a deamination product that comprises deaminated cytosines. Enzymes and kits for performing the method are also provided.
Provided herein is a polymerase-free enzyme mix (FRAG) for fragmenting double-stranded DNA. In some embodiments the enzyme mix may comprise a double-stranded DNA nickase and at least one of a DNA ligase capable of sealing a nick within a DNA, and a single-strand specific DNA nuclease. Methods for fragmenting double-stranded DNA are also provided.
Provided herein is a method for isolating high molecular weight (HMW) DNA using beads that are at least 200 μm in diameter that utilizes a device for retaining the beads and where the purified DNA eluant exits the device without shearing the HMW DNA. In some embodiments, the method comprises precipitating the DNA onto the beads, washing the beads in the device, and then eluting the DNA from the beads therein while substantially avoiding shear. Compositions and kits for practicing the method are also provided.
Compositions, methods and kits are provided that enable the detection, analysis and/or sequencing of small or large target RNA molecules whether synthetic, purified or within a biological fluid, or in cell lysate that may contain non-target RNA and other contaminating molecules without the need for depletion or purification steps that diminish what might already be low concentrations of the target molecule. The methods, compositions and kits rely on the use of a Group II Intron reverse transcriptase (Intron-RT) that have strand displacing properties and can generate concatemers in cDNA by rolling circle transcription of circRNAs that may be naturally circular or circularized in vitro from linear RNA.
C12Q 1/6848 - Nucleic acid amplification reactions characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
12.
Compositions and Methods for Detecting Molecular Targets on Chromosomal DNA
Compositions, methods and kits are provided for identifying the presence and location of a target in chromosomal DNA. A nicking endonuclease fused to a binding domain that binds to a constant region of an antibody (NEFP) is provided that may be used for binding to a target directly or via an antibody that binds to the target. The target may be a protein or structural feature of the DNA and its presence and location may correspond to a phenotype and/or pathology in a biopsy or other cell sample for diagnostic purposes. The background is reduced by the addition of a glycoaminoglycan (GAG) that reversibly inhibits binding of the NEFP to DNA. Nick translation in the presence of a strand displacing polymerase enables the incorporation of tagged nucleotides that (i) blocks re-nicking; (ii) facilitates immobilization of DNA fragments around the target for sequencing; and/or (iii) enables dye labelling of the chromosomal DNA within the cell nuclei for analysis by microscopy.
Compositions, methods and kits are provided for identifying the presence and location of a target in chromosomal DNA. A nicking endonuclease fused to a binding domain that binds to a constant region of an antibody (NEFP) is provided that may be used for binding to a target directly or via an antibody that binds to the target. The target may be a protein or structural feature of the DNA and its presence and location may correspond to a phenotype and/or pathology in a biopsy or other cell sample for diagnostic purposes. The background is reduced by the addition of a glycoaminoglycan (GAG) that reversibly inhibits binding of the NEFP to DNA. Nick translation in the presence of a strand displacing polymerase enables the incorporation of tagged nucleotides that (i) blocks re-nicking; (ii) facilitates immobilization of DNA fragments around the target for sequencing; and/or (iii) enables dye labelling of the chromosomal DNA within the cell nuclei for analysis by microscopy.
C07K 14/31 - Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F) from Staphylococcus (G)
Compositions and methods are described that are directed to specific and sensitive methods of target nucleic acid detection and more specifically detecting target nucleic acids directly from biological samples. The compositions and methods were developed to be easy to use involving a minimum number of steps and giving rapid and consistent results either at point of care or in high throughput situations. The compositions and methods are directed to labelled probes and their uses in Loop-Mediated Isothermal Amplification (LAMP) diagnostic tests to detect target DNA from the environment or from an individual and also to detect specific variants of the target DNA, both with similar sensitivity. The compositions and methods may use any single improvement or combination of improvements selected from thermolabile enzyme variants, poloxamers, various salts, indicators and one or more LAMP primer sets for detecting single and/or multiple targets, probes for detecting variants of the targets including SARS-CoV-2 variants and lateral flow devices.
C12Q 1/70 - Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
G01N 21/78 - Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
15.
Methods for Labeling a Population of RNA Molecules
A method of labeling, and optionally enriching, for a population of target RNA molecules in a mixture of RNAs is provided. In some embodiments, the method may comprise (a) adding a label to the 5′ end of 5′-diphosphorylated or 5′-triphosphorylated target RNA molecules in a sample by incubating the sample with labeled GTP and a capping enzyme; and (b) optionally enriching for target RNA comprising the affinity tag-labeled GMP using an affinity matrix that binds to the affinity tag. The label may be an oligonucleotide, which may further comprise an affinity group attached either internally or at 5′ or 3′ end of the oligonucleotide where the oligonucleotide label may be added directly, or indirectly via a reaction with a reactive group to the target RNA.
C12N 15/10 - Processes for the isolation, preparation or purification of DNA or RNA
C07H 21/02 - Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with ribosyl as saccharide radical
C12Q 1/6806 - Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
Provided herein is a reverse transcriptase mixture comprising a reverse transcriptase and a colored dye at a concentration in the range of 0.003%-1% (v/w). The colored dye may be visually observed during transfer of the mix from one vessel to another and addition of the mix to another mix can be confirmed by eye by observing the colored dye.
Methods, kits and compositions, in some embodiments, may include a thermostable DNA guided Argonaute protein for example TtAgo, a thermostable single-stranded DNA binding protein (SSB) for example, extreme thermostable single-stranded DNA binding protein (ET SSB), and, optionally, a strand-displacing polymerase. A SSB may allow (a) Argonaute/guide DNA complexes to substantially enhance cleavage efficiency of single- and double-stranded DNA substrates; (b) the use of longer guide DNAs (e.g., guide DNAs that are at least 24 nucleotides in length) and/or (c) increases in the sequence specificity of Argonaute-mediated binding and cleavage reactions.
The present disclosure relates, according to some embodiments, to compositions, methods, systems, and kits for programmable endonucleolytic cleavage of DNA (e.g., ds DNA). For example, the in vitro activity of an Argonaute (e.g., a mesophilic Argonaute CbAgo from Clostridium butyricum) may be synchronized with DNA strand unwinding activity of a helicase (e.g., a nuclease deficient RecBexo-C DNA helicase from E. coli) for a rapid and efficient cleavage of double-stranded DNA targets. Enzymatic properties of CbAgo and different aspects of ds DNA cleavage were thoroughly explored by adapting high-throughput capillary electrophoreses technique for monitoring CbAgo cleavage activity in concurrence with RecBexo-C. The present disclosure shows that in the presence of RecBexo-C, CbAgo can be programmed with guides to cleave any site of interest localized at up to 10 kb distance from the end of linear ds DNA at 37° C. temperature. CbAgo/RecBexo-C can be programmed to generate DNA fragments flanked with unique single-stranded extensions suitable for seamless ligation with compatible DNA fragments. The present disclosure relates further the compositions, methods, systems, and kits for PRC-free assembly of linear DNA molecules by using CbAgo/RecBexo-C programmable DNA endonuclease. The results presented here demonstrate that the combination of CbAgo and RecBexo-C is currently an efficient mesophilic DNA-guided DNA-cleaving programmable endonuclease which can be used to prepare synthetic biology tools that require or benefit from sequence-specific nicking/cleavage of natural DNA at otherwise inaccessible locations.
The present disclosure relates, according to some embodiments, to systems, apparatus, compositions, methods, and workflows that include DNase I variants with desirable properties including, for example, salt tolerance. A DNase I variant, in some embodiments, may have an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, and/or at least 98% identical to SEQ ID NO:1 and may be identical to SEQ ID NO:1 at one or more positions selected from the group of positions corresponding to L29, A35, D87, Q88, S94, P103, T108, P121, P132, A135, D145, E161, G172, P190, H208, and A224 of SEQ ID NO:1.
C07K 14/47 - Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from humans from vertebrates from mammals
Provided herein is a polymerase-free enzyme mix (FRAG) for fragmenting double-stranded DNA. In some embodiments the enzyme mix may comprise a double-stranded DNA nickase and at least one of a DNA ligase capable of sealing a nick within a DNA, and a single-strand specific DNA nuclease. Methods for fragmenting double-stranded DNA are also provided.
C40B 20/04 - Identifying library members by means of a tag, label, or other readable or detectable entity associated with the library members, e.g. decoding processes
C40B 50/06 - Biochemical methods, e.g. using enzymes or whole viable microorganisms
21.
Compositions and methods for detecting pyrophosphate products of enzyme reactions using pyridylazoaniline dyes
Provided herein is a composition comprising an enzyme that releases pyrophosphate from a substrate and a dye of Formula 1. A method for detecting pyrophosphate is also provided. A kit comprising a polymerase that releases pyrophosphate by hydrolysis of nucleoside triphosphates during nucleic acid replication, a divalent manganese salt, and the dye are also provided. The present composition, method and kits provide a way to detect and/or quantify substrates or products of enzyme reacted substrates associated with the release pyrophosphate (e.g., nucleic acid amplification reactions and other reactions that hydrolyze ATP) via a distinct color change without substantially affecting the sensitivity and/or specificity of the reaction.
Compositions and methods are provided for forming a single RNA polynucleotide from a plurality of DNA oligonucleotides in a single reaction chamber using combined reagents in a single step reaction. DNA polymerase, RNA polymerase and single stranded (ss) DNA oligonucleotides are combined where each DNA oligonucleotide has one or more sequence modules, wherein one sequence module in the first ss DNA oligonucleotide is complementary to a sequence module at the 3′ end of the second ss DNA oligonucleotide; and wherein a second module on the first ss DNA oligonucleotide is an RNA polymerase promoter sequence; and forming a single RNA polynucleotide, excluding the RNA promoter sequence, derived from the first and second DNA oligonucleotides
Provided herein, among other things, are various in vitro methods that involve cleaving dsDNA molecules that comprise a mismatched nucleotide using EndoMS. In some embodiments, the method may comprise ligating a T-tailed double-stranded adapter to A-tailed double-stranded fragments of nucleic acid to produce ligation products that comprise adapter-ligated fragments and double-stranded adapter dimers that comprise a T:T mismatch at the ligation junction and cleaving both strands of the adapter dimers using EndoMS.
The present disclosure relates, according to some embodiments, to methods for preparing a library for sequencing. For example, a method may comprise (a) in a coupled reaction, (i) contacting a population of nucleic acid fragments with a tailing enzyme to produce tailed fragments, and (ii) ligating to the tailed fragments a sequencing adapter with a ligase to produce adapter-tagged fragments; and/or separating adapter-tagged fragments from the tailing enzyme and the ligase to produce separated adapter-tagged fragments and, optionally, separated tailing enzyme and/or separated ligase. In some embodiments, a tailing enzyme and/or a ligase used in library preparation may be immobilized enzymes.
Provided herein, among other things, is a method for producing an RNA product that has reduced immunogenicity. In some embodiments, the method involves transcribing a template DNA with a thermostable RNA polymerase at a temperature of greater than 44° C.
A61K 48/00 - Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
C12P 19/34 - Polynucleotides, e.g. nucleic acids, oligoribonucleotides
A61K 31/713 - Double-stranded nucleic acids or oligonucleotides
A61K 31/7105 - Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
C12N 15/113 - Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides
26.
Cleavage of Single Stranded DNA Having a Modified Nucleotide
Methods are provided that, for example, include (a) combining ssDNA containing a modified nucleotide (e.g., a ssDNA with a modified nucleotide proximate to its 5′ end) with a DNA cleavage enzyme capable of cleaving the ssDNA at the modified nucleotide (e.g., to generate a first ssDNA fragment having a 3′OH and a second ssDNA fragment having the modified nucleotide); wherein the ratio of enzyme to DNA substrate is less than 1:1 molar ratio (m/m); and (b) cleaving at least 95% of the ssDNA at the modified nucleotide. In some embodiments, a method may comprise (a) combining (i) a ssDNA comprising a modified nucleotide (e.g., proximate to its 5′ end) with (ii) a DNA cleavage enzyme capable of cleaving the ssDNA at the modified nucleotide (e.g., to generate (after cleavage) a first ssDNA fragment having a 3′OH and a second ssDNA fragment comprising the modified nucleotide) wherein the ratio of enzyme to DNA substrate is less than 1:1 molar ratio and cleaving at least 95% of the ssDNA at the modified nucleotide. In some embodiments, methods provided herein may include (a) combining (i) a ssDNA (1) immobilized on a substrate and (2) comprising a modified nucleotide with (ii) a ssDNA cleaving enzyme capable of cleaving the ssDNA at the modified nucleotide (e.g., to generate (after cleavage) a first ssDNA fragment having a 3′OH and a second ssDNA fragment comprising the modified nucleotide) ; and (b) cleaving the immobilized ssDNA to release the second single stranded DNA fragment from the substrate. At least 95% (m/m) of an ssDNA comprising a modified nucleotide may be cleaved in less than 60 minutes.
A bacteriophage RNA polymerase variant is provided. In some embodiments, the variant may have increased thermostability relative to the corresponding wild type bacteriophage RNA polymerase and/or wild type T7 RNA polymerase. Compositions, kits and methods that employ the variant are also provided.
The present disclosure relates to compositions, kits, and methods of making RNA vaccines having an appropriate cap structure. Systems, apparatus, compositions, and/or methods may include and/or use, in some embodiments, non-naturally occurring single-chain RNA capping enzymes. In some embodiments, an RNA capping enzyme may include an FCE variant having (a) an amino acid sequence at least 90% identical to positions 1 to 878 of SEQ ID NO: 1, and/or (b) one or more substitutions relative to SEQ ID NO: 1 at a position selected from positions corresponding to positions 215, 337, 572, 648, and 833 (e.g., a position selected from positions corresponding to position 215, 337, and 572) of SEQ ID NO: 1.
Compositions and methods of use are provided that among other things, allow for efficient adapter ligation to small RNAs. Embodiments of the compositions include partially double stranded polynucleotides for use as 3′ adapters that contain a cleavable linker positioned between a single-stranded region and a double-stranded region. Upon ligating the 3′ adapters, the single-stranded region is released by cleaving the cleavable linker.
Kits and methods are described that are directed to specific and sensitive methods of target nucleic acid detection and more specifically detecting target nucleic acids directly from biological samples. The kits and methods were developed to be easy to use involving a minimum number of steps and giving rapid and consistent results either at point of care or in high throughput situations. The kits and methods utilize in various combinations, reversible inhibitors of kit components, thermolabile enzymes, poloxamers, various salts, indicators and one or more Loop-Mediated Isothermal Amplification (LAMP) primer sets for detecting single and/or multiple targets and variants of the targets including SARS-CoV-2 targets and variants thereof in a single reaction. The kits and methods permit detection of the target nucleic with similar sensitivity regardless of the presence of undefined mutations that may enhance the virulence of cells or viruses containing the undefined mutations.
C12Q 1/70 - Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
G01N 21/78 - Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
Provided herein is a method for chemically capping polynucleotides having a 5′ monophosphate. In some embodiments the method may comprise: combining an activated nucleoside 5′ mono- or poly-phosphate with a population of polynucleotides that comprises polynucleotides having a 5′ monophosphate, to produce a reaction mix; and incubating the reaction mix to produce reaction products that comprise a polynucleotide and a 5′ nucleoside cap, linked by a 5′ to 5′ polyphosphate linkage. The chemical capping method described herein can be incorporated into a variety of cDNA synthesis methods.
Ordered assembly of large numbers of fragments into a single large DNA have been improved in both frequency and fidelity of the assembled product. This has been achieved by novel compositions and methods that are utilized in a computer system that integrates comprehensive ligation data from multiple sources to provide optimized synthetic overhangs or overhangs from restriction endonuclease cleavage on DNA fragments for assembly by ligation. Intragenic cut sites are avoided by the use of a novel restriction endonuclease which recognizes 7 nucleotides (bases) and cuts DNA to create 4-base overhangs with the help of a synthetic activator oligonucleotide. Variations in ligation preferences by different ligases provide extra precision in assembly reactions. The use of the improved methods are exemplified by the successful assembly from 52 fragments of a viral genome and also a 52 fragment ordered assembly of a bacteria operon.
A composition and its uses and additionally a kit are provided. The composition is a synthetic self-complementary oligonucleotide that has a double-stranded region and a loop, wherein the double-stranded region contains a binding sequence for PaqCl. Additionally, the oligonucleotide includes unligatable 3′ and 5′ ends that cannot be cleaved by PaqCl. This oligonucleotide composition has been combined with PaqCl or a variant of PaqCl Type IIS restriction endonuclease in a reaction mixture, where the reaction mixture includes PaqCl or variant that can further be combined with a ligase and optionally a deadenylase, crowding molecule such as PEG and/or a repair enzyme such as Endo MS. The kit includes the oligonucleotide and Type IIS restriction endonuclease in the same or different containers.
The present disclosure relates, according to some embodiments, to methods for preparing a library for sequencing. For example, a method may comprise (a) in a coupled reaction, (i) contacting a population of nucleic acid fragments with a tailing enzyme to produce tailed fragments, and (ii) ligating to the tailed fragments a sequencing adapter with a ligase to produce adapter-tagged fragments; and/or separating adapter-tagged fragments from the tailing enzyme and the ligase to produce separated adapter-tagged fragments and, optionally, separated tailing enzyme and/or separated ligase. In some embodiments, a tailing enzyme and/or a ligase used in library preparation may be immobilized enzymes.
Provided herein, among other things, are various compositions and methods for analyzing chromatin. In some embodiments, the composition may comprise a mixture of a nicking enzyme, four dNTPs, at least one labeled dNTP and, optionally, a polymerase. In some embodiments, this method may comprise: obtaining a sample comprising chromatin, reacting the sample with the composition to selectively label the open chromatin in the sample, and analyzing the labeled sample.
C12Q 1/68 - Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
C12Q 1/6886 - Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
C12Q 1/6806 - Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
Kits and methods are described that are directed to specific and sensitive methods of target nucleic acid detection and more specifically detecting target nucleic acids directly from biological samples. The kits and methods were developed to be easy to use involving a minimum number of steps and giving rapid and consistent results either at point of care or in high throughput situations. The kits and methods utilize in various combinations, reversible inhibitors of kit components, thermolabile enzymes, poloxamers, various salts, indicators and one or more Loop-Mediated Isothermal Amplification (LAMP) primer sets for detecting single and/or multiple targets and variants of the targets including SARS-CoV-2 targets and variants thereof in a single reaction. The kits and methods permit detection of the target nucleic with similar sensitivity regardless of the presence of undefined mutations that may enhance the virulence of cells or viruses containing the undefined mutations.
G01N 21/78 - Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
C12Q 1/70 - Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
The present disclosure relates, according to some embodiments, to compositions, methods, and/or kits for producing vaccinia capping enzyme. For example, active, heterodimers of vaccinia capping enzyme may be produced as fusions comprising D1 and D12 subunits. Vaccinia capping enzyme fusion proteins may further comprise a linker.
Compositions, methods and kits are provided for identifying the presence and location of a target in chromosomal DNA. A nicking endonuclease fused to a binding domain that binds to a constant region of an antibody (NEFP) is provided that may be used for binding to a target directly or via an antibody that binds to the target. The target may be a protein or structural feature of the DNA and its presence and location may correspond to a phenotype and/or pathology in a biopsy or other cell sample for diagnostic purposes. The background is reduced by the addition of a glycoaminoglycan (GAG) that reversibly inhibits binding of the NEFP to DNA. Nick translation in the presence of a strand displacing polymerase enables the incorporation of tagged nucleotides that (i) blocks re-nicking; (ii) facilitates immobilization of DNA fragments around the target for sequencing; and/or (iii) enables dye labelling of the chromosomal DNA within the cell nuclei for analysis by microscopy.
C07K 14/31 - Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F) from Staphylococcus (G)
A method for identifying any of the presence, location and phasing of modified cytosines (C) in long stretches of nucleic acids is provided. In some embodiments, the method may comprise (a) reacting a first portion of a nucleic acid sample containing at least one C and/or at least one modified C with a DNA glucosyltransferase and a cytidine deaminase to produce a first product and/or reacting a second portion of the sample with a dioxygenase, optionally a DNA glucosyltransferase and a cytidine deaminase to produce a second product and; (b) comparing the sequences from the first and optionally the second product obtained in (a), or amplification products thereof, with each other and/or an untreated reference sequence to determine which Cs in the initial nucleic acid fragment are modified. A modified TET methylcytosine dioxygenase with improved efficiency compared to unmodified TET2 at converting methylcytosine to carboxymethylcytosine is also provided.
Methods and compositions are provided for optimizing ordered assembly of a plurality of polynucleotide fragments. The optimization involves providing sets of overhang sequences with preferred experimental conditions for high fidelity ordered assembly of polynucleotide fragments by ligation under selected experimental conditions. The methods and compositions provide the use of a computer system with inputs having a plurality of menus and outputs that include a variety of media interfaces. The computer system has access to a ligation frequency database to provide sets of overhang sequences for efficient joining of multiple fragments into the target nucleic acid. In-puts include one or more of the following: numbers and sizes of fragments, optionally a desired target polynucleotide sequence from a database, in which case one output are the recommended polynucleotide fragments for ordered assembly, and selected experimental conditions selected from any or all of ligation protocols and ligation temperature with reaction times, salt concentration in the ligation buffer, choice of ligase and restriction endonuclease and the use of DNA repair enzymes.
C12N 15/66 - General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
G16B 50/30 - Data warehousing; Computing architectures
Providing herein, among other things, are kits, compositions and methods that relate to DNA fragmentation. An embodiment of a composition provides combining: one or more enzymes capable of nick translating activity, a dNTP mix comprising at least one dNTP having a modified base, and at least one modification-sensitive nicking endonuclease that is prevented from nicking DNA if its recognition site contains the modified base. When the composition is added to a sample comprising a double-stranded DNA template that comprises recognition sites for the modification-sensitive nicking endonuclease, a reaction mix was produced which could be incubated for any time period in excess of about 5 minutes to produce fragments of a desired size of the double-stranded DNA template. In this method, the fragments produced include the modified base and, as such, are not re-nicked by the nicking endonuclease.
C12N 15/66 - General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
Kits and methods are provided for performing multiplex LAMP reactions. These kits and methods are directed to specific and sensitive methods of target nucleic acid detection and more specifically pathogen diagnostics such as detection of Coronavirus. The kits and methods utilize a plurality of sets of oligonucleotide primers for targeting the viral nucleic acid target.
C12Q 1/70 - Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
G01N 21/78 - Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
Kits and methods are provided for performing multiplex Loop-Mediated Isothermal Amplification (LAMP) reactions. These kits and methods are directed to specific and sensitive methods of target nucleic acid detection and more specifically pathogen diagnostics such as detection of Coronavirus. The kits and methods utilize a plurality of sets of oligonucleotide primers for targeting the viral nucleic acid target.
G01N 21/78 - Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
C12Q 1/70 - Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
44.
FCE mRNA capping enzyme compositions, methods and kits
The present disclosure relates to compositions, kits, and methods of making RNA vaccines having an appropriate cap structure. Systems, apparatus, compositions, and/or methods may include and/or use, in some embodiments, non-naturally occurring single-chain RNA capping enzymes. In some embodiments, an RNA capping enzyme may include an FCE variant having (a) an amino acid sequence at least 90% identical to positions 1 to 878 of SEQ ID NO: 1, and/or (b) one or more substitutions relative to SEQ ID NO: 1 at a position selected from positions corresponding to positions 215, 337, 572, 648, and 833 (e.g., a position selected from positions corresponding to position 215, 337, and 572) of SEQ ID NO: 1.
Methods and compositions are provided for engineering mutant enzymes with reduced star activity where the mutant enzymes have a fidelity index (FI) in a specified buffer that is greater than the FI of the non-mutated enzyme in the same buffer.
Compositions and methods are provided for enzymes with altered properties that involve a systematic approach to mutagenesis and a screening assay that permits selection of the desired proteins. Embodiments of the method are particularly suited for modifying specific properties of restriction endonucleases such as star activity. The compositions includes restriction endonucleases with reduced star activity as defined by an overall fidelity index improvement factor.
The present disclosure relates to compositions, kits, and methods of making RNA vaccines having an appropriate cap structure. Systems, apparatus, compositions, and/or methods may include and/or use, in some embodiments, non-naturally occurring single-chain RNA capping enzymes. In some embodiments, an RNA capping enzyme may include an FCE variant having (a) an amino acid sequence at least 90% identical to positions 1 to 878 of SEQ ID NO: 1, and/or (b) one or more substitutions relative to SEQ ID NO: 1 at a position selected from positions corresponding to positions 215, 337, 572, 648, and 833 (e.g., a position selected from positions corresponding to position 215, 337, and 572) of SEQ ID NO: 1.
Provided herein, among other things, are various in vitro methods that involve cleaving dsDNA molecules that comprise a mismatched nucleotide using EndoMS. In some embodiments, the method may comprise ligating a T-tailed double-stranded adapter to A-tailed double-stranded fragments of nucleic acid to produce ligation products that comprise adapter-ligated fragments and double-stranded adapter dimers that comprise a T:T mismatch at the ligation junction and cleaving both strands of the adapter dimers using EndoMS.
Variants of the bacteriophage B103 DNA polymerase are described herein. The variant has improved properties, that include when compared to wild-type Phi29 DNA polymerase, at least one of the following: increased thermostability, improved reaction rate for DNA amplification, reduced background and a reduction of bias. Methods of using the DNA polymerase variant are also described herein.
Provided herein, among other things, is a method for producing an RNA product that has reduced immunogenicity. In some embodiments, the method involves transcribing a template DNA with a thermostable RNA polymerase at a temperature of greater than 44° C.
A61K 48/00 - Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
C12P 19/34 - Polynucleotides, e.g. nucleic acids, oligoribonucleotides
A61K 31/713 - Double-stranded nucleic acids or oligonucleotides
A61K 31/7105 - Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
C12N 15/113 - Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides
Kits and methods are provided for performing multiplex LAMP reactions. These kits and methods are directed to specific and sensitive methods of target nucleic acid detection and more specifically pathogen diagnostics such as detection of Coronavirus. The kits and methods utilize a plurality of sets of oligonucleotide primers for targeting the viral nucleic acid target.
G01N 21/78 - Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
C12Q 1/70 - Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
Provided herein is a thermolabile proteinase and methods of using the same. In some embodiments, the thermolabile proteinase may comprise an amino acid sequence that is at least 90% identical to any of SEQ ID NOs:1-11 and at least one amino acid substitution in helix 3. The thermolabile proteinase is active at a temperature in the range of 4° C.-40° C. and is inactivated by raising the temperature to above 50° C., where the proteinase is substantially inactive at 65° C.
Kits and methods are provided for performing multiplex LAMP reactions. These kits and methods are directed to specific and sensitive methods of target nucleic acid detection and more specifically pathogen diagnostics such as detection of Coronavirus. The kits and methods utilize a plurality of sets of oligonucleotide primers for targeting different template sequences in a single nucleic acid target. The kits and methods also include guanidium salts that enhance the sensitivity of the assay.
G01N 21/78 - Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
C12Q 1/70 - Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
G16B 25/20 - Polymerase chain reaction [PCR]; Primer or probe design; Probe optimisation
Variants of the bacteriophage B103 DNA polymerase are described herein. The variant has improved properties, that include when compared to wild-type Phi29 DNA polymerase, at least one of the following: increased thermostability, improved reaction rate for DNA amplification, reduced background and a reduction of bias. Methods of using the DNA polymerase variant are also described herein.
Provided herein is a method for efficiently capping RNA in vitro. In some embodiments the capping reaction may be done at high temperature using Vaccinia capping enzyme or a variant thereof. In other embodiments, the capping reactions may comprise a capping enzyme from a large virus of amoeba, e.g., Faustovirus, mimivirus or moumouvirus, or a variant thereof. Compositions and kits for practicing the method are also provided.
C07H 19/20 - Purine radicals with the saccharide radical being esterified by phosphoric or polyphosphoric acids
C07H 21/04 - Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
57.
Isolation of high molecular weight DNA using beads
Provided herein is a method for isolating high molecular weight (HMW) DNA using beads that are at least 200 μm in diameter that utilizes a device for retaining the beads and where the purified DNA eluant exits the device without shearing the HMW DNA. In some embodiments, the method comprises precipitating the DNA onto the beads, washing the beads in the device, and then eluting the DNA from the beads therein while substantially avoiding shear. Compositions and kits for practicing the method are also provided.
Mutant bacteriophage DNA ligases that have increased tolerance to salt and/or heat is provided. Methods, compositions and kits that employ the same are also provided.
Compositions are provided for 3′ adapters and methods of use are provided that include methods requiring a plurality of ligation steps involving a single-stranded target polynucleotide and 3′ and 5′ adapters. Embodiments of the 3′ adapters comprise a cleavable linker positioned between a single-stranded region and a double-stranded region. Upon ligating the 3′ adapters, the single-stranded region is released by cleaving the cleavable linker.
Providing herein, among other things, is a method comprising incubating a double-stranded nucleic acid having a nick with a nick translating activity, a ligase, and a nucleotide mix comprising at least one modified nucleotide, to generate a product comprising a patch of a newly synthesized strand of a duplex nucleic acid containing a plurality of modified nucleoside monophosphates that are at or adjacent to the site of the nick. In some embodiments, the method may be used to map damaged nucleoside monophosphates in a nucleic acid. Compositions and kits for use in performing the method are also provided.
The present disclosure provides, among other things, a way to amplify and sequence target sequences in a low-input sample. In some embodiments, the method comprises ligating a double-stranded adaptor onto a population of fragments to produce tagged fragments, and linearly amplifying the tagged fragments.
C12N 15/10 - Processes for the isolation, preparation or purification of DNA or RNA
C40B 50/16 - Solid phase synthesis, i.e. wherein one or more library building blocks are bound to a solid support during library creation; Particular methods of cleavage from the solid support involving encoding steps
C40B 50/18 - Solid phase synthesis, i.e. wherein one or more library building blocks are bound to a solid support during library creation; Particular methods of cleavage from the solid support using a particular method of attachment to the solid support
C40B 70/00 - Tags or labels specially adapted for combinatorial chemistry or libraries, e.g. fluorescent tags or barcodes
62.
β-lactamase targeted photosensitizer for pesticide and pest detection
A01N 43/90 - Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having two or more relevant hetero rings, condensed among themselves or with a common carbocyclic ring system
A01N 25/00 - Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
C07D 501/16 - Compounds having a nitrogen atom directly attached in position 7 with a double bond between positions 2 and 3
Provided herein is a thermolabile proteinase and methods of using the same. In some embodiments, the thermolabile proteinase may comprise an amino acid sequence that is at least 90% identical to any of SEQ ID NOs:1-11 and at least one amino acid substitution in helix 3. The thermolabile proteinase is active at a temperature in the range of 4° C.-40° C. and is inactivated by raising the temperature to above 50° C., where the proteinase is substantially inactive at 65° C.
A bacteriophage RNA polymerase variant is provided. In some embodiments, the variant may have increased thermostability relative to the corresponding wild type bacteriophage RNA polymerase and/or wild type T7 RNA polymerase. Compositions, kits and methods that employ the variant are also provided.
Methods and compositions for capping RNA in an in vitro transcription mixture are provided that include a thermostable RNA polymerase variant and a cap analog such that when a DNA template is added to the mixture, and the mixture is then incubated under conditions for in vitro transcription, capped RNA is produced.
A probe qPCR master mix is provided. In some embodiments, the master mix comprises nucleotides, an enzyme comprising a polymerase activity and a flap endonuclease activity, a chelating agent at a concentration greater than 5 μM, and a divalent cation. The relatively high concentration of chelating agent stabilizes the flap endonuclease activity during storage. As such, the polymerase and flap endonuclease activities may be substantially the same before and after storing the master mix for 7 days at 37° C.
C12Q 1/6848 - Nucleic acid amplification reactions characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
C12N 15/00 - Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
Methods and compositions are provided for engineering mutant enzymes with reduced star activity where the mutant enzymes have a fidelity index (FI) in a specified buffer that is greater than the FI of the non-mutated enzyme in the same buffer.
Compositions and methods are provided for forming a single RNA polynucleotide from a plurality of DNA oligonucleotides in a single reaction chamber using combined reagents in a single step reaction. DNA polymerase, RNA polymerase and single stranded (ss) DNA oligonucleotides are combined where each DNA oligonucleotide has one or more sequence modules, wherein one sequence module in the first ss DNA oligonucleotide is complementary to a sequence module at the 3′ end of the second ss DNA oligonucleotide; and wherein a second module on the first ss DNA oligonucleotide is an RNA polymerase promoter sequence; and forming a single RNA polynucleotide, excluding the RNA promoter sequence, derived from the first and second DNA oligonucleotides.
Provided herein, among other things, are various compositions and methods for analyzing chromatin. In some embodiments, the composition may comprise a mixture of a nicking enzyme, four dNTPs, at least one labeled dNTP and, optionally, a polymerase. In some embodiments, this method may comprise: obtaining a sample comprising chromatin, reacting the sample with the composition to selectively label the open chromatin in the sample, and analyzing the labeled sample.
C12Q 1/68 - Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
C12Q 1/6886 - Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
C12Q 1/6806 - Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
Methods are provided for a rapid, low cost approach to monitoring an amplification reaction. This includes monitoring the progress of isothermal or PCR amplification reactions to completion using pH-sensitive dyes that are either colored or fluorescent. Compositions are described that include a mixture of a DNA polymerase, deoxyribonucleotide triphosphate and Tris buffer in the range of 1.5 mM Tris to 5 mM Tris or equivalent.
The United States of America, as represented by the Secretary, Department of Health and Human Services (USA)
The University of Tokyo (Japan)
New England Biolabs, Inc. (USA)
Inventor
Inglese, James
Dranchak, Patricia
Macarthur, Ryan
Suga, Hiroaki
Yu, Hao
Carlow, Clotilde
Li, Zhiru
Abstract
Disclosed herein are isolated peptides inhibit activity of a cofactor-independent phosphoglycerate mutase. In some examples, the isolated peptide is 6-20 amino acids long and includes the amino acid sequence of any one of SEQ ID NOs: 1-22 or 54, an analog or derivative thereof, or a pharmaceutically acceptable salt or ester thereof. In some examples, the peptide is a cyclic peptide with an N-terminal ring of 6-15 amino acids (for example, 6-10 amino acids) and a C-terminal linear portion of 1-9 amino acids (for example, 3-8 amino acids. Also disclosed h are methods of treating or inhibiting an infection in a subject, including administering to the subject an effective amount of a composition including one of more of the disclosed peptides, or analogs or derivative thereof, or pharmaceutically acceptable salts or esters thereof.
C07K 7/64 - Cyclic peptides containing only normal peptide links
C07K 7/08 - Linear peptides containing only normal peptide links having 12 to 20 amino acids
C07K 7/06 - Linear peptides containing only normal peptide links having 5 to 11 amino acids
C07K 7/56 - Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring the cyclisation not occurring through 2,4-diamino-butanoic acid
C07K 14/00 - Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
Providing herein, among other things, are kits, compositions and methods that relate to DNA fragmentation. An embodiment of a composition provides combining: one or more enzymes capable of nick translating activity, a dNTP mix comprising at least one dNTP having a modified base, and at least one modification-sensitive nicking endonuclease that is prevented from nicking DNA if its recognition site contains the modified base. When the composition is added to a sample comprising a double-stranded DNA template that comprises recognition sites for the modification-sensitive nicking endonuclease, a reaction mix was produced which could be incubated for any time period in excess of about 5 minutes to produce fragments of a desired size of the double-stranded DNA template. In this method, the fragments produced include the modified base and, as such, are not re-nicked by the nicking endonuclease.
C12N 15/66 - General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
A method for identifying any of the presence, location and phasing of modified cytosines (C) in long stretches of nucleic acids is provided. In some embodiments, the method may comprise (a) reacting a first portion of a nucleic acid sample containing at least one C and/or at least one modified C with a DNA glucosyltransferase and a cytidine deaminase to produce a first product and/or reacting a second portion of the sample with a dioxygenase, optionally a DNA glucosyltransferase and a cytidine deaminase to produce a second product and; (b) comparing the sequences from the first and optionally the second product obtained in (a), or amplification products thereof, with each other and/or an untreated reference sequence to determine which Cs in the initial nucleic acid fragment are modified. A modified TET methylcytosine dioxygenase with improved efficiency compared to unmodified TET2 at converting methylcytosine to carboxymethylcytosine is also provided.
Methods of capturing N-glycan linked glycomolecules including N-glycans, N-glycopeptides and N-glycoproteins are described. The methods provide substantially unbiased capture of charged and uncharged N-glycans and/or N-glycan linked glycomoleules. Binding reagents for substantially unbiased binding of N-glycans and/or N-glycan linked glycomolecules are also described.
C07H 21/04 - Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
Methods, kits and compositions, in some embodiments, may include a thermostable DNA guided Argonaute protein for example TtAgo, a thermostable single-stranded DNA binding protein (SSB) for example, ET SSB, and, optionally, a strand-displacing polymerase. A SSB may allow (a) Argonaute/guide DNA complexes to substantially enhance cleavage efficiency of single- and double-stranded DNA substrates; (b) the use of longer guide DNAs (e.g., guide DNAs that are at least 24 nucleotides in length) and/or (c) increases in the sequence specificity of Argonaute-mediated binding and cleavage reactions.
Among other things, a method for performing multiple enzyme reactions in a single tube is provided. In some embodiments, the method may comprise producing a reaction mix comprising a thermolabile UDG, an AP lyase and DNA fragments that comprise one or more uracil residues, incubating the reaction mix at a relatively low temperature to cleave fragments at the one or more uracil residues, raising the temperature of the reaction mix to a relatively high temperature to inactivate the thermolabile UDG; and deaminating the fragments, thereby converting any cytosine in the fragments of DNA to uracil.
This disclosure provides, among other things, a composition comprising: comprising a fusion protein comprising: (a) a DNA polymerase; and (b) a heterologous sequence-specific DNA binding domain. A method for copying a DNA template, as well as a kit for performing the same, are also described.
C12P 19/34 - Polynucleotides, e.g. nucleic acids, oligoribonucleotides
C12N 15/10 - Processes for the isolation, preparation or purification of DNA or RNA
C12N 15/64 - General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
C12N 15/66 - General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
A method for identifying the location and phasing of modified cytosines (C) in long stretches of nucleic acids is provided. In some embodiments, the method may comprise (a) reacting a first portion of a nucleic acid sample containing at least one C and/or at least one modified C with a DNA glucosyltransferase and a cytidine deaminase to produce a first product and optionally reacting a second portion of the sample with a dioxygenase and a cytidine deaminase to produce a second product and; (b) comparing the sequences from the first and optionally the second product obtained in (a), or amplification products thereof, with each other and/or an untreated reference sequence to determine which Cs in the initial nucleic acid fragment are modified. A modified TET methylcytosine dioxygenase that is more efficient at converting methylcytosine to carboxymethylcytosine is also provided.
Provided herein is an α-fucosidase that can cleave a conjugate comprising an N-glycan and a label where the label is added by amine reactive chemistry. The α-fucosidase also has an accelerated reaction time using Schiff base labeled N-glycans compared with bovine kidney fucosidase. A reaction mix, enzyme mix and kit comprising the α-fucosidase are provided, as well as a method for analyzing glycoproteins. The α-fucosidase finds particular use in analyzing the N-glycans of therapeutic glycoproteins.
Compositions and methods for performing a template-switching reaction are provided that may include reducing or eliminating concatemerization of the template-switching oligonucleotide (TSO). In some embodiments, the composition may comprise: a reverse transcriptase; a TSO that includes a recognition sequence for a site-specific double strand nucleic acid cleaving enzyme, wherein the TSO has at its 3′ end at least one nucleotide capable of hybridizing to at least one or more non-templated nucleotides added to a templated cDNA strand by the reverse transcriptase; and a site-specific double strand nucleic acid cleaving enzyme that cleaves the TSO at the recognition sequence.
C12N 15/10 - Processes for the isolation, preparation or purification of DNA or RNA
C12Q 1/6806 - Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
C12Q 1/6848 - Nucleic acid amplification reactions characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
83.
Use of thermostable RNA polymerases to produce RNAs having reduced immunogenicity
Provided herein, among other things, is a method for producing an RNA product that has reduced immunogenicity without requiring removal of any dsRNA from the RNA product. In some embodiments, the method involves transcribing a template DNA with a thermostable RNA polymerase at a temperature of greater than 44° C.
C12Q 1/68 - Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
A61K 45/06 - Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
A61K 48/00 - Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
C12P 19/34 - Polynucleotides, e.g. nucleic acids, oligoribonucleotides
84.
Methods for labeling a population of RNA molecules
A method of labeling, and optionally enriching, for a population of target RNA molecules in a mixture of RNAs is provided. In some embodiments, the method may comprise (a) adding a label to the 5′ end of 5′-diphosphorylated or 5′-triphosphorylated target RNA molecules in a sample by incubating the sample with labeled GTP and a capping enzyme; and (b) optionally enriching for target RNA comprising the affinity tag-labeled GMP using an affinity matrix that binds to the affinity tag. The label may be an oligonucleotide, which may further comprise an affinity group attached either internally or at 5′ or 3′ end of the oligonucleotide where the oligonucleotide label may be added directly, or indirectly via a reaction with a reactive group to the target RNA.
C12N 15/10 - Processes for the isolation, preparation or purification of DNA or RNA
C12Q 1/6806 - Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
C07H 21/02 - Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with ribosyl as saccharide radical
Compositions and methods are provided for enzymes with altered properties that involve a systematic approach to mutagenesis and a screening assay that permits selection of the desired proteins. Embodiments of the method are particularly suited for modifying specific properties of restriction endonucleases such as star activity. The compositions includes restriction endonucleases with reduced star activity as defined by an overall fidelity index improvement factor.
Provided herein is a method for reducing amplification of non-template molecules in a nucleic acid sample. In certain embodiments, the method involves adding a helicase to a reaction mixture for non-helicase-dependent amplification of target nucleic acid.
C12P 19/34 - Polynucleotides, e.g. nucleic acids, oligoribonucleotides
C12Q 1/6853 - Nucleic acid amplification reactions using modified primers or templates
C12Q 1/6848 - Nucleic acid amplification reactions characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
87.
Compositions and methods for analyzing modified nucleotides
A composition comprising a variant BbsI restriction endonuclease having reduced star activity is provided, as well as kits and methods employing the same.
Methods and compositions are provided for engineering mutant enzymes with reduced star activity where the mutant enzymes have a fidelity index (FI) in a specified buffer that is greater than the FI of the non-mutated enzyme in the same buffer.
A mutant MMLV reverse transcriptase that may have an improvement in one or more properties is provided. For example, the present reverse transcriptase is believed to be more efficient relative to other commercially available MMLV reverse transcriptase variants, particularly for templates with a higher GC content.
Compositions and methods for performing a template-switching reaction are provided that may include reducing or eliminating concatemerization of the template-switching oligonucleotide (TSO). In some embodiments, the composition may comprise: a reverse transcriptase; a TSO that includes a recognition sequence for a site-specific double strand nucleic acid cleaving enzyme, wherein the TSO has at its 3′ end at least one nucleotide capable of hybridizing to at least one or more non-templated nucleotides added to a templated cDNA strand by the reverse transcriptase; and a site-specific double strand nucleic acid cleaving enzyme that cleaves the TSO at the recognition sequence.
C12Q 1/6806 - Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
C12Q 1/6848 - Nucleic acid amplification reactions characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
C07H 21/04 - Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
Provided herein is a method for making an cDNA library, comprising adding an affinity tag-labeled GMP to the 5′ end of targeted RNA species in a sample by optionally decapping followed by incubating the sample with an affinity tag-labeled GTP and a capping enzyme, enriching for RNA comprising the affinity tag-labeled GMP using an affinity matrix that binds to the affinity tag, reverse transcribing the enriched RNA to produce a population of cDNAs, and adding a tail to the 3′ end of the population of cDNAs using a terminal transferase, to produce an cDNA library.
C07H 21/00 - Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
93.
Compositions and methods relating to synthetic RNA polynucleotides created from synthetic DNA oligonucleotides
Compositions and methods are provided for forming a single RNA polynucleotide from a plurality of DNA oligonucleotides in a single reaction chamber using combined reagents in a single step reaction. DNA polymerase, RNA polymerase and single stranded (ss) DNA oligonucleotides are combined where each DNA oligonucleotide has one or more sequence modules, wherein one sequence module in the first ss DNA oligonucleotide is complementary to a sequence module at the 3′ end of the second ss DNA oligonucleotide; and wherein a second module on the first ss DNA oligonucleotide is an RNA polymerase promoter sequence; and forming a single RNA polynucleotide, excluding the RNA promoter sequence, derived from the first and second DNA oligonucleotides.
A method of enriching for a population of RNA molecules in a mixture of RNAs is provided. In some embodiments, the method may comprise (a) adding an affinity tag to the 5′ end of 5′-diphosphorylated or 5′-triphosphorylated RNA molecules in a sample by incubating the sample with an affinity tag-labeled GTP and a capping enzyme; and (b) enriching for RNA comprising the affinity tag-labeled GMP using an affinity matrix that binds to the affinity tag.
A bacteriophage RNA polymerase variant is provided. In some embodiments, the variant may have increased thermostability relative to the corresponding wild type bacteriophage RNA polymerase and/or wild type T7 RNA polymerase. Compositions, kits and methods that employ the variant are also provided.
A method for identifying any of the presence, location and phasing of modified cytosines (C) in long stretches of nucleic acids is provided. In some embodiments, the method may comprise (a) reacting a first portion of a nucleic acid sample containing at least one C and/or at least one modified C with a DNA glucosyltransferase and a cytidine deaminase to produce a first product and/or reacting a second portion of the sample with a dioxygenase, optionally a DNA glucosyltransferase and a cytidine deaminase to produce a second product and; (b) comparing the sequences from the first and optionally the second product obtained in (a), or amplification products thereof, with each other and/or an untreated reference sequence to determine which Cs in the initial nucleic acid fragment are modified. A modified TET methylcytosine dioxygenase with improved efficiency compared to unmodified TET2 at converting methylcytosine to carboxymethylcytosine is also provided.
Compositions and methods are provided for storing prokaryotic cells including competent prokaryotic cells at −20° C. in a buffer so that the cells are suitable for transformation at 0° C. with a foreign molecule.
This disclosure provides, among other things, a composition comprising: comprising a fusion protein comprising: (a) a DNA polymerase; and (b) a heterologous sequence-specific DNA binding domain. A method for copying a DNA template, as well as a kit for performing the same, are also described.
This disclosure provides, among other things, a composition comprising: comprising a fusion protein comprising: (a) a DNA polymerase; and (b) a heterologous sequence-specific DNA binding domain. A method for copying a DNA template, as well as a kit for performing the same, are also described.
C12N 15/10 - Processes for the isolation, preparation or purification of DNA or RNA
C12P 19/34 - Polynucleotides, e.g. nucleic acids, oligoribonucleotides
C12N 15/64 - General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
C12N 15/66 - General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
C12N 9/20 - Triglyceride splitting, e.g. by means of lipase
Compositions and methods are provided for efficiently preparing a completely deglycosylated antibody where efficiency is measured in relative amounts of reagents in soluble or lyophilized form, and time and temperature of the reaction. Compositions and methods are also provided for separating substantially all N-linked glycans from a glycosylated antibody and for preserving functionality of the antibody. The methods are compatible with glycan labeling and protease digestion without the need for prior purification steps.
G01N 33/68 - Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
C07K 1/12 - General processes for the preparation of peptides by hydrolysis
C07K 16/00 - Immunoglobulins, e.g. monoclonal or polyclonal antibodies
C07K 16/28 - Immunoglobulins, e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
C07K 16/24 - Immunoglobulins, e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
patents
