25 results about "Double strand dna" patented technology

The disclosure provides, for example, double stranded DNA (dsDNA) molecules comprising one or more chemically modified nucleobases. In some embodiments, the dsDNA molecule is circular and comprises a first strand and a second strand, wherein the first strand comprises one or more chemically modified nucleobases, and the second strand is free of chemically modified nucleobases. In some embodiments, the dsDNA molecule comprises a promoter sequence and an effector sequence that encodes an effector.

The disclosure provides, for example, double stranded DNA (dsDNA) molecules comprising one or more chemically modified nucleobases. In some embodiments, the dsDNA molecule is circular and comprises a first strand and a second strand, wherein the first strand comprises one or more chemically modified nucleobases, and the second strand is free of chemically modified nucleobases. In some embodiments, the dsDNA molecule comprises a promoter sequence and an effector sequence that encodes an effector.

The methods, compositions, and kits of the disclosure provide a novel approach for a whole genome, unbiased DNA analysis method that can be performed on limited amounts of DNA can be used to analyze DNA to determine its modification status. Aspects of the disclosure relate to a method for amplifying bisulfite-treated deoxyribonucleic acid (DNA) molecules comprising: (a) ligating an adaptor to the DNA molecules, wherein the adaptor comprises a RNA polymerase promoter comprising bisulfite-protected cytosines; (b) treating the ligated DNA molecules with bisulfite; (c) hybridizing the bisulfite-treated DNA molecules with a primer; (d) extending the hybridized primer to make double stranded DNA; and (e) in vitro transcribing the double-stranded DNA to make RNA.

The present invention relates to enzymes having DNA polymerase and 3′-5′ exonuclease activities. In particular, the present invention relates to a heat labile enzyme possessing a DNA polymerase II activity and a 3′-5′ exonuclease activity of marine origin. Furthermore, the present invention relates to a DNA polymerase primarily exerting a 3′-5′ activity, i.e. where the polymerase activity is absent. The present invention furthermore relates to the use of the exonuclease activity to degrade the 3′-5′ strand of double stranded DNA to perform single stranded overhang, e.g. in recombinant cloning processes, or in processes for removal of contaminating nucleic acid molecules.

The disclosure provides, for example, double stranded DNA (dsDNA) molecules comprising one or more chemically modified nucleobases. In some embodiments, the dsDNA molecule is circular and comprises a first strand and a second strand, wherein the first strand comprises one or more chemically modified nucleobases, and the second strand is free of chemically modified nucleobases. In some embodiments, the dsDNA molecule comprises a promoter sequence and an effector sequence that encodes an effector.

Described herein are, among other things, in vitro methods of identifying double stranded DNA sequences that are cleaved by a nuclease. For example, by providing a library of defined linear dsDNA oligonucleotides of known sequences, incubating the library in the presence of a nuclease, ligating DNA adapters, amplifying cleaved oligonucleotides, and determining the sequence(s) of the amplified fragments.

The methods, compositions, and kits of the disclosure provide a novel approach for a whole genome, unbiased DNA analysis method that can be performed on limited amounts of DNA can be used to analyze DNA to determine its modification status. Aspects of the disclosure relate to a method for amplifying bisulfite-treated deoxyribonucleic acid (DNA) molecules comprising: (a) ligating an adaptor to the DNA molecules, wherein the adaptor comprises a RNA polymerase promoter comprising bisulfite-protected cytosines; (b) treating the ligated DNA molecules with bisulfite; (c) hybridizing the bisulfite-treated DNA molecules with a primer; (d) extending the hybridized primer to make double stranded DNA; and (e) in vitro transcribing the double-stranded DNA to make RNA.

Mismatch anchoring compounds (MACs) are disclosed that recognize and bind to specific base-pair mismatches (mmBP) present within single stranded or double stranded DNA, RNA, and oligonucleotide sequences, and enable the ex vivo identification of DNA / RNA point mutations (DNAPM / RNAPM), including disease-associated, rare, and low abundance DNAPM / RNAPM, and the isolation and elimination of mmBP-positive cells. The use of MACs in vitro and in vivo (a) blocks mmBP-positive DNA replication and gene transcription / expression, (b) inhibits mmBP-positive cell proliferation, (c) reverses, prevents, and / or treats mmBP-rnediated disease, aging, and age-related disorders, (d) blocks mmBP-positive RNA and / or RNA-DNA duplex translation and inhibits the production of abnormal disease-causing proteins, (e) silence mmBP-positive genes, and (f) blocks intracellular pathogen replication. MAC-conjugates and their uses are disclosed, including MACs radiolabeled with SPECT / PET / particle-emitting isotopes for radioimaging and / or radiotherapy of mmBP-positive diseases.

Described is a linear double stranded DNA product comprising a first adaptor molecule ligated to a first end and a second adapter ligated to a second end. The first adaptor is a linear nucleic acid co

Current minicircle production methods are slow, expensive, and difficult to perform under GMP conditions because, in most cases, the product is derived from bacteria. In contrast, HTLA-and CHTLA-based synthetic circular supercoiled DNA production can be done completely in a test tube, using chemically or enzymatically synthesized oligonucleotides, long single stranded DNA, and / or double stranded DNA.

A primer for detection of a mutation in a microsatellite contained in a target sequence of a double stranded DNA molecule, wherein the mutation is a frameshift of the microsatellite as compared with the corresponding wild type microsatellite sequence. The primer comprises a region of at least 10 nucleotides that is complementary to the target sequence of the antisense or the sense strand of the DNA molecule containing the microsatellite having a frameshift mutation, except that the primer includes between one and four, or one and three, nucleotides which are mismatched to the target sequence containing the mutation in the microsatellite and which are also mismatched to a corresponding sequence containing the wild type microsatellite.

Described herein are various methods of removing uracils due to the deamination of unmethylated cytosines in an assay using engineered cytosine deaminases to deaminate methylated cytosines. The Chemoenzymatic Uracil Replacement of Nucleobases (ChURN) method includes providing a sample comprising single stranded DNA library fragments in which a cytosine deaminase has deaminated methylated cytosines; contacting the sample with an uracil DNA glycosylase (UDG) to deglycosylate uracil residues to form abasic sites having a hemiacetal formation within the single stranded DNA library fragments; contacting the sample with a reactive cytosine nucleobase analog to install a cytosine at abasic sites thru a noncanonical linkage; and subjecting the sample to polymerase chain reaction (PCR) amplification resulting in double stranded DNA corrected library fragments. The Uracil Enzymatic Removal and Substitution at Errors (U-ERASE) method includes providing a sample comprising single stranded DNA library fragments in which a cytosine deaminase has deaminated methylated cytosines, synthesizing double stranded DNA library fragments in which the second strand is tagged to facilitate its downstream degradation, treating the double stranded DNA library fragments with an uracil DNA glycosylase and an endonuclease resulting in the removal of uracil bases and single nucleotide gaps at those sites, and repairing the single nucleotide gaps through treatment with a polymerase, dCTP, and a ligase, resulting in replacement of false positive uracil bases with a mismatched base, such as cytosine. Subsequently, the second strand is selectively degraded, allowing for selective amplification of the original DNA strand via PCR.

The methods, compositions, and kits of the present disclosure provide a new method for whole genome, i.e., unbiased DNA analysis, that can be performed on limited amounts of DNA. It can be used to analyze DNA to determine its modification status. Aspects of the present disclosure relate to methods for amplifying a bisulfite treated deoxyribonucleic acid (DNA) molecule, the method comprising: (a) ligating an adaptor to the DNA molecule, wherein the adaptor comprises a RNA polymerase promoter comprising bisulfite protected cytosines; (b) treating the ligated DNA molecule with bisulfite; (c) hybridizing the bisulfite treated DNA molecule to a primer; (d) extending the hybridized primer to produce double stranded DNA; and (e) in vitro transcribing the double stranded DNA to produce RNA.

The disclosed invention relates to a linear double stranded DNA product with enhanced exonuclease resistance due to the presence of protected nucleotides, preferably phosphorothioated nucleotides. The

The invention relates to an in vitro method for modifying double stranded DNA (dsDNA) in a eukaryotic cell. The method comprises: a) introducing a base editor system into the cell, said system comprising i) a base editor, comprising an RNA guided DNA nickase linked to a single-stranded DNA nucleobase modifying enzyme, or a nucleic acid encoding said base editor, and ii) at least two guide RNA (sgRNA) molecules, comprising a first sgRNA that hybridizes to a first target sequence in the dsDNA, and a second sgRNA that hybridizes to a second target sequence in an opposing strand of the dsDNA, b) generating at least two single-strand breaks (SSBs), in opposing strands, of the dsDNA, wherein a first base editor complex, comprising the first sgRNA, introduces a nick cleavage of one strand of the dsDNA in the first target sequence, and a second base editor complex, comprising the second sgRNA, introduces a nick cleavage of the opposite strand of the dsDNA in the second target sequence, thereby producing a 5' or 3' overhang, c) inserting an exogenous DNA repair template sequence in proximity to the two single strand breaks (SSBs). The invention further relates to a genetically modified eukaryotic cell obtained by the method according to the present invention. The invention also relates to a kit for use in the in vitro method.

Mismatch anchoring compounds (MACs) are disclosed that recognize and bind to specific base-pair mismatches (mmBP) present within single stranded or double stranded DNA, RNA, and oligonucleotide sequences, and enable the ex vivo identification of DNA / RNA point mutations (DNAPM / RNAPM), including disease-associated, rare, and low abundance DNAPM / RNAPM, and the isolation and elimination of mmBP-positive cells. The use of MACs in vitro and in vivo (a) blocks mmBP-positive DNA replication and gene transcription / expression, (b) inhibits mmBP-positive cell proliferation, (c) reverses, prevents, and / or treats mmBP-rnediated disease, aging, and age-related disorders, (d) blocks mmBP-positive RNA and / or RNA-DNA duplex translation and inhibits the production of abnormal disease-causing proteins, (e) silence mmBP-positive genes, and (f) blocks intracellular pathogen replication. MAC-conjugates and their uses are disclosed, including MACs radiolabeled with SPECT / PET / particle-emitting isotopes for radioimaging and / or radiotherapy of mmBP-positive diseases.

Described is a linear double stranded DNA product comprising a first adaptor molecule ligated to a first end and a second adapter ligated to a second end. The double stranded DNA molecule is closed at

An effective gene therapy technique that can transfect a gene to a cell in an organ or a tissue in a subject in a safe, effective, and durable manner is disclosed herein. The methods disclosed herein can induce expression of a nucleic acid payload in target cell(s) of a mammalian subject by administering to the subject a linear double stranded DNA (dsDNA) construct covalently closed at each of its ends by DNA loops, the linear dsDNA construct comprising the nucleic acid payload; administering to the subject a plurality of microbubbles; and administering to the subject, in proximity to the target cell(s) an effective amount of an ultrasound energy, thereby inducing the formation of pores the target cell(s) by disrupting the plurality of microbubbles to result in introduction of the dsDNA into the target cell(s) and expression of the nucleic acid payload in the target cell(s).

The present invention relates to a method for producing large quantities of double stranded DNA material in an amount suitable for downstream applications, such as for in vitro transcription of RNA for therapeutic use. In particular, the present invention relates to a method of performing polymerase chain reaction (PCR) using a microwave synthesis reactor.

Provided herein are methods for determining a sequence of a double stranded DNA molecule of an immune cell receptor (e.g., T cell receptor, B cell receptor).

The present disclosure provides compositions and methods for inhibiting herpesvirus replication in a cell infected with herpesvirus. The composition includes a double stranded DNA molecule including a sequence of a cis regulatory sequence (crs) of a herpesvirus, wherein the crs is flanked on 5′ end by a first sequence of at least 2 base pairs and on the 3′ end by a second sequence of at least 2 base pairs.

The disclosed invention relates to a linear double stranded DNA product with enhanced exonuclease resistance due to the presence of protected nucleotides, preferably phosphorothioated nucleotides. The linear double stranded DNA comprises: a sense and antisense strand, at least two protected nucleotides at internal positions in the antisense strand, a single cassette comprising a coding sequence, and wherein the antisense strand has a protected nucleotide upstream and downstream of the cassette. By flanking the cassette, the protected nucleotides protect the cassette from exonuclease degradation. In preferred embodiments, the cassette comprises a promoter, and the sense strand also comprises protected nucleotides upstream and downstream of the cassette. A method of manufacturing the DNA product is disclosed, comprising rolling circle amplification (RCA) of a template in the presence of phosphorothioated nucleotides thereby inserting said nucleotides into the product in a sequence and concentration dependent manner, and restriction digesting the RCA product such that each fragment comprises a single cassette. The DNA products of the invention are transfected into cells, resulting in expression of recombinant GFP, and also used for in vitro transcription (IVT) reactions.

The present invention relates to a method for generating a single stranded DNA (ssDNA) molecule from a cyclic double stranded DNA (dsDNA) molecule.

A composition comprising a complex containing a polynucleotide of interest and a carrier is provided. The surface of the complex is coated covalently or non-covalently with a CD22 receptor ligand and can target CD22 on cells. The carrier may contain a polymer, a lipid, or a protein. The polynucleotide of interest may contain, for example, plasmid DNA (pDNA), linear double stranded DNA (dsDNA), linear single stranded DNA (ssDNA), messenger RNA (mRNA), small hairpin RNA (shRNA), or small interfering RNA (siRNA) that alters level or activity of a target molecule in B cells. Methods of delivering a polynucleotide of interest to a CD22 expressing cell, and methods of treating a B cell-associated disease (such as acute lymphoblastic leukemia) in a subject using the composition are also provided.

Described herein are, among other things, methods of identifying double stranded DNA sequences that are modified by a base editing enzyme. For example, by providing a plurality of linear dsDNA oligonucleotides of known sequences, incubating the library in the presence of a base editing enzyme, amplifying the oliognucleotides with a polymerase that converts edited base pairs to equal mixtures of canonical base pairs during DNA synthesis, and determining the sequences of the amplified oligonucleotides.