14 results about "Nuclease digestion" patented technology

The present invention relates to methods for producing a protected deoxyribonucleic acid (DNA) product with enhanced resistance to nuclease digestion. The methods comprise digesting a double-stranded DNA molecule with an endonuclease to generate a digested double-stranded DNA molecule comprising a first strand and a second strand, wherein the first strand has a 5' overhang; and extending the second strand 5' to 3' using a polymerase in the presence of one or more nuclease-resistant deoxynucleotide triphosphates to generate the protected DNA product, wherein the protected DNA product comprises an extended second strand comprising at least one nuclease-resistant nucleotide incorporated into the extended second strand by the polymerase using the 5' overhang of the first strand as template. Also provided are protected DNA products and uses thereof.

The present invention is directed to methods and kits for template-free enzymatic synthesis of polynucleotides employing hybridization stringency and / or nuclease digestion for removing failure sequences.

Linear DNA with improved resistance against exonucleases and methods for its production. The present invention relates to methods for producing a linear deoxyribonucleic acid (DNA) product (e.g., a closed linear DNA product) with improved resistance to nuclease digestion. The present invention relates to methods comprising the steps of: (a) contacting a double-stranded DNA molecule with an endonuclease, a ligase, and first and second terminal adaptor molecules and intermediate adaptor molecules to form a single contiguous aqueous volume; and (b) incubating the single contiguous aqueous volume to generate a linear DNA product (e.g., a closed linear DNA product). The present invention also relates to linear deoxyribonucleic acid (DNA) products (e.g., a closed linear DNA product) with improved resistance to nuclease digestion and uses thereof.

The invention discloses a method for analyzing a random hexamer primer degradation product, and relates to the technical field of molecular biology. Nuclease is used for digesting the random hexamer primer, the high performance liquid chromatography technology is combined, the degradation condition of the random hexamer primer can be rapidly and accurately analyzed, and the method has the technical advantages of being easy to operate, high in liquid phase method specificity, low in analysis cost and high in sensitivity.

The present invention relates to methods for producing a linear deoxyribonucleic acid (DNA) product (e.g. a closed linear DNA product) with enhanced resistance to nuclease digestion using a method comprising a cas nuclease. The present invention relates to methods comprising contacting a doublestranded DNA molecule with an RNP complex comprising a single stranded guide RNA (gRNA) and a cas nuclease, a ligase and first and second adaptor molecules to generate a linear DNA product. The step of contacting the DNA molecule with the RNP complex may be carried out prior to or simultaneously with the step of contacting with a ligase and first and second adaptor molecules.

The present disclosure provides a method for scalable and high-purity cell-free synthesis of DNA vectors, particularly closed-end DNA vectors (e.g., ceDNA vectors) with a linear and continuous structure for transgene delivery and expression. The cell-free synthesis involves digesting a double-stranded DNA construct with at least one restriction endonuclease that can cleave the construct at a cleavage site that is different from the recognition site to release an insert with a unique overhang that regulates the high specificity of the subsequent ligation reaction. The insert is then ligated with an inverted terminal repeat (ITR) oligonucleotide to form a closed-end DNA vector. The corresponding DNA vectors and related products prepared by these methods, as well as other bases associated with the methods and intermediate vectors and constructs, are also provided in the present disclosure.

In certain embodiments, the present invention provides a detection composition comprising a picodroplet comprising (a) an aqueous solution, and (b) a substrate probe comprising (i) an oligonucleotide of 2 to 75 nucleotides in length, (ii) a fluorophore operably linked to the oligonucleotide, and (iii) a quencher operably linked to the oligonucleotide. As used herein, the term “picodroplet” comprises a liquid droplet that has a volume of 0.014 to 2.6 picoliters. In certain embodiments, the present invention provides a method of detecting at least one individual nuclease molecule present in a sample, comprising contacting an aqueous sample suspected of containing at least one nuclease with at least one detection composition comprising a picodroplet comprising (a) an aqueous solution, and (b) a substrate probe comprising (i) an oligonucleotide of 2 to 75 nucleotides in length, (ii) a fluorophore operably linked to the oligonucleotide, and (iii) a quencher operably linked to the oligonucleotide to form an aqueous reaction mixture; emulsifying the aqueous mixture in oil to form picoliter-scale droplets in an emulsion, (c) incubating the picoliter-scale droplets in the emulsion in order for the nuclease, if present, to digest the substrate probes linked to the microbeads; recovering the microbeads; and detecting fluorescence emitting from the microbeads.

The present invention discloses a method and a kit for detecting HBV cccDNA in a single liver cell based on droplet digital PCR. The detection method provided by the present invention comprises the following steps: 1) preparing a single cell suspension by enzymatic hydrolysis; 2) DNase I nuclease digestion; 3) laminar flow washing; 4) ddPCR amplification and detecting the droplet fluorescence signal; according to the droplet fluorescence signal, obtaining the proportion of HBV infected cells in the sample to be tested and / or the viral load of HBV infected cells; the method or kit for detecting HBV cccDNA by digital PCR of the present invention has the following beneficial effects: the sample to be tested is a patient's liver biopsy tissue, and the absolute quantification of HBV cccDNA in the liver can be achieved at the single cell level, and the proportion of infected cells can be obtained; the detection sensitivity is high, the specificity is strong, the reaction system is small, the reaction is rapid, and the throughput is high, which is convenient for clinical large sample detection.

The invention relates to methods for producing a linear deoxyribonucleic acid (DNA) product with enhanced resistance to nuclease digestion. The linear DNA product may be a closed linear DNA product, a linear DNA product comprising nuclease-resistant nucleotides, or a partially closed DNA product.

The present invention relates to methods of enriching target single-stranded nucleic acids in a mixed population of single-stranded nucleic acids. The method involves protecting the target single-stranded nucleic acids and using a 5′ exonuclease to digest the non-target single-stranded nucleic acids. The invention also relates to methods of cloning target single-stranded nucleic acids into vectors, and to associated compositions and kits.

The invention relates to a preparation method and application of pseudoviral particles containing genome I / genome II (GI / GII) type norovirus nucleic acid fragments, and effectively solves the problems of application in preparation of drugs for inhibiting noroviruses and application in preparation of kits for detecting noroviruses, the preparation method comprises the following steps: constructing pET28a / CP recombinant plasmids, constructing pET28a / CP-NGI / NGII recombinant plasmids, constructing pET28a / CP-NGI / NGII recombinant plasmids, and constructing a pET28a / CP-NGI / NGII recombinant plasmids, thereby obtaining the pseudoviral particles containing genome I / genome II (GI / GII) type norovirus nucleic acid fragments. Transforming the expression vector into escherichia coli BL21, and inducing to express virus-like particles; adding lysate, performing ultrasonic treatment, crushing, adding ammonium sulfate, and performing coarse precipitation; filtering, chromatography and refining, loading a sample at a flow rate, and collecting an effluent; and adding Mg < + > and DNase I nuclease, and digesting to obtain the product. The method is scientific and reasonable, easy to operate, stable in performance, free of missing detection, accurate in detection, resistant to RNase attack and capable of monitoring operation in the whole process, a new way for detecting the norovirus is developed, application to preparation of drugs for inhibiting the norovirus and application to preparation of kits for detecting the norovirus are achieved, and the norovirus positive quality control product is suitable for being used for detecting the norovirus. The economic and social benefits are obvious.

The present invention relates to methods for producing a linear deoxyribonucleic acid (DNA) product (e.g. a closed linear DNA product) with enhanced resistance to nuclease digestion using a method comprising a Cas nuclease. The present invention relates to methods comprising contacting a double-stranded DNA molecule with an RNP complex comprising a guide RNA (gRNA) (e.g. a single stranded gRNA) and a Cas nuclease, a ligase and first and second adaptor molecules to generate a linear DNA product. The step of contacting the DNA molecule with the RNP complex may be carried out prior to or simultaneously with the step of contacting with a ligase and first and second adaptor molecules.

Disclosed herein are dual-expression polynucleotide vectors including a first polynucleotide sequence including, in the 5' to 3' direction, a bacteriophage promoter sequence operatively linked to a sequence encoding a plurality of first RNA hairpin structures, a multiple cloning site sequence, a sequence encoding a plurality of second RNA hairpin structures, and a transcription terminator sequence; and a second polynucleotide sequence including, in the 5' to 3' direction, a bacteriophage promoter sequence operatively linked to a ribosome binding site sequence, a viral coat protein sequence, and the transcription terminator sequence. RNA detection and / or quantification standards or controls produced from these dual-expression vectors, along with methods for producing the RNA detection and / or quantification standards or controls, and methods of detecting the presence or quantity of nucleic acid using the RNA detection and / or quantification standards, are also disclosed.

The invention discloses an information storage, encryption and decryption method based on a DNA nano switch, and belongs to the technical field of biomolecule self-assembly material information storage and encryption. According to the method, a key sequence is protected by means of an RNA chain, formation of an annular DNA nano-switch is prevented, and information storage and encryption are achieved, a target chain Tx chain is added in advance, then a secret key RNaseA nuclease is added to digest the RNA protection chain, the target chain Tx and a detection chain Dx form the annular DNA nano-switch based on base complementary pairing, RNaseA can digest all the RNA protection chains in a solution, and the RNA protection chains in the solution can be separated from the target chain Tx. The method has the advantages that the RNA protection chain Rx and the detection chain Dx are connected to form the annular DNA nano switch Sx, secondary connection of the residual RNA protection chain Rx and the detection chain Dx is avoided, the target chain Tx and the detection chain Dx are connected to form the annular DNA nano switch Sx after RNAseA nuclease digests the RNA protection chain, the agarose gel imaging analysis technology is combined, rapid decryption and accurate reading of molecular-level information can be achieved, and the accuracy of information encryption is improved. And a basic strategy is provided for promoting the development of a nano-scale biological information encryption technology.