137 results about "Reverse transcriptase" patented technology

The invention belongs to the field of biological detection, and discloses an exosome RNA detection composition based on positive and negative targets and a use method thereof, the composition comprises two positive targets and one negative target: the positive target A is miRNA stably expressed in exosome and is used for reflecting the expression level of small RNA; the positive target B is typical mRNA and is used for evaluating the quality of the long-fragment RNA; the negative target C is bacterial 16S rRNA and is used for identifying bacterial nucleic acid pollution in a sample. The method combines III-generation reverse transcriptase and HotStart Taq enzyme to construct a qPCR system, has high sensitivity and specificity, and is suitable for rapid detection of trace RNA. Whether the exosome RNA is suitable for downstream analysis or not can be judged by acquiring the Cq values of the three types of targets and setting a reference range. According to the invention, the integration of quality control, quantification and pollution identification of the exosome RNA is realized.

The present disclosure provides evolved and engineered reverse transcriptase variants and Cas9 variants with improved properties (e.g., improved editing efficiency when used in a guided editor context). The disclosure also provides fusion proteins comprising the reverse transcriptase variants and Cas9 variants described herein, including, for example, a guided editor. The disclosure also provides polynucleotides encoding the reverse transcriptase variants, Cas9 variants, and guide editors provided herein, as well as vectors comprising such polynucleotides. The disclosure also provides pharmaceutical compositions and cells comprising the reverse transcriptase variants, Cas9 variants, and a guided editor described herein. The present disclosure also provides methods and uses relating to the reverse transcriptase variants, Cas9 variants, and guide editors described herein.

Provided is a reverse prime editing system, which is used for achieving site-specific and precise small-fragment DNA editing upstream of a target site. In the reverse prime editing system, a targeted strand nickase (such as D10A-Cas9) is used to cleave a nick on a targeted strand, thereby enabling precise small-fragment DNA base insertion, deletion and substitution upstream of the target site under the action of a reverse transcriptase.

The invention discloses application of novel reverse transcriptase and related fusion protein of the novel reverse transcriptase in plant-guided editing. Candidate RT protein is obtained through systematic mining from a public database, novel reverse transcriptase cl1 with activity is screened out in combination with a fluorescence report system, and a guided editing system suitable for plants is constructed. The system tests in wheat protoplast, and verifies the editing activity of cl1 to a plurality of endogenous targets. Further combining with protein structure prediction and rational design point mutation optimization, the cl1 mutant with higher editing efficiency is obtained, and the method can be applied to accurate and effective editing of plant genomes and plant breeding and improvement.

The invention discloses a reverse transcriptase, wherein a coding sequence of the reverse transcriptase is derived from a Rattus norvegicus genome. The protein sequence of the protein comprises a protein sequence of a wild type Rattus norveicus, or a protein sequence which is subjected to engineering modification on the basis of the wild type Rattus norveicus, or a protein sequence which is subjected to engineering modification on the basis of the wild type Rattus norveicus. Furthermore, the protein sequence of the gene is as shown in any one of SEQ No.1-55. In a rice genome site and a human HEK 293T cell genome site, the system shows efficient editing efficiency of a guide editor. The invention provides a series of efficient reverse transcriptase components which can be carried on a guide editing system, important bottom-layer technical support is provided for research of point mutation in a genome, creation of a special site disease model and correction of genetic mutation sites, and the reverse transcriptase component has a wide application prospect in the field of precision medicine and precision agriculture breeding.

The present disclosure relates to crystalline and solvate forms of nucleoside reverse transcriptase translocation inhibitor (NRTTI) and pharmaceutical compositions thereof for use in the treatment and prevention of retroviridae viral infections, including infections caused by HIV virus.

The invention belongs to the technical field of biology, and relates to a pathogen detection technology based on a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) system, in particular to a tubercle bacillus detection and activity determination method based on fluorescent T7-CRISPR. The first purpose is to provide a reagent combination for detecting viable tubercle bacillus, and the reagent combination comprises a) a T7 reverse transcription reaction reagent and b) a CRISPR-Cas13a reaction system; a) the T7 reverse transcription reaction reagent comprises a T7 reverse transcription primer and a T7 reverse transcriptase; and b) the CRISPR-Cas13a reaction system comprises specific crRNA, a fluorescence labeled ssRNA reporter, a reaction buffer solution, a Cas13a protein, RNase-Free H2O and a T7 reverse transcriptase. The invention also provides application of the reagent combination in detection of viable tubercle bacillus. The primer combination disclosed by the invention has the advantages of strong specificity and high sensitivity in detection application, can realize rapid detection and visual detection, and is low in detection cost, simple and convenient to operate and suitable for large-scale clinical application.

The disclosure provides Moloney murine leukemia virus (MMLV) reverse transcriptase (RTase) mutants. The disclosure as provides suitable amino acid positions in MMLV RTase for mutagenesis and methods and kits for using MMLV RTase mutants to synthesize cDNA from RNA templates.

Disclosed herein is a chimeric nucleic acid including a human telomerase reverse transcriptase (hTERT) enhancer or a fragment thereof and an SV40 enhancer or a fragment thereof and methods of using the same.

The present disclosure provides Moloney murine leukemia virus (MMLV) reverse transcriptase (RTase) variants. The present disclosure further provides amino acid positions for mutagenesis of MMLV RTase as well as nucleic acids, kits, compositions, fusion proteins, and methods including MMLV RTase variants.

The present invention provides a method for screening for a telomerase reverse transcriptase (TERT) phosphorylation inhibitor using TERT or a fragment thereof, and a TERT kinase.

The invention provides a chain-specific library preparation method and a high-throughput sequencing method for rapidly detecting multiple types of RNAs (Ribonucleic Acid). The method comprises the following steps: artificially adding poly A tails at 3'tail ends of multiple RNAs by utilizing poly A polymerase; meanwhile, a polydeoxythymine ribonucleotide primer with deoxyuracil is used for synthesizing single-stranded cDNA under the action of reverse transcriptase, obtained single-stranded cDNA molecules are subjected to a series of reactions and finally subjected to PCR amplification to obtain strand specific libraries of multiple types of RNA, and samples of the libraries can be subjected to computer sequencing.

This invention pertains to fusion protein mutants comprising a Prime Editing enzyme having a first amino acid sequence and a second amino acid sequence, wherein the first amino acid sequence comprises a SpCas9 H840A nickase mutant protein of SEQ ID NO:152 and the second amino acid sequence comprises a Moloney Murine Leukemia Virus reverse transcriptase protein mutant (MMLV RTase mutant), wherein the fusion protein mutant displays at least the equivalent or greater activity of a reference Prime Editing enzyme in genome editing.

The invention discloses a kit and a method for preparing cDNA (complementary deoxyribonucleic acid) by reverse recording of a single B cell of a mouse. The kit comprises a B cell lysis solution and a reverse transcription buffer solution system, wherein the B cell lysis solution is prepared from a nonionic detergent, dNTP (deoxyribonucleoside triphosphate), dCTP (deoxycytidine triphosphate), a secondary structure stabilizer, a reducing agent, BioIS-mCH2-RT (reverse transcriptase), BioIS-CK-RT, a mouse RNA (Ribonucleic Acid) enzyme inhibitor and nuclease-free water; the reverse transcription buffer solution system comprises 5X RT Buffer, a secondary structure stabilizer, Mg < 2 + >, a reducing agent, a mouse RNA enzyme inhibitor, a Bio-Adaptor primer, reverse transcriptase and nuclease-free water. The kit can significantly improve the success rate, accuracy and sensitivity of reverse transcription of a single B cell, and simplify the operation process.

(1) An artificial nucleic acid molecule comprising (a) at least one of a 3'-untranslated region (3'UTR) element and / or (b) a 5'-untranslated region (5'UTR) element, and (2) a poly(A) tail is described. The artificial nucleic acid molecule may further comprise a polypeptide comprising a reverse transcriptase (RT) domain and optionally an endonuclease domain for genome modification. A system comprising the artificial nucleic acid molecule and a method of using the system are also described.

The invention belongs to the technical field of medicines, and particularly relates to pyrrolo [2, 3-d] pyrimidine compounds of aryl fragments and piperidine fragments as well as a preparation method and application of the pyrrolo [2, 3-d] pyrimidine compounds. The pyrrolo [2, 3-d] pyrimidine compound containing the aryl fragment and the piperidine fragment is an HIV-1 reverse transcriptase inhibitor, and the pyrrolo [2, 3-d] pyrimidine compound further comprises pharmaceutical salt, hydrate and solvate of the pyrrolo [2, 3-d] pyrimidine compound, polycrystal or eutectic of the pyrrolo [2, 3-d] pyrimidine compound, and precursors and derivatives with the same biological functions of the pyrrolo [2, 3-d] pyrimidine compound. The invention also relates to a preparation method of the compound and application of a composition containing one or more of the compounds in related medicines for treating AIDS and the like. The result of an in-vitro cellular level anti-HIV-1 activity experiment shows that the micromolecule has relatively strong anti-HIV-1 biological activity, can remarkably inhibit virus replication in MT-4 cells infected by HIV-1 viruses, and has relatively low cytotoxicity.

The application belongs to the technical field of biology, and specifically provides a reverse transcriptase UCRT vZ mutant with improved thermal stability, the reverse transcriptase UCRT vZ mutant includes four single-point mutants, six double mutants and four triple mutants, compared with a wild-type reverse transcriptase UCRT vZ, the half-life of the mutants is longer at 65 DEG C, the effect of the double mutants is better, and the half-life is about 4 times that of the wild-type reverse transcriptase UCRT vZ. The application further provides a genetically engineered bacterium containing the mutant. The reverse transcriptase UCRT vZ mutant obtained by the construction method has better thermal stability, and when used for RNA reverse transcription into cDNA at a higher temperature, the reverse transcriptase UCRT vZ mutant has higher thermal stability and great application potential.

Provided are methods comprising expressing in a recombinant cell comprising a Cas gene a recombinant error-prone reverse transcriptase (RT) and a recombinant spacer RNA comprising a target sequence for mutagenesis of a DNA sequence in the Cas gene; making a mutagenized cDNA polynucleotide homologous to the DNA sequence in the recombinant cell; expressing a recombinant recombineering system in the recombinant cell; and recombining the mutagenized cDNA with the homologous DNA sequence of the Cas gene in the recombinant cell. Also provided are recombinant cells comprising recombinant coding sequences for a recombinant Cas protein, recombinant error-prone reverse transcriptase (RT), recombinant spacer RNA comprising the target sequence, and recombinant recombineering system.

According to the invention, reverse transcriptase Marathon or a mutant thereof is fused with Cas nuclease, a small RNA binding exonuclease protection factor La and a positioning signal peptide to form a fusion protein capable of guiding editing. Experiments show that a guide editing system based on the fusion protein can realize base replacement operation in rice callus, and has important application value in the field of biological breeding.

A method of immortalizing a human keratinocyte. Also provided are immortalized human keratinocytes, for example immortalized human keratinocytes that have been immortalized using the presently disclosed method. Further provided are organotypic cultures, co-cultures, assays and kits comprising one or more immortalized human keratinocytes. In one embodiment, the immortalized human keratinocyte is produced by sequentially deleting the p16 locus, using CRISPR-Cas9 based gene editing, followed by lentiviral-based introduction of the gene encoding human telomerase reverse transcriptase (hTERT).

Cell culture media comprising luteinizing hormone, follicle-stimulating hormone and at least one histone acetyltransferase inhibitor or reverse transcriptase inhibitor are provided. Methods of in vitro maturing a human oocyte, determining suitability for in vitro maturation, as well as kits comprising media, luteinizing hormone, follicle-stimulating hormone and at least one histone acetyltransferase inhibitor or reverse transcriptase inhibitor are also provided.

Disclosed herein include systems, methods, compositions, and kits for prevention of antibody oligonucleotide extension. There are provided, in some embodiments, blocking oligonucleotides. In some embodiments, the blocking oligonucleotide comprises a binding region capable of hybridizing barcoding oligonucleotides to form a protected duplex. The blocking oligonucleotide can be a locked nucleic acid (LNA), a peptide nucleic acid (PNA), a DNA, an LNA / PNA chimera, an LNA / DNA chimera, or a PNA / DNA chimera. In some embodiments, the 3′ end of a blocking oligonucleotide is incapable of being extended by a reverse transcriptase or a polymerase. In some embodiments, the blocking oligonucleotide of a protected duplex is incapable of being removed by the strand displacement activity of a reverse transcriptase or a polymerase.

Provided is a novel compound that is useful as an antiviral drug, exhibits strong drug efficacy in low dosages, can be used safely, and has reverse transcriptase inhibitory activity. This compound is expressed by formula (I) (wherein ring A is (i-a) or (i-b) (where R1 represents a halogen or the like, R2 represents a halogen or the like, R3 represents hydrogen or the like, L represents –O– or the like, R4 represents hydrogen or the like, ring B represents an optionally substituted five-membered or six-membered aromatic heterocycle ring or the like, and n represents an integer of 1-3), and Z is (Z) (where each R6 independently represents a halogen or the like, and m represents an integer of 0-2)).

Disclosed is a cDNA amplification method capable of comprehensively amplifying mRNA with a high efficiency and a low bias even from a very small amount of cell sample. The method of amplifying a complementary DNA strand of the present invention comprises steps of capturing a target RNA and synthesizing a complementary DNA strand using a reverse transcriptase having terminal transferase activity in a liquid-phase system or a solid-phase system such that bases such as CCC are added to an end of the complementary DNA strand, and thereafter carrying out homopolymer tailing by the TdT reaction in the presence of a chain-terminating nucleotide triphosphate. By combination with a template-switching reaction, the method enables amplification of the complementary DNA strand with a higher efficiency and a lower bias.

The application discloses a circRNA detection method based on Argonaute protein and rolling circle amplification. The method comprises the following steps: mixing a sample containing target circRNA to be detected with a specific reverse transcription primer and reverse transcriptase to obtain long-chain cDNA; mixing the obtained long-chain cDNA with pfAgo, gDNA and a DNA nucleic acid probe to carry out a reaction, and calculating the content of the target circRNA according to the fluorescence signal of the reaction mixture; the nucleotide sequence of the gDNA is specifically complementary to the cDNA sequence obtained by reverse transcription of the circRNA; the sequence of the DNA nucleic acid probe is specifically complementary to the sequence of the cDNA after one cutting of the cDNA by the pfAgo; and the DNA nucleic acid probe is a single-stranded DNA probe with a fluorescent label. The application can realize high-sensitivity, high-specificity and multiple target simultaneous analysis and detection of potential diagnostic biomarkers of liver cancer such as hsa_circ_0001445 and hsa_circ_0001141.

Described herein are recombinant viral genomes comprising a nucleotide sequence encoding a telomerase reverse transcriptase (TERT) operably linked to a tissue-specific and / or organ-specific promoter. Aspects described herein may be used in the treatment of conditions associated with shortened telomere length, such as pulmonary fibrosis, myocardial infarction and conditions associated therewith.

Compositions and methods are provided herein for conducting prime editing of a target DNA molecule (e.g., a genome) that enables the incorporation of a nucleotide change and / or targeted mutagenesis. The compositions include fusion proteins comprising nucleic acid programmable DNA binding proteins (napDNAbp) and a polymerase (e.g., reverse transcriptase), which is guided to a specific DNA sequence by a modified guide RNA, named an PEgRNA. The PEgRNA has been altered (relative to a standard guide RNA) to comprise an extended portion that provides a DNA synthesis template sequence which encodes a single strand DNA flap which is synthesized by the polymerase of the fusion protein and which becomes incorporated into the target DNA molecule.

The present invention provides novel engineered reverse transcriptase enzymes that afford beneficial improvements in thermal stability, processivity, cDNA yields and elimination of secondary enzymatic activity. The present invention also provides methods for amplifying template nucleic acids using such reverse transcriptase enzymes. This invention addresses deficiencies in the current state of the art reverse transcriptase enzymes in RNA detection and analysis including deficiencies in detection sensitivity, specificity, side enzyme activities, enzyme stability and synthesis capacity, especially when using template nucleic acids ranging in length, secondary structure and nucleotide content.

The present invention relates to a method for recording a transcriptome of a cell by: providing a test cell that includes a first transgene nucleic acid sequence encoding a fusion protein that is a reverse transcriptase polypeptide and a Cas1 polypeptide and a second transgene nucleic acid sequence encoding a Cas2 polypeptide, wherein the first transgene nucleic acid sequence and the second transgene nucleic acid sequence are under transcriptional control of an inducible promoter sequence, and a third transgene nucleic acid sequence including a CRISPR direct repeat (DR) sequence; wherein the CRISPR direct repeat sequence is specifically recognizable by a RT-Cas1-Cas2 complex formed by the expression products of the first transgene nucleic acid sequence and the second transgene nucleic acid sequence.

Methods and compositions for modulating a target genome are disclosed. The composition may comprise a first RNA encoding a polypeptide comprising a retrotransposase reverse transcriptase domain and a retrotransposase endonuclease domain. The composition may also comprise a second RNA comprising a sequence that binds the polypeptide and a heterologous object sequence. The composition may insert the sequence of the heterologous object sequence into a target DNA.