231 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 invention belongs to the technical field of molecular diagnosis, and particularly discloses a high-risk HPV16 rapid detection system based on RPA-CRISPR / Cas12a, which is prepared by the following steps: (1) mixing a nucleic acid lysis solution containing Tris (pH = 8.0), NaHCO3, polyvinylpyrrolidone and Tween-20 with Proteinase K to treat a sample, and performing pyrolysis at room temperature for 5 minutes to obtain a nucleic acid template; (2) an RPA reaction system contains an RNA inhibitor, a reverse transcriptase and an RPA reaction essential enzyme, and after the template and the starting agent in the step (1) are added, RPA amplification is carried out at 37 DEG C; (3) dropping the reaction liquid on a colloidal gold test strip subjected to position exchange and transformation of a sample pad and a water absorption pad, and carrying out result interpretation; and (4) adding the RPA product into a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) reaction system containing the LbCas12a protein, the specific gRNA and the probe. According to the scheme, a rapid, high-sensitivity and cross-reaction-free detection system of the HPV16 is realized by optimizing a nucleic acid lysis solution formula, designing a specific primer and gRNA and modifying a test strip for detection.

The invention discloses a polypeptide ligand for improving the thermal stability of MMLV reverse transcriptase and application of the polypeptide ligand, and relates to the technical field of biology. The invention specifically discloses a polypeptide ligand with an amino acid sequence as shown in SEQ ID NO: 1. The polypeptide ligand can obviously improve the thermal stability of MMLV RT. After being combined with the polypeptide ligand, the MMLV RT can effectively maintain the activity of the MMLV RT at 45-60 DEG C and maintain good activity in a freeze-thaw cycle, and the Ct value change is small after the MMLV RT is placed at 37 DEG C for more than 15 days, so that the polypeptide has a very good application prospect in industrial production and commercial application.

A gene editing system comprising (a) a fusion polypeptide comprising an RNA-guided nuclease and a reverse transcriptase, or a nucleic acid encoding the fusion polypeptide, and (b) an RNA molecule comprising a guide RNA and a reverse transcription donor RNA, or a nucleic acid encoding the RNA molecule. Also provided herein are methods of using the gene editing system for modifying target genes of interest.

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.

This invention relates to recombinant nucleic constructs comprising a DNA binding domain, an endonuclease and a reverse transcriptase and methods of use thereof for modifying nucleic acids in plants.

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 application discloses a method for synthesizing reverse transcriptase: obtaining a target reverse transcriptase expression sequence based on database primary screening; obtaining a candidate reverse transcriptase expression sequence by using a fluorescent reporter system to rescreen the target reverse transcriptase expression sequence; and determining a synthetic reverse transcriptase expression sequence by using the fluorescent reporter system after optimizing the candidate reverse transcriptase expression sequence. The application further screens a plurality of natural or engineered small reverse transcriptases in view of the bottleneck of the prior art, and verifies the applicability of the reverse transcriptases in prime editing. The technology is expected to break through the application limitation of the existing prime editing system, and provide more efficient and safer precise editing tools for the fields of genetic disease treatment, agricultural breeding and synthetic biology.

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.

Disclosed are DNA aptamers. Related compositions and kits are also provided. Also disclosed are methods for amplifying a target DNA sequence using the DNA aptamers, and methods of identifying the DNA aptamers.

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.

Disclosed are DNA aptamers. Related compositions and kits are also provided. Also disclosed are methods for amplifying a target DNA sequence using the DNA aptamers, and methods of identifying the DNA aptamers.

(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.

The present invention relates to a reverse transcriptase comprising a modified Moloney murine leukemia virus reverse transcriptase amino acid sequence wherein the amino acid sequence comprises a mutation relative to the wild type Moloney murine leukemia virus reverse transcriptase and the enzyme has enhanced inhibitor resistance compared to the wild type Moloney murine leukemia virus reverse transcriptase. The invention also relates to a composition, a reaction mixture and a kit comprising the reverse transcriptase. The reverse transcriptase is used for synthesizing cDNA from RNA.

The invention discloses a KIM-1 detection kit, which is designed based on an RT-LAMP method of Taqman, and comprises: a specific primer group comprising an outer primer and an inner primer; a TaqMan probe; the reaction enzyme mixture comprises BstDNA polymerase with strand displacement activity and AMV reverse transcriptase; the reaction buffer solution is prepared from Tris. HCl, KCl, (NH4) 2SO4 and Tween20; the quality control product comprises a positive control group, a negative control group and a blank plasmid control group. Compared with the prior art, the kit has the advantages that the KIM-1 detection kit is designed based on the RT-LAMP technology of Taqman, and quantitative detection of KIM-1 mRNA is achieved through the integrated reaction of reverse transcription, amplification and real-time fluorescence detection.

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.