26 results about "Reverse transcriptase activity" patented technology

The invention discloses a polypeptide ligand 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 stability of MMLV reverse transcriptase can be remarkably improved, the MMLV reverse transcriptase can be placed at 37 DEG C for more than 15 days, and the Ct value difference is lower than 2; in addition, the MMLV reverse transcriptase activity can be maintained under a high-temperature condition (45-60 DEG C); the activity loss of the MMLV reverse transcriptase in a freeze-thaw cycle is reduced, and the industrial production and commercial application of the MMLV reverse transcriptase are effectively promoted.

To provide a novel divided-type reverse transcriptase that shows activity only in the case where all constituting fragments are present.SOLUTION: Provided is a divided-type reverse transcriptase including an N terminal fragment and a C terminal fragment in which a reverse transcriptase is divided into two, where the N terminal fragment and the C terminal fragment are at least one kind selected from the group consisting of an N terminal fragment and a C terminal fragment which are divided in any one place in a specific region of a specific amino acid sequence, and because the N terminal fragment and the C terminal fragment are present, reverse transcriptase activity is reproduced.SELECTED DRAWING: Figure 9

This split-type reverse transcriptase includes an N-terminal fragment and a C-terminal fragment obtained by dividing a reverse transcriptase into two. The N-terminal fragment and the C-terminal fragment are at least one type selected from the group consisting of: (a) an N-terminal fragment and a C-terminal fragment obtained by splitting a polypeptide containing an amino acid sequence specified by SEQ ID NO: 1 at one location in the region of positions 114-133 in the amino acid sequence specified by SEQ ID NO: 1 or at one location in the region of positions 167-186 in the amino acid sequence specified by SEQ ID NO: 1; (b) an N-terminal fragment and a C-terminal fragment obtained by splitting a polypeptide, which contains the amino acid sequence specified by SEQ ID NO: 1 with one or a plurality of amino acid residues having been substituted, deleted, inserted, and / or added, at one location in a region corresponding to positions 114-133 in the amino acid sequence specified by SEQ ID NO: 1 or at one location in a region corresponding to positions 167-186 in the amino acid sequence specified by SEQ ID NO: 1; and (C) an N-terminal fragment and a C-terminal fragment obtained by splitting a polypeptide, which contains an amino acid sequence having at least 90% homology with the amino acid sequence specified by SEQ ID NO: 1, at one location in a region corresponding to positions 114-133 in the amino acid sequence specified by SEQ ID NO: 1 or at one location in a region corresponding to positions 167-186 in the amino acid sequence specified by SEQ ID NO: 1. The reverse transcriptase activity is regenerated owing to the presence of the N-terminal fragment and the C-terminal fragment.

Substituted cyclo-oxymethylene phosphonamidates and related compounds, pharmaceutical compositions, their use for inhibiting LINE1 reverse transcriptase and / or HERV-K reverse transcriptase activity, and their use in the treatment of medical disorders, such as cancer, are disclosed herein.

The present application relates to the technical field of genetic engineering, and specifically provides a multi-link quality control product for novel coronavirus nucleic acid detection laboratory quality control and a preparation method thereof. The multi-link quality control product is a multi-component composite system, which is formed by mixing three substances of a pseudo virus, a target gene RNA molecule and an internal standard plasmid DNA. The pseudo virus is a pseudo virus particle of a vesicular stomatitis virus vector wrapping a novel coronavirus ORF1ab gene RNA, which is used for monitoring virus nucleic acid extraction; the target gene RNA molecule is a novel coronavirus N gene RNA molecule, which can monitor reverse transcriptase activity; and the internal standard plasmid is a plasmid DNA of a human source housekeeping gene RPP30, which monitors whether there is inhibition or failure in the amplification link. The present application realizes synchronous layered monitoring of the whole process of nucleic acid extraction, reverse transcription and amplification, solves the monitoring blind area of traditional single-component quality control products, and significantly improves the accuracy and efficiency of laboratory detection quality control.

The invention relates to the technical field of traditional Chinese medicine extract fermentation, and provides a microspore ganoderma lucidum immune protein and herba houttuyniae fermentation method for treating AIDS and application of microspore ganoderma lucidum immune protein and herba houttuyniae fermentation method.The microspore ganoderma lucidum immune protein and herba houttuyniae fermentation method comprises the steps that microspore ganoderma lucidum strains and herba houttuyniae fermentation liquor are co-fermented, beta-cyclodextrin-selenocystine of a specially-made additive is combined, and the microspore ganoderma lucidum immune protein is obtained; the problems that in traditional AIDS treatment, latent infection cells are difficult to remove, toxic and side effects of drugs are remarkable, and the reconstruction efficiency of an immune system is low are effectively solved, experiments prove that the fermentation product can activate the immune system, the cell number and the dendritic cell maturity are remarkably improved, and the curative effect is good. Meanwhile, the multi-target antiviral effect is achieved by blocking virus envelope combination, inhibiting reverse transcriptase activity and inducing latent infection cell apoptosis; the anti-oxidation and anti-inflammatory characteristics can reduce the risk of opportunistic infection, reduce the toxic damage of antiviral drugs to liver, kidney and intestinal tracts, and provide a new scheme for comprehensive treatment of AIDS and nutrition support of patients.

The present disclosure relates to a method for analyzing mutations, insertions, deletions and / or single or nucleotide variations in an RNA sample via nick filling, the method comprising the steps of: (a) providing an RNA sample comprising at least one nucleotide extension to be identified having an unknown identity, the nucleotide extension segment with unknown identity is laterally connected with a nucleotide extension segment with known identity; (b) contacting the RNA sample with at least one DNA probe, such as at least two linear probes or at least one DNA padlock probe having a first end and a second end, under conditions and reagents that allow hybridization, the first end and the second end are designed to hybridize with a sequence having a known identity that flanks the at least one nucleotide extension having an unknown identity, thereby creating a gap between the hybridized first end and second end of the at least one DNA probe; (c) adding a polymerase having reverse transcriptase activity and optionally having nick translation activity and optionally having limited strand displacement under conditions and reagents allowing reverse transcription, thereby allowing reverse transcription of the at least one DNA probe from the first end to the second end using the RNA sample as a template, such that the reverse transcribed portion of the at least one DNA probe comprises a complementary sequence of the nucleotide extension having the unknown identity in the RNA sample; (d) adding a ligase which can use RNA as a splint molecule under conditions and reagents which allow ligation, thereby allowing the blocking of the at least one reverse transcribed DNA probe; (e) the probe containing the nucleotide subjected to reverse transcription can be amplified; and (f) sequencing the at least one amplified cyclized oligonucleotide, thereby revealing the identity of the at least one nucleotide extension having an unknown identity of the RNA sample. The present disclosure further relates to a kit for use in a method of analyzing mutations for both in situ and in vitro applications.

Taq DNA polymerase mutants exhibiting reverse transcriptase activity compared to wild type polymerase were engineered, characterized, and selected via polymerase chain reactions visualized via electrophoresis on agarose gels. Initial screening was followed up with probe-based qualitative, real-time PCR (qPCR) with a typical reverse transcription cycling protocol to detect specified ribonucleic acid (RNA) target sequences. The engineered variants can render robust cDNA from RNA target substrates and amplify that cDNA under standard reaction conditions without the assistance of added reverse transcriptase enzymes.

Provided herein are compositions and methods for use in amplification assays. In particular, provided herein are Bst polymerases with enhanced reverse transcriptase (RT) activity for use in amplification assays such as, for example, loop-mediated isothermal amplification (LAMP) assays.

Taq DNA polymerase mutants exhibiting reverse transcriptase activity compared to wild type polymerase were engineered, characterized, and selected via polymerase chain reactions visualized via electrophoresis on agarose gels. Initial screening was followed up with probe-based qualitative, real-time PCR (qPCR) with a typical reverse transcription cycling protocol to detect specified ribonucleic acid (RNA) target sequences. The engineered variants can render robust cDNA from RNA target substrates and amplify that cDNA under standard reaction conditions without the assistance of added reverse transcriptase enzymes.

The invention belongs to the technical field of virus detection, and discloses a reaction solution for DNA virus and RNA virus detection, the reaction solution is a reaction solution containing acetate, acetate salt is introduced as an ionic reaction system, the specificity of a one-step method RT-PCR amplification system is greatly improved, and the specificity of the one-step method RT-PCR amplification system is greatly improved. The amplification inhibition phenomenon of the residual reverse transcriptase activity on a subsequent DNA template is greatly reduced, so that the kit has a good effect on DNA detection while being capable of detecting the RNA virus.

Compounds, pharmaceutical compositions, their use for inhibiting LINE1 reverse transcriptase activity, and their use in the treatment of medical disorders, such as cancer, are disclosed herein.

The application belongs to the field of genetic engineering and enzyme engineering, and particularly relates to a MMLV reverse transcriptase mutant, a preparation method, products and applications thereof. The mutant is based on the sequence shown in SEQ ID NO. 1, contains a Q374R mutation, and contains at least two mutations selected from L362P, R657C, N131D, G138S and L499Q, preferably L362P, Q374R and R657C, N131D, Q374R and L499Q, N131D, G138S, L362P, Q374R and L499Q. The mutant can be used for cDNA synthesis and RNA reverse transcription, quantitative detection. Compared with the wild type, the mutant has higher reverse transcriptase activity and thermal stability, can tolerate higher reaction temperature to open the secondary structure of RNA, improve the cDNA synthesis effect, has better detection sensitivity in RT-qPCR, and meets the efficient reverse transcription requirements in multiple fields.

The application discloses a heat-resistant reverse transcriptase mutant and a preparation method and application thereof, and relates to the technical fields of genetic engineering and enzyme engineering. The amino acid sequence of the reverse transcriptase mutant is compared with the amino acid sequence shown in SEQ ID NO:1, and one or more of the positions of the amino acid sequence shown in SEQ ID NO:1 are mutated, that is, the 8th position, the 204th position, the 249th position, the 289th position, the 306th position, the 309th position, the 524th position, the 562th position, the 571th position, the 583th position and the 664th position. The purity of the reverse transcriptase mutant can reach more than 98 %, the reverse transcription temperature of 50 DEG C and 55 DEG C is tolerated, more than 50 % of reverse transcriptase activity is retained, one-step RT-PCR is realized, and the application efficiency and prospect of the reverse transcriptase are expanded.

Method and system for detecting reverse transcriptase activity of a sample by digital assay. In an exemplary method, a reaction mixture including the sample, an RNA polymer, and reagents for reverse transcription may be prepared. Complementary DNA (cDNA) may be synthesized in the reaction mixture using the RNA polymer as a template. An amount of the cDNA synthesized may be proportional to the reverse transcriptase activity of the sample. Partitions may be formed after synthesizing the cDNA. The partitions may contain copies of the cDNA at partial occupancy. Each partition may include a portion of the reaction mixture. A target representing the cDNA may be amplified in the partitions. Amplification data for the target may be collected from the partitions.

This invention discloses a Taq DNA polymerase mutant and its applications. The Taq DNA polymerase mutant is derived from the amino acid sequence SEQ ID NO.1 and contains the following mutations: E9K, L15S, D18R, H20E, H21E, K31E, and R37D. This invention obtains a Taq DNA polymerase mutant through point mutagenesis. Unlike the wild-type Taq DNA polymerase, it possesses highly efficient and stable reverse transcriptase activity, enabling efficient conversion of cDNA using RNA substrates and amplification of the cDNA under standard reaction conditions without the need for additional reverse transcriptase. This significantly improves the efficiency of detecting target ribonucleic acid (RNA) by real-time quantitative PCR.

The present invention provides a composition comprising one or more polynucleotides capable of upregulating expression of telomerase wherein telomerase consists of a protein component with reverse transcriptase activity encoded by telomerase reverse transcriptase (TERT) gene and a non-coding RNA encoded by telomerase RNA component (TERC) gene, In an embodiment, the one or more polynucleotides of the present invention upregulates the telomerase by downregulating antisense RNA that regulates telomerase gene. In an embodiment, the one or more polynucleotides comprises an siRNA or an shRNA. In an embodiment, one or more polynucleotides of the present invention is delivered by a nanoparticle such as an exosome that encapsulates the one or more polynucleotides. The present invention also provides a method of treatment of telomerase deficiency related diseases or disorders using any embodiment of the one or more polynucleotides of the present invention.

The invention relates to the technical field of virus nucleic acid detection, and provides a dengue virus rapid detection kit and a dengue virus rapid detection method. By constructing a novel fusion enzyme with both reverse transcriptase activity and DNA polymerase activity, developing a rapid nucleic acid extraction technology based on high-performance magnetic beads, and establishing an optimized freeze-drying formula and a rapid amplification program, the whole process from sample treatment to result interpretation is completed within 25-30 minutes, the detection sensitivity reaches 500 copies / mL, the reagent can be stored at normal temperature for 12 months, and the detection time is greatly shortened. The operation is simple and convenient. Clinical samples prove that the sensitivity is 99.2%, the specificity is 100%, compared with an existing method, the detection time is shortened to 1 / 5 of the original detection time, the sensitivity is doubled, and the reagent stability is remarkably improved. The method is suitable for port quarantine, basic medical institutions and on-site rapid screening of epidemic situations, and has important practical significance on prevention and control of dengue fever epidemic situations.

The application discloses a recombinant Bst DNA polymerase with high DNA polymerase activity and reverse transcriptase activity, and preparation and application thereof. Sulfolobus tokodaii The application carries out sequence rearrangement on a Headpiece domain truncated body Hp47 derived from chicken villin and a Sto7d domain derived from a thermophilic archaea, and fuses the amino terminal end of the Bst DNA polymerase large fragment to construct a novel Bst DNA polymerase rearrangement mutant HpSt-G23. The recombinant polymerase HpSt-G23 obtained by the application has high polymerase activity and reverse transcriptase activity, and can resist inhibitors such as urea and sodium chloride. Tth The HpSt-G23 can be used in a RecA detection system, and only two primers are needed to complete DNA or RNA amplification in the same system, and the detection sensitivity of the RNA template is greatly improved, so that the HpSt-G23 has a good development prospect in the field of nucleic acid detection.

The invention belongs to the field of gene engineering and enzyme engineering, and particularly relates to an MMLV reverse transcriptase mutant as well as a preparation method, a product and application thereof. The mutant is based on a sequence as shown in SEQ ID NO.1, must contain Q374R mutation, and contains at least two mutations selected from L362P, R657C, N131D, G138S and L499Q, preferably L362P, Q374R and R657C, N131D, Q374R and L499Q, and N131D, G138S, L362P, Q374R and L499Q. The mutant can be used for cDNA synthesis and RNA reverse transcription and quantitative detection. Compared with a wild type, the mutant has higher reverse transcriptase activity and thermal stability, can tolerate a higher reaction temperature to open an RNA secondary structure and improve the cDNA synthesis effect, has better detection sensitivity in RT-qPCR, and meets the requirements of efficient reverse transcription in multiple fields.

This invention discloses a method for detecting reverse transcriptase activity. The method includes: S1. mixing the reverse transcriptase to be tested, RNA template, reverse transcription primers, and dNTPs to perform a reverse transcription reaction to obtain a reverse transcription product; S2. preparing a nanopore sequencing library using the reverse transcription product; S3. sequencing the nanopore sequencing library using nanopores to obtain sequencing data; S4. determining the activity of the reverse transcriptase to be tested using the sequencing data. This method can accurately and comprehensively evaluate the transcriptional ability of different reverse transcriptases for RNA of various lengths, thus providing precise and detailed data support for the evaluation and optimization of reverse transcriptases.

The application discloses a high-temperature-resistant reverse transcriptase which can be used for nucleic acid detection, and a preparation method and application thereof. The application carries out random mutation on a reverse transcriptase gene, and carries out screening of high-temperature-resistant reverse transcriptase, and finally successfully screens a high-temperature-resistant M-MLV reverse transcriptase mutant M-MLV-M3, the amino acid sequence of which is shown in sequence 8. Compared with natural M-MLV and commercial high-temperature-resistant M-MLV reverse transcriptase, the M-MLV-M3 has better thermal stability, still has good reverse transcriptase activity at 65 DEG C, can be used for conventional reverse transcription reaction, and can also be used for fluorescence PCR detection, and is especially suitable for high-GC-content or complex-structure RNA templates. The application effectively improves the thermal stability of the reverse transcriptase and the performance of the reverse transcriptase product by optimizing and reforming the reverse transcriptase, and especially improves the research on complex-structure RNA and the nucleic acid detection level.

Pfu DNA polymerase mutants with reverse transcriptase activity and their applications are provided. The Pfu DNA polymerase mutants include K467R / F588L / W769R, R382H / Y385H / V390I, I38L / R97M, E665K / E735K, K118I / N713V, with K118I / N713V showing the strongest reverse transcription activity. These Pfu DNA polymerase mutants still retain the polymerase activity of wild-type enzymes, thus providing higher assurance compared to traditional reverse transcriptase. Meanwhile, these Pfu DNA polymerase mutants exhibit high heat tolerance. Therefore, these Pfu DNA polymerase mutants can efficiently and high fidelity generate cDNA using RNA as a substrate, and further amplify the cDNA under conventional PCR reaction conditions without the need for additional reverse transcriptase, achieving a “one-step” RT-PCR process.

The present invention relates to a nucleic acid molecule encoding a Group II intron wherein the Group II intron encodes a reverse transcriptase activation marker (RAM) and a reverse selection marker. The invention further relates to a nucleic acid molecule encoding a first promoter operably linked to a Group II intron wherein the Group II intron does not encode a protein comprising reverse transcriptase activity, and a second promoter operably linked to a Group II intron wherein the Group II intron does not encode a protein comprising reverse transcriptase activity. The second promoter is operably linked to a nucleic acid sequence encoding an introne encoding protein (IEP), wherein the Group II introns encode: a reverse transcription transposon activation marker (RAM), a reverse selection marker, and a targeting sequence; and related kits and methods.

The invention relates to the technical field of protein engineering and molecular detection, in particular to a fusion protein with DNA polymerase activity, reverse transcriptase activity and RNase inhibitory activity and application thereof. The fusion protein provided by the invention comprises a DNA polymerase functional domain and an RNase inhibitory protein functional domain, the amino acid sequence of the RNase inhibitory protein functional domain is as shown in SEQ ID NO: 2. Through an innovative protein fusion strategy, multiple key enzyme activities such as reverse transcription and nucleic acid amplification are integrated into a single protein, and the problems that a traditional multi-enzyme system is poor in compatibility and conditions are difficult to unify are fundamentally solved. The fusion protein shows excellent tolerance to various known PCR inhibitors such as high-concentration chaotropic agents, RNase and heparin, and the reaction stability and the success rate in complex samples are remarkably improved.

The present disclosure relates generally to engineered nucleic acid processing enzymes, based on DNA polymerases (e.g., engineered family B polymerases), and derivatives thereof having reverse transcriptase activity and substantially lacking strand displacement activity; kits comprising the engineered family B polymerases; and methods of generating and using the engineered family B polymerases.