57 results about "Reverse transcriptase activity" patented technology

A method is described for the direct, exponential amplification and sequencing ("DEXAS") of a DNA molecule from a complex mixture of nucleic acids, wherein truncated DNA molecules as well as DNA molecules of full length are synthesized simultaneously and exponentially between two positions on the said DNA molecule, which initially contains a DNA molecule in a thermocycling reaction, a first primer, a second primer, a reaction buffer, a thermostable DNA polymerase, a thermostable pyrophosphatase (optionally), deoxynucleotides or derivatives thereof and a dideoxynucleotide or derivatives thereof. In a preferred embodiment of the method of the invention, direct sequencing of RNA can be performed using one polymerase having a Tabor-Richardson mutation, or a functional derivative thereof, and reverse transcriptase activity. In a more preferred embodiment of the method of the invention, direct sequencing of RNA can be performed in one step, in one vessel.

The invention provides antibodies specific for HIV env, including monoclonal antibodies and related hybridomas. The antibodies block CD4 / g120 binding and reduce reverse transcriptase activity in vitro.

Non-infectious, retrovirus-like particles contain mutations to reduce gag-dependent RNA-packaging of the gag gene product, eliminate reverse transcriptase activity of the pol gene product, eliminate integrase activity of the pol gene product and eliminate RNase H activity of the pol gene product through genetic manipulation of the gag and pol genes. The corresponding nucleic acid molecules are described. The non-infectious, retrovirus-like particles have utility in diagnosis.

The present invention provides polypeptides having a nucleotide polymerase activity and method of enhancing polymerase activity. The polypeptides of the present invention may posses both a DNA-dependent DNA polymerase activity and an RNA-dependent DNA polymerase activity, i.e., a reverse transcriptase activity. The polypeptides of the present invention may be used in any application including, but not limited to, DNA sequencing reactions, amplification reactions, cDNA synthesis reactions, and combined cDNA synthesis and amplification reactions, e.g., RT-PCR.

The invention relates to a cervical exfoliated cell preservative liquid, the preservative liquid comprises the following components with the contents (by weight): 20 percent to 50 percent of alcohols; 15 percent to 50 percent of anti-aggregation reagent; 5 percent to 10 percent of buffer solution; 1 percent to 20 percent of ion strength maintaining reagent; 0.01 percent to 0.5 percent of anti-microbial reagent; 0.1 to 5 percent of mucus dissolving reagent; and 0 to 0.5 percent of cleaning agent. Compared with the prior art, the preservative liquid can not only lead cells to maintain the shape in an in vitro liquid suspension environment, minimize the protein precipitation, dissolve larger protein substances, such as blood and mucus, and reduce the cell aggregation, but can also selectively eliminate or reduce red cells, effectively kill microbes, prevent the activity of reverse transcriptase and retain the integrity of nucleic acids and proteins for facilitating the later analysis; in addition, the preservative liquid can greatly reduce the costs of consumptive materials for the TCT detection, improve the sensitivity and the specificity of the cervical cancer screening and accelerate the promotion and the popularization of the TCT technology in a medical system.

The invention relates to the generation and characterization of Archaeal DNA polymerase mutants with deficient 3′-5′ exonuclease activity and reduced base analog detection activity. The invention further provides for Archaeal DNA polymerase mutants with deficient 3′-5′ exonuclease activity and reduced base analog detection activity containing additional mutations that modulate other DNA polymerase activities including DNA polymerization or reverse transcriptase activity. The invention also discloses methods and applications of DNA polymerases with deficient 3′-5′ exonuclease activity and reduced base analog detection activity.

The present invention relates to reverse transcription of RNA, and in particular to reverse transcription by thermostable DNA polymerases. Thermoactinomyces vulgaris and Bacillus stearothermophilus possess reverse transcriptase activity in the presence of magnesium or manganese ions. Methods, compositions, and kits for reverse transcription and RT-PCR are also provided.

The present invention provides an improved method for cDNA preparation. The method of the present invention comprises the following steps: (1) contacting mRNA with a cDNA synthesis primer which can anneal to RNA and a suitable enzyme which possesses reverse transcriptase activity under conditions sufficient to permit the template-dependent extension of the primer to generate an mRNA-cDNA intermediates; (2) contacting a mixture from step 1 with a deoxyribonucleotide adapter in the presence of Mn2+-ions, wherein said oligonucleotide adapter has a pre-selected arbitrary nucleotide sequence at its 5′-end, and a short dG stretch at its 3′-end. The 3′-end nucleotide of the adapter is a terminator nucleotide, e.g., a nucleotide with a modified 3′-OH group of a deoxyribose residue.

Non-infectious, retrovirus-like particles contain mutations to reduce gag-dependent RNA-packaging of the gag gene product, eliminate reverse transcriptase activity of the pol gene product, eliminate integrase activity of the pol gene, product and eliminate RNase H activity of the pol gene product through genetic manipulation of the gag and pol genes. The corresponding nucleic acid molecules are described. The non-infectious, retrovirus-like particles have utility in in vivo administration including to humans and in diagnosis.

Thermostable viral polymerases exhibiting a combination of activities selected from, proofreading (3′-5′) exonuclease activity, nick translating (5′-3′) nuclease activity, synthetic primer-initiated polymerase activity, nick-initiated polymerase activity, reverse transcriptase activity, strand displacement activity, and / or decreased discrimination against incorporation of nucleotide analogs. Also provided are compositions including the polymerases, polynucleotides encoding the polymerases and methods of using the polymerases.

The invention relates to an isolated polynucleotide sequence from the genome of Bacillus stearothermophilus (Geobacillus stearothermophilus) and an amino acid sequence encoded by the polynucleotide sequence, the corresponding amino acid sequence comprising a novel enzyme, Tirt (thermostable intron reverse transcriptase), having reverse transcriptase activity and retaining that activity at temperatures of up to about 75° C.

Thermostable viral and microbial polymerases exhibiting a combination of activities selected from proofreading (3′-5′) exonuclease activity, nick translating (5′-3′) nuclease activity, synthetic primer-initiated polymerase activity, nick-initiated polymerase activity, reverse transcriptase activity, strand displacement activity, terminal transferase activity, primase activity, and / or efficient incorporation of chain terminating analogs. Some of the polymerases provided herein include a first motif and a second motif. The first motif preferably has the sequence X1X2X3DX4PX5IELRX6X7X8, wherein X1 is I or V; X4 is F or Y; X8 is G or A; and X2, X3, X5, X6, and X7 are any amino acid. The second motif preferably has the sequence RX9X10X11KSANX12GX13X14YG, wherein X11 is G or A; X12 is F, L, or Y; X13 is L or V; X14 is I or L; and X9 and X10 are any amino acid. Also provided are reagents for expressing the polymerases, polynucleotides encoding the polymerases, host cells expressing the polymerases, and methods of using the polymerases.

The present invention provides compositions and methods for high fidelity cDNA synthesis. In particular, the composition of the present invention contains a first enzyme exhibiting a reverse transcriptase activity and a second enzyme comprising a 3′-5′ exonuclease activity.

Provided are mutant polymerases having DNA polymerase activity and reverse transcriptase activity or strand displacement activity, along with target nucleic acid amplification methods employing such mutant polymerases.

Thermostable viral polymerases exhibiting a combination of activities selected from, proofreading (3′-5′) exonuclease activity, nick translating (5′-3′) nuclease activity, synthetic primer-initiated polymerase activity, nick-initiated polymerase activity, reverse transcriptase activity, strand displacement activity, and / or decreased discrimination against incorporation of nucleotide analogs. Also provided are compositions including the polymerases, polynucleotides encoding the polymerases and methods of using the polymerases.

The invention discloses an application of Bst DNA polymerase in RNA amplification. The Bst DNA polymerase has reverse transcriptase activity, and thus RNA is subjected to inverse transcription into cDNA under action of the Bst DNA polymerase. A new RNA amplification detection method is established on the basis of the new discovery that the Bst DNA polymerase has reverse transcriptase activity. The method integrates inverse transcription and subsequent nucleic acid amplification, temperature adjustment is not needed during the whole process, addition of extra reverse transcriptases is not needed, operation is simple, reaction time is shortened, and new methodology reference and technical support are provided for RNA inverse transcription and amplification detection. The discovered Bst DNA polymerase can perform an inverse transcription reaction at a high temperature, which facilitates to open and combine RNAs with complex secondary structures, and therefore the problem is solved that reverse transcriptases of AMV or the like are difficult to amplify RNA advanced structure areas.

According to the invention, mutation of several sites of Ser358Asp, Thr480Asn, Asp533Glu and Ala539Gly is carried out on a wild type Bst DNA polymerase sequence, then 292-305 amino acid EGLLKVVRPDTKKV of the Bst DNA polymerase subjected to point mutation is replaced with DPLPDLIHPRTLRL, a DNA binding protein is fused at the C end of the mutated Bst DNA polymerase sequence, an HP47 polypeptide sequence (SEQ ID No.17) is fused at the N end of the mutated Bst DNA polymerase sequence, a CL7-SUMO-Tag is fused in front of the HP47 polypeptide sequence, and the recombinant mutant Super-Bst (SEQ ID No.16) of the Bst DNA polymerase with high activity and thermal stability is obtained. The thermal stability, specificity, strand displacement capacity, extension capacity and reverse transcriptase activity of Super-Bst are remarkably improved, and Super-Bst can tolerate high salt and various inhibitors and can be massively obtained through prokaryotic expression and affinity purification. The invention also discloses coding DNA of the gene and an ultrafast magnetic bead LAMP detection method.

The invention discloses coprinus comatus laccase with activity of inhibiting tumor cell proliferation and a preparation method thereof. The coprinus comatus laccase provided in the invention is prepared by the following steps: 1) breaking up cells; 2) collecting a crude extract of proteins; 3) separating and purifying laccase from the crude extract of proteins collected in the step 2), wherein the obtained laccase is an extract of coprinus comatus, the molecular weight of the laccase is 64KD, and N terminal sequence of the laccase is described by sequence 1 in a sequence table. The coprinus comatus extract obtained through the preparation method in the invention has an obvious inhabiting effect on cancer cell proliferation and HIV reverse transcriptase activity and has an important value in the field of research on novel anti-cancer and anti-AIDS medicines.

The invention discloses a trifluoromethyl-containing indole ketone compound, and a preparation method and application thereof. The compound has the chemical structural general formula I, wherein Y is carbon or oxygen; or the compound has the chemical structural general formula II, wherein Y is carbon or oxygen and X is oxygen or sulfur; or the compound has the chemical structural general formula III, wherein Y is hydrogen or halogen and X is oxygen or sulfur; or the compound has the chemical structural general formula IV, wherein Y is hydrogen or halogen. The compound is prepared by reacting substituted aniline with methyl trifluoropyruvate or ethyl trifluoropyruvate in a microwave reactor. The trifluoromethyl-containing indole ketone compound has anti-HIV reverse transcriptase activity, basically has no toxicity on cells, is expected to serve as an HIV reverse transcriptase inhibitor, and has important value on research of the HIV reverse transcriptase inhibitor.

The invention relates to a novel molecule with a relative structure, which is designed according to the bioisosteric principle and the theories of hydrogen bonding interaction and the like by using a non nucleoside human immunodeficiency virus-1 (HIV-1) reverse transcriptase inhibitor 4-(cyclohexyl methoxyl)-6-phenethyl-2(1H)-pyridone-3-carboxylic acid ethyl ester as a primer. In a general formula I, the definitions of groups are described in claims. Meanwhile, the invention relates to reverse transcriptase activity evaluation of a compound and application of the compound as the HIV-1 reverse transcriptase inhibitor. The conformation of the synthesized novel compound is more easily combined with HIV-1 reverse transcriptase, so that the novel compound is more favorable for inhibiting the activity of the reverse transcriptase and becomes the novel high-activity low-toxicity HIV-1 reverse transcriptase inhibitor.

A method for isothermal amplification of a target nucleic acid sequence is disclosed. The target nucleic acid is amplified by an enzyme with helicase activity and an enzyme with reverse transcriptase activity and DNA-dependant DNA polymerase activity. Also disclosed is a kit for isothermal amplification of a target nucleic acid sequence, including HPV nucleic acids. The kit comprises a first enzyme with helicase activity and a second enzyme having both reverse transcriptase activity and DNA-dependant DNA polymerase activity.

A kit for amplifying deoxynucleic acid (DNA) from ribonucleic acid (RNA) template is provided. The kit for amplifying a RNA comprises at least one inosine-containing primer; and at least one enzyme comprising a reverse transcriptase activity, a strand displacement DNA polymerase activity, a nuclease acitivity for nicking DNA 3′ to an inosine residue of the primer or combinations thereof. The kit further comprises one or more quantifying reagents to detect the presence of RNA in a sample or quantify the RNA present in a sample.

Pharmaceutical compositions which comprise HIV Pol DNA vaccines are disclosed, along with the production and use of these DNA vaccines. The pol-based DNA vaccines of the invention are administered directly introduced into living vertebrate tissue, preferably humans, and preferably express inactivated versions of the HIV Pol protein devoid of protease, reverse transcriptase activity, RNase H activity and integrase activity, inducing a cellular immune response which specifically recognizes human immunodeficiency virus-1 (HIV-1). The DNA molecules which comprise the open reading frame of these DNA vaccines are synthetic DNA molecules encoding codon optimized HIV-1 Pol and codon optimized inactive derivatives of optimized HIV-1 Pol, including DNA molecules which encode inactive Pol proteins which comprise an amino terminal leader peptide.

The present invention is generally related to compositions and methods for the reverse transcription of nucleic acid molecules, especially messenger RNA molecules. Specifically, the invention relates to compositions comprising mixtures of polypeptides having reverse transcriptase (RT) activity, and to methods of producing, amplifying or sequencing nucleic acid molecules (particularly cDNA molecules) using these compositions or polypeptides, particularly at temperatures above about 55° C. The invention also relates to nucleic acid molecules produced by these methods, to vectors and host cells comprising these nucleic acid molecules, and to the use of such nucleic acid molecules to produce desired polypeptides. The invention also relates to methods for producing Rous Sarcoma Virus (RSV) and Avian Myeloblastosis Virus (AMV) RTs or other Avian Sarcoma-Leukosis Virus (ASLV) RTs (α and / or β subunits thereof), to isolated nucleic acid molecules encoding such RSV RT, AMV RT or other ASLV RT subunits, to vectors and host cells comprising these isolated nucleic acid molecules and to RSV RT, AMV RT and other ASLV RT subunits produced by these methods. The invention further relates to nucleic acid molecules encoding recombinant heterodimeric RT holoenzymes, particularly heterodimeric RSV RTs, AMV RTs or other ASLV RTs (which may be αβ RTs, ββ RTs, or α RTs), vectors (particularly baculovirus vectors) and host cells (particularly insect and yeast cells) comprising these nucleic acid molecules, methods for producing these heterodimeric RTs and heterodimeric RTs produced by these methods. The invention also relates to kits comprising the compositions, polypeptides, or RSV RTs, AMV RTs or other ASLV RTs of the invention.

In one aspect, the present invention provides methods for synthesizing multiple copies of antisense cDNA molecules from an RNA molecule, comprising using an RNA molecule as a template for synthesizing multiple copies of antisense cDNA molecules. In some embodiments of the methods, the RNA molecule is incubated with a primer and with an enzyme possessing reverse transcriptase activity under suitable conditions for synthesizing multiple copies of antisense cDNA molecules. In some embodiments, the methods produce multiple copies of double-stranded cDNA from a template RNA molecule. In further embodiments, the methods produce multiple copies of cRNA from a template RNA molecule.

A method is described for the direct, exponential amplification and sequencing ("DEXAS") of a DNA molecule from a complex mixture of nucleic acids, wherein truncated DNA molecules as well as DNA molecules of full length are synthesized simultaneously and exponentially between two positions on the said DNA molecule, which initially contains a DNA molecule in a thermocycling reaction, a first primer, a second primer, a reaction buffer, a thermostable DNA polymerase, a thermostable pyrophosphatase (optionally), deoxynucleotides or derivatives thereof and a dideoxynucleotide or derivatives thereof. In a preferred embodiment of the method of the invention, direct sequencing of RNA can be performed using one polymerase having a Tabor-Richardson mutation, or a functional derivative thereof, and reverse transcriptase activity. In a more preferred embodiment of the method of the invention, direct sequencing of RNA can be performed in one step, in one vessel.

Provided are: a DNA polymerase mutant having reverse transcriptase activity, the DNA polymerase mutant including a sequence consisting of twelve specific amino acids A1-A12, wherein the DNA polymerase mutant having reverse transcriptase activity is characterized in that the A3 and / or A10 amino acid is substituted by a basic amino acid residue different from that prior to the introduction of mutation; a kit and a composition including the DNA polymerase; a method for producing the DNA polymerase; and a method for modifying an existing DNA polymerase having reverse transcriptase activity.