115 results about "Nucleoside triphosphate synthesis" patented technology

Processes are disclosed that use 3′-reversibly terminated nucleoside triphosphates to analyze DNA for purposes other than sequencing using cyclic reversible termination. These processes are based on the unexpected ability of terminal transferase to accept these triphosphates as substrates, the unexpected ability of polymerases to add reversibly and irreversibly terminated triphosphates in competition with each other, the development of cleavage conditions to remove the terminating group rapidly, in high yield, and without substantial damage to the terminated oligonucleotide product, and the ability of reversibly terminated primer extension products to capture groups. The presently preferred embodiments of the disclosed processes use a triphosphate having its 3′-OH group blocked as a 3′-ONH₂ group, which can be removed in buffered NaNO₂ and use variants of Taq DNA polymerase, including one that has a replacement (L616A).

The invention provides methods for sequencing a nucleic acid, and particularly methods for synthesizing fluorescently labeled nucleoside triphosphates and related analogs for sequencing nucleic acids.

Disclosed are devices for amplifying a preselected polynucleotide in a sample by conducting a polynucleotide polymerization reaction. The devices comprise a substrate microfabricated to define a sample inlet port and a mesoscale flow system, which extends from the inlet port. The mesoscale flow system includes a polynucleotide polymerization reaction chamber in fluid communication with the inlet port which is provided with reagents required for polymerization and amplification of a preselected polynucleotide. In one embodiment the devices may be utilized to implement a polymerase chain reaction (PCR) in the reaction chamber (PCR chamber). The PCR chamber is provided with the sample polynucleotide, polymerase, nucleoside triphosphates, primers and other reagents required for the polymerase chain reaction, and the device is provided with means for thermally controlling the temperature of the contents of the reaction chamber at a temperature controlled to dehybridize double stranded polynucleotide, to anneal the primers, and to polymerize and amplify the polynucleotide.

Improved methods are provided in vitro synthesis of biological molecules, providing for improved yields, lowered costs, and enhanced utility. Improved yield and lowered cost is obtained by the use of a phosphate free energy source in the presence of exogenous phosphate, and optionally in the absence of exogenous nucleoside triphosphates.

The present invention provides a simplified method for identifying differences in nucleic acid abundances (e.g., expression levels) between two or more samples. The methods involve providing an array containing a large number (e.g. greater than 1,000) of arbitrarily selected different oligonucleotide probes where the sequence and location of each different probe is known. Nucleic acid samples (e.g. mRNA) from two or more samples are hybridized to the probe arrays and the pattern of hybridization is detected. Differences in the hybridization patterns between the samples indicates differences in expression of various genes between those samples. This invention also provides a method of end-labeling a nucleic acid. In one embodiment, the method involves providing a nucleic acid, providing a labeled oligonucleotide and then enzymatically ligating the oligonucleotide to the nucleic acid. Thus, for example, where the nucleic acid is an RNA, a labeled oligoribonucleotide can be ligated using an RNA ligase. In another embodiment, the end labeling can be accomplished by providing a nucleic acid, providing labeled nucleoside triphosphates, and attaching the nucleoside triphosphates to the nucleic acid using a terminal transferase.

The present invention relates to methods and compositions having trehalose and DNA polymerase for facilitating the rapid and efficient amplification of nucleic acid molecules and the detection and quantitation of RNA molecules, and for increasing the detection sensitivity and reliability through generation of secure cDNA molecules prior to gene-specific primer dependent amplification. The reagent mixture comprises a ready to use reagent solution, wherein the solution comprises: (a) trehalose in a concentration between about 5% and about 35%; (b) a viral reverse transcriptase; and (c) at least one DNA polymerases, in a buffer suitable for use in a reverse transcription reaction, wherein the buffer comprises a co-factor metal ion and nucleoside triphosphates.

This invention claims aqueous compositions that comprise triphosphates of 2′-deoxynucleoside derivatives that have, instead of a 3′-OH moiety, a 3′-ONH₂ moiety; wherein said compositions contain less than 0.5 mole percent contaminating triphosphate having a 3′-OH moiety, as well as processes for providing such compositions. The compositions further must contain insubstantial amounts of hydroxylamine.

The present invention relates to a DNA sequencing method using a nucleoside triphosphate with a fluorescent blocking group on its 3′-OH end as a reversible terminator. Further, the present invention relates to sequencing-by-synthesis method using the mono-modified reversible terminator (MRT), the novel nucleotide monomer having a reversible fluorescent blocking group removable chemically or enzymatically at its 3′-OH end. The sequencing method of the present invention facilitates sequencing of bases inserted by terminating extension of a nucleotide chain by the nucleotide monomer and then detecting fluorescence signal from 3′-OH end. At this time, after analyzing the fluorescence signal, the blocking group conjugated to the 3′-OH end can be effectively removed, indicating that a free 3′-OH functional group can be successfully restored, so that the next monomer insertion is possible, making continuous sequencing possible.

The invention provides a nucleic-acid fluorescent quantitative RT-PCR detection kit of H1N1 influenza virus A and a detection method thereof. The detection kit mainly comprises a specific primer, a fluorescent probe, a PCR buffer solution, a deoxidized nucleoside triphosphate compound, a DNA polymerase, a reverse transcriptase and an RNA enzyme inhibitor, wherein the specific primer and the fluorescent probe have the following sequences: an upstream primer H1N1 A-FP:5'-AGGTTTGAGATATTCCCCAAGACA-3'; a downstream primer H1N1 A-RP:5'-AATTTTTGTAGAAGCTTTTTGCTCC-3'; and a specific probe H1N1 A-P:5'-F-CATGGCCCAATCATGACTCGAACA-Q-3', wherein F is a fluorescent reporter group; and Q is a fluorescent quenching group. The invention has the advantages that the nucleic-acid fluorescent quantitative RT-PCR detection kit of the H1N1 influenza virus A and the detection method thereof can be applied to the emergent detection and the monitoring of a lab where the H1N1 influenza virus A causes an outbreakepidemic.

The invention discloses a nucleotide sequence for detecting listeria monocytogenes and a detection method and a detection kit, wherein the nucleotide sequence is expressed as SEQ ID NO. 1, the detection method is as follows: firstly, extracting the gene group DNA of the sample to be detected; secondly, taking the gene group DNA as the template, mixing with the specific primer Lm16, PCR buffer solution, MgCl2 solution, deoxidation nucleoside triphosphate mixture and Taq-DNA polymerase for preparing the PCR reaction system, carrying out the PCR reaction; finally, detecting whether the PCR product is the single amplification product, the size of which is 1623bp. The kit comprises the specific primer, PCR buffer solution, MgCl2 solution, deoxidation nucleoside triphosphate mixture and Taq-DNA polymerase. The kit can effectively detect the listeria monocytogenes in the food, and the detection sensitivity for the bacteria is high, the specificity is good and the antijamming capability is strong.

The invention provides a real-time fluorescent PCR reagent kit for screening active Listeria monocytogenes. The real-time fluorescent PCR reagent kit mainly comprises specific primers, a fluorescent probe, PCR buffer solution, deoxidant nucleoside triphosphate mixture and DNA polymerase. The sequence of the specific primers and the fluorescent probe is as follows: an upstream primer: 5'- CGGAGGTTCCGCAAAAGATG-3', a downstream primer: 5' -CCTCCAGAGTGATCGATGTT-3' and the fluorescent probe: 5' FAM-AAATCATCGACGGCAACCTC-TAMRA-3', wherein, the FAM is a fluorescent reporter group, and the TAMRA is a fluorescent quenching group. The real-time fluorescent PCR reagent kit has the beneficial effects that the real-time fluorescent PCR reagent kit can effectively screen the life and death state of bacteria and has the high sensitivity and the good specificity of the detection of bacteria, thereby reducing the false positive rate of regular PCR amplification; and the real-time fluorescent PCR reagent kit can realize fast, exact and specific detection towards the active Listeria monocytogenes.

The present invention provides fluorescent cyanine dye-based nucleotide analogues, in which the cyanine dye is coupled to the γ-phosphate group of a nucleoside triphosphate. Preferred embodiments of the invention are directed to fluorescent cyanine dye-based GTP analogues which may be employed in an homogeneous FRET-based assay to measure the binding of guanine nucleotides to GPCR polypeptides, or alternatively, to measure the effect of an exogenous ligand on GPCR protein binding.

The invention provides a T7 RNA polymerase mutant by introducing a novel mutation. The T7 RNA polymerase mutant is selected from the mutant (R632C) with arginine at the position 632 in the amino acid sequence as shown in SEQ ID NO: 1 constituting a wild type T7 RNA polymerase substituted by cysteine. The T7 RNA polymerase mutant has DNA-dependent RNA polymerase activity, and can use various 2'-modified nucleoside triphosphates as a synthetic substrate compared with the wild type T7 RNA polymerase. The invention further provides methods and kits for synthesizing the mutant and a nucleic acid containing one or more modified nucleotides.

A method and kit for synthesizing a nucleic acid molecule comprising at least one non-canonical nucleoside triphosphate using a double-mutant polymerase having a reduced discrimination between canonical and non-canonical substrates is disclosed. The method comprises incubating a template nucleic acid in a reaction mixture comprising the mutant nucleic acid polymerase and the appropriate canonical and non-canonical nucleoside triphosphates which are desired substrates for the mutant nucleic acid polymerase. The present invention is also a method of determining the sequence of a nucleic acid molecule using the mutant polymerase to create a nucleic acid molecule comprising at least one non-canonical nucleoside triphosphate.