3085 results about "Polymerase" patented technology

The identification of mutations that are present in a small fraction of DNA templates is essential for progress in several areas of biomedical research. Though massively parallel sequencing instruments are in principle well-suited to this task, the error rates in such instruments are generally too high to allow confident identification of rare variants. We here describe an approach that can substantially increase the sensitivity of massively parallel sequencing instruments for this purpose. One example of this approach, called “Safe-SeqS” for (Safe-Sequencing System) includes (i) assignment of a unique identifier (UID) to each template molecule; (ii) amplification of each uniquely tagged template molecule to create UID-families; and (iii) redundant sequencing of the amplification products. PCR fragments with the same UID are truly mutant (“super-mutants”) if ≧95% of them contain the identical mutation. We illustrate the utility of this approach for determining the fidelity of a polymerase, the accuracy of oligonucleotides synthesized in vitro, and the prevalence of mutations in the nuclear and mitochondrial genomes of normal cells.

System, including methods, apparatus, compositions, and kits, for making and using compound droplets of a multiple emulsion to supply an amplification reagent, such as a heat-stable DNA polymerase or DNA ligase, to an aqueous phase in which the compound droplets are disposed. The compound droplets may be induced to supply the amplification reagent by heating the multiple emulsion, to achieve hot-start amplification.

Disclosed herein are nucleoside phosphoramidates and their use as agents for treating viral diseases. These compounds are inhibitors of RNA-dependent RNA viral replication and are useful as inhibitors of HCV NS5B polymerase, as inhibitors of HCV replication and for treatment of hepatitis C infection in mammals.

Methods and compositions are provided for detecting single nucleotide polymorphisms using a pair of oligonucleotides, a primer and a snp detection sequence, where the snp detection sequence hybridizes to the target DNA downstream from the primer and in the direction of primer extension. The snp detection sequence is characterized by having a nucleotide complementary to the snp and adjacent nucleotide complementary to adjacent nucleotides in the target and an electophoretic tag bonded to the 5'-nucleotide. The pair of oligonucleotides is combined with the target DNA under primer extension conditions, where the polymerase has 5'-3' exonuclease activity. When the snp is present, the electophoretic tag is released from the snp detection sequence, and can be detected by electrophoresis as indicative of the presence of the snp in the target DNA.

In its many embodiments, the present invention provides a novel class of pyrazolo[1,5-a]pyrimidine compounds as inhibitors of protein and/or checkpoint kinases, methods of preparing such compounds, pharmaceutical compositions including one or more such compounds, methods of preparing pharmaceutical formulations including one or more such compounds, and methods of treatment, prevention, inhibition, or amelioration of one or more diseases associated with the protein or checkpoint kinases using such compounds or pharmaceutical compositions. The invention also relates to the inhibition of hepatitis C virus (HCV) replication. In particular, embodiments of the invention provide compounds and methods for inhibiting HCV RNA-dependent RNA polymerase enzymatic activity. The invention also provides compositions and methods for the prophylaxis and treatment of HCV infection.

The present invention provides a method for identifying one or more low abundance sequences differing by one or more single-base changes, insertions, or deletions, from a high abundance sequence in a plurality of target nucleotide sequences. The high abundance wild-type sequence is selectively removed using high fidelity polymerase chain reaction analog conversion, facilitated by optimal buffer conditions, to create a restriction endonuclease site in the high abundance wild-type gene, but not in the low abundance mutant gene. This allows for digestion of the high abundance DNA. Subsequently the low abundant mutant DNA is amplified and detected by the ligase detection reaction assay. The present invention also relates to a kit for carrying out this procedure.

The present invention relates to a novel method for analyzing nucleic acid sequences based on real-time detection of DNA polymerase-catalyzed incorporation of each of the four nucleotide bases, supplied individually and serially in a microfluidic system, to a reaction cell containing a template system comprising a DNA fragment of unknown sequence and an oligonucleotide primer. Incorporation of a nucleotide base into the template system can be detected by any of a variety of methods including but not limited to fluorescence and chemiluminescence detection. Alternatively, microcalorimetic detection of the heat generated by the incorporation of a nucleotide into the extending template system using thermopile, thermistor and refractive index measurements can be used to detect extension reactions.

An instrument for performing highly accurate PCR employing a sample block in microtiter tray format. The sample block has local balance and local symmetry. A three zone film heater controlled by a computer and ramp cooling solenoid valves also controlled by the computer for gating coolant flow through the block controls the block temperature. Constant bias cooling is used for small changes. Sample temperature is calculated instead of measured. A platen deforms plastic caps to apply a minimum acceptable threshold force for seating the tubes and thermally isolates them. A cover isolates the block. The control software includes diagnostics. An install program tests and characterizes the instrument. A new user interface is used. Disposable, multipiece plastic microtiter trays to give individual freedom to sample tubes are taught.

The invention provides a method of making measurements on individual cells of a population by forming reactors containing single cells and a predetermined number, usually one, homogeneous sequence tag. In one aspect, the invention provides a method of making multiparameter measurements on individual cells of such a population by carrying out a polymerase cycling assembly (PCA) reaction to link their identifying nucleic acid sequences, such as sequence tag copies derived from a homogeneous sequence tag, to other cellular nucleic acids of interest, thereby forming fusion products. The fusion products of such PCA reactions are then sequenced and tabulated to generate multiparameter data for cells of the population.

The present invention relates, e.g., to in vitro method, using isolated protein reagents, for joining two double stranded (ds) DNA molecules of interest, wherein the distal region of the first DNA molecule and the proximal region of the second DNA molecule share a region of sequence identity, comprising contacting the two DNA molecules in a reaction mixture with (a) a non-processive 5′ exonculease; (b) a single stranded DNA binding protein (SSB) which accelerates nucleic acid annealing; (c) a non strand-displacing DNA polymerase; and (d) a ligase, under conditions effective to join the two DNA molecules to form an intact double stranded DNA molecule, in which a single copy of the region of sequence identity is retained. The method allows the joining of a number of DNA fragments, in a predetermined order and orientation, without the use of restriction enzymes.

Methods and a kit are provided for selectively and exponentially amplifying nucleic acids and include the use of a single strand helicase preparation or a thermostable helicase in the absence of a single strand binding protein and a DNA polymerase such that the amplification can be performed isothermally.

Contemplated dinucleotide compounds have a general structure of A-B and inhibit synthesis of an RNA-dependent polymerase that initiates RNA replication de novo. In preferred dinucleotides, A comprises a purine or modified purine heterocyclic base and B comprises a pyrimidine or modified pyrimidine heterocyclic base.

Compositions, methods and systems are provided for the isolation of polymerase-nucleic acid complexes. Complexes can be separated from free enzyme by using hook molecules to target single stranded regions on the nucleic acid. Active complexes can be isolated from mixtures having both active and inactive complexes by initiating nucleic acid synthesis so as to open up a portion of a double stranded region rendering that region single stranded. Hook molecules are targeted to bind the sequences that are thus exposed. The hook molecules bound to active polymerase-nucleic acid complex are isolated, and the active polymerase-nucleic acid complexes released.

Compounds having the formula I wherein A, m and R¹ are herein defined are Hepatitis C virus NS5b polymerase inhibitors. Also disclosed are compositions and methods for inhibiting hepatitis replication, processes for making the compounds and synthetic intermediates used in the process

The present invention relates generally to systems and methods for the rapid serial processing of multiple nucleic acid assays. More particularly, the present invention provides for the real time processing of nucleic acid during polymerase chain reaction (PCR) and thermal melt applications. According to an aspect of the invention, a system for the rapid serial processing of multiple nucleic acid assays is provided. In one embodiment, the system includes, but is not limited to: a microfluidic cartridge having microfluidic (flow-through) channels, a fluorescence imaging system, a temperature measurement and control system; a pressure measurement and control system for applying variable pneumatic pressures to the microfluidic cartridge; a storage device for holding multiple reagents (e.g., a well-plate); a liquid handling system comprising at least one robotic pipettor for aspirating, mixing, and dispensing reagent mixtures to the microfluidic cartridge; systems for data storage, processing, and output; and a system controller to coordinate the various devices and functions.

The present invention relates to a method for synthesizing an RNA molecule of a given sequence, comprising the step of determining the fraction (1) for each of the four nucleotides G, A, C and U in said RNA molecule, and the step of synthesizing said RNA molecule by in vitro transcription in a sequence-optimized reaction mix, wherein said sequence-optimized reaction mix comprises the four ribonucleoside triphosphates GTP, ATP, CTP and UTP, wherein the fraction (2) of each of the four ribonucleoside triphosphates in the sequence-optimized reaction mix corresponds to the fraction (1) of the respective nucleotide in said RNA molecule, a buffer, a DNA template, and an RNA polymerase. Further, the present invention relates to a bioreactor (1) for synthesizing RNA molecules of a given sequence, the bioreactor (1) having a reaction module (2) for carrying out in vitro RNA transcription reactions in a sequence-optimized reaction mix, a capture module (3) for temporarily capturing the transcribed RNA molecules, and a control module (4) for controlling the infeed of components of the sequence-optimized reaction mix into the reaction module (2), wherein the reaction module (2) comprises a filtration membrane (21) for separating nucleotides from the reaction mix, and the control of the infeed of components of the sequence-optimized reaction mix by the control module (4) is based on a measured concentration of separated nucleotides.

The present invention provides novel cleavage agents and polymerases for the cleavage and modification of nucleic acid. The cleavage agents and polymerases find use, for example, for the detection and characterization of nucleic acid sequences and variations in nucleic acid sequences. In some embodiments, the 5′ nuclease activity of a variety of enzymes is used to cleave a target-dependent cleavage structure, thereby indicating the presence of specific nucleic acid sequences or specific variations thereof.