2919 results about "Polymerase chain reaction" patented technology

Embodiments of the present invention are directed to methods and devices/systems for amplifying genetic material and may include providing a water-in-oil emulsion in a continuous flow. The emulsion may include a plurality of water droplets comprising microreactors. Each of the plurality of microreactors may include a single bead capable of capturing a nucleic acid template, a single species nucleic acid template and sufficient reagents to amplify the copy number of the nucleic acid template. The method also includes flowing the emulsion across a first temperature zone and a second lower temperature zone to thermally process the microreactors to amplify the nucleic acid template by polymerase chain reaction.

This invention relates to methods and apparatus for performing microanalytic and microsynthetic analyses and procedures. The invention provides a microsystem platform and a micromanipulation device for manipulating the platform that utilizes the centripetal force resulting from rotation of the platform to motivate fluid movement through microchannels. The invention specifically provides devices and methods for performing miniaturized in vitro amplification assays such as the polymerase chain reaction. Methods specific for the apparatus of the invention for performing PCR are provided.

The invention encompasses a method for amplifying at least two different nucleic acid sequences. In particular, the method encompasses a multiplexed nucleic acid patch polymerase chain reaction.

Methods and apparatus for implementing microfluidic analysis devices are provided. A monolithic elastomer membrane associated with an integrated pneumatic manifold allows the placement and actuation of dense arrays of a variety of fluid control structures, such as structures for isolating, routing, merging, splitting, and storing volumes of fluid. The fluid control structures can be used to implement a pathogen detection and analysis system including integrated immunoaffinity capture and analysis, such as polymerase chain reaction (PCR) and capillary electrophoresis (CE) analysis. An analyte solution can be input into the device and pumped through a series of immunoaffinity capture matrices in microfabricated chambers having antibodies targeted to the various classes of microbiological organisms such as bacteria, viruses and bacterial spores. The immunoaffinity chambers can capture, purify, and concentrate the target for further analysis steps.

An apparatus having within or as part of a housing; a sample port; a microarray port; a lysis module; a purification module for containing a solid phase for binding of oligonucleotides; a thermocycling module for containing a polymerase chain reaction; a fragmentation module; and a microarray module for holding a microarray and a liquid in contact with the microarray. The apparatus is configured to be coupled to a device for: pumping a liquid through, in order, the lysis, purification, thermocycling, fragmentation, and microarray modules; sonicating any contents of the lysis module; thermocycling the thermocycling module to perform the polymerase chain reaction; heating the fragmentation module to fragment any oligonucleotides contained therein; circulating a fluid over the surface of the microarray; and performing one or more washing or staining steps on the microarray.

A method for quantitatively measuring nucleic acid amplification reactions, especially the polymerase chain reaction, employing microparticles as hybridization solid phase, a probe sequence labeled with a fluorescent label and a fluorescence detection system which is based on two-photon fluorescence excitation, contacting all the amplification reaction components and the solid phase simultaneously in a closed cuvette, performing the amplification reactions in the same cuvette, focusing a two-photon exciting laser beam into the cuvette during the amplification cycles and measuring the fluorescence signal emitted by the microparticles from one particle at a time when they randomly float through the focal volume of the laser beam. The features of this invention allow a method and device for performing a fast quantitative nucleic acid amplification assay of single or multiple target sequences in a very small closed sample volume.

The present invention relates to a process for detecting the presence of at least one specific nucleic acid sequence in a sample containing a nucleic acid or a mixture of nucleic acids by amplifying the nucleic acid using polymerase chain reaction, cleaving the amplified products with uracil DNA glycosylase to obtain short DNA segments and detecting the DNA fragments by using reverse blot hybridization.

Methods for classifying and for evaluating the prognosis of a subject having breast cancer are provided. The methods include prediction of breast cancer subtype using a supervised algorithm trained to stratify subjects on the basis of breast cancer intrinsic subtype. The prediction model is based on the gene expression profile of the intrinsic genes listed in Table 1. This prediction model can be used to accurately predict the intrinsic subtype of a subject diagnosed with or suspected of having breast cancer. Further provided are compositions and methods for predicting outcome or response to therapy of a subject diagnosed with or suspected of having breast cancer. These methods are useful for guiding or determining treatment options for a subject afflicted with breast cancer. Methods of the invention further include means for evaluating gene expression profiles, including microarrays and quantitative polymerase chain reaction assays, as well as kits comprising reagents for practicing the methods of the invention.

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.

Disclosed are integrated devices capable of performing a polymerase chain reaction within a single vessel. Also disclosed are related methods of sample analysis.

RNA prepared by in vitro transcription using a polymerase chain reaction (PCR)-generated template can be introduced into a cell to modulate cell activity. This method is useful in de-differentiating somatic cells to pluripotent, multipotent, or unipotent cells; re-differentiating stem cells into differentiated cells; or reprogramming of somatic cells to modulate cell activities such as metabolism. Cells can also be transfected with inhibitory RNAs, such as small interfering RNA (siRNA) or micro RNA (miRNA), or combinations thereof to induce reprogramming of somatic cells. For example, target cells are isolated from a donor, contacted with one or more RNA's causing the cells to be de-differentiated, re-differentiated, or reprogrammed in vitro, and administered to a patient in need thereof. The resulting cells are useful for treating one or more symptoms of a variety of diseases and disorders, for organ regeneration, and for restoration of the immune system.

An integrated heat exchange system on a microfluidic card. According to one aspect of the invention, the portable microfluidic card has a heating, cooling and heat cycling system on-board such that the card can be used portably. The microfluidic card includes one or more reservoirs containing exothermic or endothermic material. Once the chemical process of the reservoir material is activated, the reservoir provides heat or cooling to specific locations of the microfluidic card. Multiple reservoirs may be included on a single card to provide varying temperatures. The assay chemicals can be moved to the various reservoirs to create a thermal cycle useful in many biological reactions, for example, Polymerase Chain Reaction (PCR) or rtPCR. According to another aspect of the invention, the integrated heat exchanger is an adjacent microfluidic circuit containing fluid that is either independently heated or cooled, or is an exothermic or endothermic material, such that the fluid in the adjacent circuit imparts a change in temperature to the assay fluid in an independent circuit. According to yet another aspect of the invention, a thermal electric cooler (TEC) is used for thermocycling the amplification chamber of a disposable microfluidic card.

The subject invention relates to a set of DNA primers which, when utilized in conjunction with the polymerase chain reaction (PCR) assay, can amplify and speciate DNA from five medically important Candida species. Furthermore, the PCR amplified products, generated by the primers, can also be used to create species specific probes which can also detect and confirm the five species of Candida. Thus, the present invention allows for early diagnosis and treatment of an infection. The assay is useful in the context of monitoring antifungal treatment regimens, screening potential antifungal agents, and similar applications requiring quantitative determinations.

Clinical management of human cancer is dependent on the accurate monitoring of residual and recurrent tumors. We have developed a method, called personalized analysis of rearranged ends (PARE), which can identify translocations in solid tumors. Analysis of four colorectal and two breast cancers revealed an average of nine rearranged sequences (range 4 to 15) per tumor. Polymerase chain reaction with primers spanning the breakpoints were able to detect mutant DNA molecules present at levels lower than 0.001% and readily identified mutated circulating DNA in patient plasma samples. This approach provides an exquisitely sensitive and broadly applicable approach for the development of personalized biomarkers to enhance the clinical management of cancer patients.

The invention relates to the sequencing of a target polynucleotide sequence, immobilized on a solid support, using the polymerase reaction to extend a suitable primer and characterizing the sequential addition of labelled bases. The present invention further relates to the presence of a polymerase enzyme that retains a 3' to 5' exonuclease function, which is induced to remove an incorporated labelled base after detection of incorporation. A corresponding non-labelled base may then be incorporated into the complementary strand to allow further sequence determinations to be made. Repeating the procedure allows the sequence of the complement to be identified, and thereby the target sequence.

An integrated microfabricated instrument for manipulation, reaction and detection of microliter to picoliter samples. The instrument is suited for biochemical reactions, particularly DNA-based reactions such as the polymerase chain reaction, that require thermal cycling since the inherently small size of the instrument facilitates rapid cycle times. The integrated nature of the instrument provides accurate, contamination-free processing. The instrument may include reagent reservoirs, agitators and mixers, heaters, pumps, and optical or electromechanical sensors. Ultrasonic Lamb-wave devices may be used as sensors, pumps and agitators.

The present invention provides a method and apparatus for economically filtering air into swine farms and other animal enclosures. Advantageously, the invention substantially reduces the risk to airborne transfer of disease into animal enclosures. In one embodiment, a method for providing clean air to an animal enclosure includes providing an animal enclosure having an animal containing volume for containing a plurality of animals, removing air from the internal volume via at least one exhaust fan, and filtering air being pulled into the enclosure by the at least one exhaust fan using a fully sealed filter, the fully sealed filter having a particle separation sufficiently efficient to prevent detection of live (PRRS-) virus downstream the of filter, using polymerase chain reaction (PCR) analysis of samples collected with a cyclonic aerosol collector.

Polymerase chain reaction primers and methods directed to detecting the EGFR mutant C→T at the position corresponding to base 2369 of EGFR cDNA. The invention provides a PCR primer that hybridizes under suitable PCR conditions to a polynucleotide sequence 5′ in each respective strand to a mutation of an EGFR gene that encodes a substitution of threonine by methionine at position 790 of the EGFR polypeptide. The invention also provides a PCR primer hybridizes to a sequence that includes a mutant T at the position corresponding to base 2369 of EGFR cDNA but not to a second EGFR polynucleotide containing a wild type C. The invention provides several methods and kits for detecting a mutant epidermal growth factor receptor (EGFR) gene in a sample comprising probing the sample with a means for selectively detecting a nucleotide sequence comprising a mutant T at the position corresponding to base 2369 of EGFR cDNA, and identifying that the base at said position is T. These methods and kits are also useful to predict resistance to the therapeutic effects of gefitinib or erlotinib in a subject suffering from or suspected of having a cancer.

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.

The invention discloses a construction method and an application of a high-throughout sequencing library. The construction method of the high-throughout sequencing library comprises the following steps: fragmenting genome DNA (deoxyribonucleic acid); repairing terminals of DNA fragments; adding a base A to 3' terminals of the DNA fragments with the repaired terminals; connecting the DNA fragments with sticky ends A to methylation linkers; carrying out hybrid capture on connecting products by using a specificity probe so as to obtain target fragments; carrying out bisulfite treatment on the target fragments so as to convert non-methylated cytosine in the target fragments to uracil; carrying out PCR (polymerase chain reaction) amplification on the converted target fragments; separating and purifying amplification products which form the high-throughout sequencing library. According to the method and application of the high-throughout sequencing library, the high-throughout sequencing library of a genome specific zone of a sample can be conveniently and efficiently constructed.

The invention provides an overexpression porcine co-stimulatory 4-1BB vector and application thereof. PCR (polymerase chain reaction) amplification is performed on a left homologous arm and a right homologous arm of an intron 1 of a rosa26 gene, a 4-1BB regulatory sequence and an OCT4 specific promoter; the left homologous arm, a 4-1BB expression cassette, LoxP locus-contained Cre and Neo expression cassettes, the right homologous arm and negative selection DTA diphtheria toxin are connected in sequence to obtain a 4-1BB homologous recombinant vector p4BOCNDR; the vector and a CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated) targeting vector of sgRNA (small guide ribonucleic acid) containing the intron 1 of the specific targeting porcine rosa26 gene are transferred together into a porcine fetus fibroblast; by taking a positive cell as a donor cell and an oocyte as a recipient cell, a cloned embryo is obtained through a somatic cell nuclear transfer technique; the cloned embryo is transplanted into a porcine uterus for fetation to obtain a transgenic pig integrating a 4-1BB gene at the fixed point of a first intron of the rosa26 gene and automatically deleting a marker gene.

The present invention provides methods for classifying and for evaluating the prognosis of a subject having breast cancer are provided. The methods include prediction of breast cancer subtype using a supervised algorithm trained to stratify subjects on the basis of breast cancer intrinsic subtype. The prediction model is based on the gene expression profile of the intrinsic genes listed in Table 1. Further provided are compositions and methods for predicting outcome or response to therapy of a subject diagnosed with or suspected of having breast cancer. These methods are useful for guiding or determining treatment options for a subject afflicted with breast cancer. Methods of the invention further include means for evaluating gene expression profiles, including microarrays and quantitative polymerase chain reaction assays, as well as kits comprising reagents for practicing the methods of the invention.

The present invention provides thermostable or thermoactive DNA polymerases with attenuated 3′-5′ exonuclease activity, methods for their synthesis, methods for their use, kits comprising the polymerases, nucleic acids encoding the polymerases and cells comprising such a nucleic acid. The DNA polymerases of the invention are useful in many recombinant DNA techniques, such as nucleic acid amplification by the polymerase chain reaction. The DNA polymerases of the invention allow higher fidelity replication and amplification of a template DNA sequence, allow less degradation of primers and/or more efficient use of deoxynucleotide triphosphates and are in general more efficient and less costly to make and use.

The invention discloses 'a method for reducing dimers of a pair of primers with part of sequence being identical'. According to the discovery of the inventor, in a primer pair without continuous complementary bases, the resultant force of multiple spaced hydrogen bonds formed by parallel pairing in a direction from 5' to 3' promotes some complementary bases at the 3' terminals of the primer pair to form hydrogen bonds that bond and extend the terminals to form a dimer; and a single primer does not form a dimer by itself. Similar single primer of a polymerase chain reaction (PCR) primer pair is designed according to a proportion as high as possible. The method is characterized in that: two fragments of identical sequences containing 4 to 10 bases each are present in the middle of the PCR primer pair along the direction from 5' to 3', wherein the 1 to 5 bases in the 3' terminal of each primer do not contain spaced complementary bases; each 5' terminal region may have not more than three spaced complementary bases; and preferably, the sequences at the 5' terminal are identical completely. The method can greatly reduce the formation of the dimers of the primers and optimize the quality of PCR, particularly real-time PCR of a fluorescent dye.

The present invention relates to new methods and uses for the detection and quantification of microbial nucleic acids employing an internal quantitative reference. Preferred methods are based on the amplification of nucleic acids, preferably the polymerase chain reaction. Further provided are kits comprising components for performing said methods and uses. Moreover, an analytical system for advantageously performing the method according to the invention is disclosed.

The present invention provides a molecular level of DNA information code which uses a base pair sequence as an information code unit. Also, the present invention provides a molecular code system which includes designing and coding DNA which is an information code unit; stabilizing the DNA information code by encapsulating it with an inorganic capsule and coating the DNA-inorganic capsule to a medium; taking and extracting the coated DNA information code which is present in a trace amount, collecting the DNA information code using a polypyrrole-maghemite nanohybrid; and amplifying the collected DNA information code using a polymerase chain reaction and reading the amplified DNA information code. According to the present invention, the DNA information code having high security is prepared by assigning a security unit to a DNA which has an excellent accumulating capacity, and then the DNA information code is stabilized so as to be coated to a medium. Only the DNA information code may be extracted, collected, and read, if necessary. Thus, a unified molecular code system can be established.

A reaction vessel includes a receiving portion and a capillary portion. The capillary portion has a first end having an opening in fluid connection with the receiving portion, and a second end closed by a plug portion formed as an integral one-piece member with the capillary portion. The capillary portion is optionally detachable from the receiving portion of the reaction vessel. The reaction vessel simplifies many processes involving small volume chemical reactions, such as Polymerase Chain Reaction (PCR) determinations.

The present invention is related to genomic nucleotide sequencing. In particular, the invention describes a single reaction method to co-amplify multiple subsequences of a nucleic acid fragment sequence (i.e., for example, at least two read pairs from a single library insert sequence). Nucleic acid fragment sequences may include, but are not limited to, localizing library insert sequences and/or unique read pair sequences in specific orientations on a single emulsion polymerase chain reaction bead. Methods may include, but are not limited to, annealing, melting, digesting, and/or reannealing high throughput sequencing primers to high throughput sequencing primer binding sites. The compositions and methods disclosed herein contemplate sequencing complex genomes, amplified genomic regions, as well as detecting chromosomal structural rearrangements that are compatible with massively parallel high throughput sequencing platforms as well as ion semiconductor matching sequencing platforms (i.e., for example, Ion Torrent platforms).

The invention discloses a method for detecting mutation information in a multiplex amplification sequencing product of a genome. The method comprises steps as follows: sequencing data are subjected to quality assessment and preprocessing; a recognizable sequencing sequence is selected for sequence assembling; the recognizable sequencing sequence or a sequence obtained through assembling is compared with a reference gene sequence, and preliminary variation information is obtained; fine calibration of sequence variation is performed according to different types of conditions; a calibrated sequencing fragment is obtained; the homozygosis or heterozygosis state of a target fragment is obtained according to the type of the sequencing fragment with the highest abundance; finally, the mutation information in the multiplex amplification sequencing product of the genome is obtained. By means of the method, the amplification product can be rapidly, efficiently and accurately recognized, and the calculation resources are saved; the sequence assembling process is compatible, and the problem of reduction of the quality value of basic groups produced in the sequencing process can be effectively solved; the homozygosis/heterozygosis state of variation information can be more effectively and stably judged, and random errors introduced in the PCR (polymerase chain reaction) process and the sequencing process are eliminated.

The invention provides a fluorescently-labeled gene multiplex amplification technology for detecting sources of goat, sheep, pig and duck meat simultaneously. A method comprises the steps that mitochondria 16S rRNA (ribosomal Robonucleic Acid) and Cyt (Cytosine) b genes of a goat, sheep, a pig and a duck serve as target genes, and are subjected to PCR (Polymerase Chain Reaction) multiplex amplification by two pairs of fluorescently-labeled primers; PCR amplification products are detected by a 3730xl full automatic gene analysis meter; and species identification is performed according to 16S rRNA gene amplified fragment length polymorphism. The method fills in a gap of a detection method of the four kinds of meat.