3347 results about "Pcr dgge" patented technology

The invention concerns methods and instruments for fast, selective replication of deoxyribonucleic acid (DNA) from biomaterial through the known polymerase chain reaction (PCR), working in individual duplication thermocycles. The invention consists of extremely brief cycle times of only a few seconds for the PCR reactions, generated, on the one hand, by reaction chambers for the reception of the reaction solution constructed of a pattern of fine capillaries in close proximity to heating and cooling elements in order to optimally accelerate the temperature setting in the reaction solution for the three temperature phases of the PCR duplication cycles and, on the other hand, by keeping the flow rates in the capillaries to a minimum during the amplification phase so that the polymerase reaction is not disturbed. The capillary pattern can be simply produced by means of microsystern technology.

A method for balancing multiplexed PCR methods is provided. In the method, two or more sequential temporal PCR stages are used to effectively separate two or more PCR reactions in a single tube as an alternative to primer limiting to modulate the relative rate of production of a first amplicon by a first primer set and a second amplicon by a second primer set during the first and second amplification stages. Also provided are rapid RT-PCR methods that find particular use in intraoperative diagnoses and prognoses, for instance in diagnosing malignant esophageal adenocarcenoma by determining expression levels of carcinoembryonic antigen (CEA) in sentinel lymph nodes.

The present invention provides an efficient way for high throughput haplotype analysis. Several polymorphic nucleic acid markers, such as SNPs, can be simultaneously and reliably determined through multiplex PCR of single nucleic acid molecules in several parallel single molecule dilutions and the consequent statistical analysis of the results from these parallel single molecule multiplex PCR reactions results in reliable determination of haplotypes present in the subject. The nucleic acid markers can be of any distance to each other on the chromosome. In addition, an approach wherein overlapping DNA markers are analyzed can be used to link smaller haplotypes into larger haplotypes. Consequently, the invention provides a powerful new tool for diagnostic haplotyping and identifying novel haplotypes.

A method for obtaining from genomic material genomic copy number information unaffected by amplification distortion, comprising obtaining segments of the genomic material, tagging the segments with substantially unique tags to generate tagged nucleic acid molecules, such that each tagged nucleic acid molecule comprises one segment of the genomic material and a tag, subjecting the tagged nucleic acid molecules to polymerase chain reaction (PCR) amplification, generating tag associated sequence reads by sequencing the product of the PCR reaction, assigning each tagged nucleic acid molecule to a location on a genome associated with the genomic material by mapping the subsequence of each tag associated sequence read corresponding to a segment of the genomic material to a location on the genome, and counting the number of tagged nucleic acid molecules assigned to the same location on the genome having a different tag, thereby obtaining genomic copy number information unaffected by amplification distortion.

Sieving mediums comprising less than about 0.5% polymer, less than about 0.4% polymer, and 0.35% polymer or less are used to perform nucleic acid separations and PCR. The low polymer concentration does not inhibit PCR reactions and is sufficient for performing nucleic acids separations. Microfluidic devices are used to perform nucleic acids separations and PCR reactions in the sieving mediums described.

The present invention concerns a method for the quantification of a target nucleic acid in a sample comprising the following steps: (i) determination of the amplification efficiency of the target nucleic acid under defined amplification conditions, (ii) amplification of the target nucleic acid contained in the sample under the same defined reaction conditions, (iii) measuring the amplification in real-time, (iv) quantification of the original amount of target nucleic acid in the sample by correction of the original amount derived from step (iii) with the aid of the determined amplification efficiency. The efficiency correction of PCR reactions according to the invention for the quantification of nucleic acids can be used for absolute quantification with the aid of an external or internal standard as well as for relative quantification compared to the expression of housekeeping genes.

The present invention is directed to a new composition for performing a nucleic acid amplification reaction comprising (i) a thermostable DNA-Polymerase, (ii) a thermostable 3'-5' Exonuclease, and (iii) at least one primer for nucleic acid amplification with a modified 3' terminal residue which is not elongated by said thermostable DNA-Polymerase as well as methods for performing a PCR reaction using this composition. Furthermore, the method is directed to kits comprising such a composition.

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

The invention provides methods and compositions for detecting a mutation in a target gene in a sample of blood or a fraction thereof, including in certain examples, a fraction that includes circulating tumor DNA. The methods can include a tiling PCR reaction, for example a one-sided multiplex tiling reaction. Virtually any type of mutation can be detected with the methods and compositions. In certain embodiments, gene fusions are detected. Improved PCR methods, especially for performing nested multiplex PCR reactions are provided.

A double-stranded nucleic acid hybridization probe and methods of using the same are described. The probe described is particularly suited for real-time RT-PCR reactions and has high tolerance to mismatches.

The invention provides a full-automatic nucleic acid extraction and PCR amplification micro-fluidic chip which comprises at least one main body, wherein the main body is distributed in equal distance around the chip; the main body comprises a nucleic acid extraction unit, a PCR amplification unit, a waste liquid unit and an exhaust unit, wherein the nucleic acid extraction unit is positioned on the main body and is used for extracting, washing and eluting nucleic acid in sequence under the driving of centrifugal force and capillary force; the PCR amplification unit is adopted for PCR reaction amplification; the waste liquid unit comprises a waste liquid cavity which is used for storing a waste liquid; the exhaust unit comprises exhaust holes and a plurality of exhaust channels; and the plurality of exhaust channels are communicated with the nucleic acid extraction unit, the PCR amplification unit and the waste liquid unit and are used for exhausting air. The invention discloses a micro-fluidic chip for achieving full-automatic nucleic acid extraction and PCR reaction through centrifugal force, capillary force and siphon phenomenon, the micro-fluidic chip has no integrated equipment such as pumps or valves, so that the manufacturing difficulty and cost of the chip are greatly reduced, and moreover the reliability of the chip is improved.

The invention discloses a full automatic instrument for extracting nucleic acids, which is capable of fully automating the processes of acquiring sample information, automatically adding samples, automatically loading consumptive materials, automatically monitoring the extraction process, and establishing a PCR reaction system. The full automatic instrument for extracting nucleic acids comprises a working platform, a sample adding system which is mounted on the working platform, a deep-well multiwell plate movement device and a nucleic acid extraction device, wherein a plurality of working positions are arranged on the working platform and comprise a sample area, a reagent area and an extract product storage area. The sample adding system respectively adds the sample and a PCR reaction reagent into the deep-well multiwell plate movement device and the extract product storage area from the sample area and the reagent area respectively, the sample is moved below the nucleic acid extraction device by the deep-well multiwell plate movement device and allowed to perform the nucleic acid extraction reaction, and the extraction products are stored in the extract product storage area. The impact of human factor on experimental result can be effectively reduced, and the purity the extracted nucleic acid and the nucleic acid extraction efficiency can be improved.

The invention provides methods for detecting contamination in a PCR reaction. Methods of the invention are especially useful for detection of contamination in heterogeneous samples containing a rare nucleic acid to be detected.

A method for inspecting hepatitis and AIDS nucleic acid by synchronized amplification and its reagent knit are disclosed. The process is carried out by taking magnetic ball as automatic medium, specific synchronized capturing HBV, CHV and HIV nucleic acid, accelerating biotin primer construction and purification by RNA external label and internal label, real-time synchronized inspecting and T-PCR amplifying based on Tagman probe. The reagent knit consists of dis-inhibitor, cracking liquid, magnetic ball suspension, washing liquor, internal check, RT-PCR reactive liquor, enzyme mixture, fluorescent mixture, positive check and negative check. It's accurate and automatic, has single-tube operation, closed inspection AND synchronized extraction, and it has better sensitivity and specific performance and can be used for large-scale blood screening and large-capacity clinical inspection.

The present invention discloses novel blends of chimeric and non-chimeric thermostable DNA polymerases for use in PCR, DNA sequencing and mutagenesis protocols. The invention allows for PCR reactions with shorter extension times that will facilitate PCR amplification of genomic DNA templates and improve the efficacy of long PCR.

A method of obtaining DNA amplification of a nucleic acid target from a volume of whole blood comprising performing DNA amplification in a PCR assay mixture with a blood-resistant polymerase.

The invention relates to an apparatus and methods for preventing condensation on the interior surfaces of sample tubes which are being exposed to temperature cycles, such as during a PCR amplification reaction. In particular, the invention relates to apparatus comprising a sample block comprising a plurality of sample wells for receiving sample tubes and heating elements disposed in the sample wells for heating at least a portion of the sides of sample tubes (e.g., at least the portion which forms the head space after a tube is filled with a PCR reaction mixture). In a preferred aspect, the sample block is part of a thermocycling device for performing PCR and the side-wall heater is used to enhance uniformity and speed of amplification reactions. For example, by decreasing or eliminating condensation, signal strength jumps in a real-time PCR assay can be minimized as can reaction non-homogeneity.

The present invention concerns a method for the quantification of a target nucleic acid in a sample comprising the following steps: (i) determination of the amplification efficiency of the target nucleic acid under defined amplification conditions, (ii) amplification of the target nucleic acid contained in the sample under the same defined reaction conditions, (iii) measuring the amplification in real-time, (iv) quantification of the original amount of target nucleic acid in the sample by correction of the original amount derived from step (iii) with the aid of the determined amplification efficiency. The efficiency correction of PCR reactions according to the invention for the quantification of nucleic acids can be used for absolute quantification with the aid of an external or internal standard as well as for relative quantification compared to the expression of housekeeping genes.

The invention provides unique index sequences with a length of 7bp. The index sequences can be respectively imported into a DNA(deoxyribonucleic acid) index library through adapter link and PCR (polymerase chain reaction). The invention provides a method for building the DNA index library by using the index sequences based on a solexa sequencing platform of the current illumina company, and the method is applied to solexa DNA sequencing and has the effects on improving the preparation efficiency of the DNA index library, increasing the sequencing throughput of the DNA samples and lowering thesolexa sequencing cost of a single sample.

The invention discloses a fluorescence quantitative PCR detection kit of hepatitis B virus and an application thereof. The kit is composed of the following independent components: DNA extraction solution I, DNA extraction solution II, DNA extraction solution III, DNA extraction solution IV, positive control interior label, PCR reaction liquid, probe HBV-SP, enzyme mixed liquor containing heat resistant DNA polyase and uracil DNA glycosylase, quantitative hepatitis B virus reference material, hepatitis B virus positive control serum and hepatitis B virus negative control serum, wherein DNA extraction solution I contains 0.2-1.0% of lauryl sodium sulphate (mass / volume), 1.0-4.0% of Triton (volume / volume) and 0.2-1.0mol / L of guanidinium isothiocyanate; DNA extraction solution II contains 100-300mmol / L of 4-HEPES, 100-300mmol / L of sodium chloride with pH of 6.5+ / -0.2 and 100-400 mu g / ml of magnetic beads; DNA extraction solution III contains 0.1-1.0% of Triton (volume / volume) and 100-300mmol / L of sodium chloride; DNA extraction solution IV contains mineral oil. The fluorescence quantitative PCR detection kit of hepatitis B virus of the invention can be used for detecting the HBV-DNA concentration in samples of serum, blood plasma or latex and the like.

The invention designs 161 unique tag sequences with the lengths of 8 bp. Tags are embedded into a deoxyribonucleic acid (DNA) joint so as to form a DNA polymerase chain reaction (PCR)-free tag joint. The invention also provides a DNA tag library which introduces the tag sequence by connecting the DNA PCR-free tag joint without a PCR and is applied to solexa DNA sequencing. After a library construction method is optimized, the DNA tag library is constructed without the PCR.

The invention relates to a method for manufacturing a digital PCR (polymerase chain reaction) chip based on a mineral-oil saturated PDMS (polydimethylsiloxane) material. The method is characterized in that the digital PCR chip based on PDMS is prepared from a PDMS monomer of a certain amount of mineral oil (liquid paraffin) and comprises an emulsion droplet generation structure and an emulsion droplet collection structure. After the emulsion droplets are made and collected on the same chip, the emulsion droplets are subjected to PCR amplification on the same chip. The phagocytosis to the oil phase in the digital PCR system by the PDMS of the chip can be avoided, the emulsion droplets can be kept stable during PCR, and the stability of the PCR can be guaranteed. In addition, compared with the existing technology of the digital PCR chip, the method provided by the invention is low in cost, is convenient to operate and has a very wide application prospect.

The invention provides a real-time fluorescence quantitative PCR (Polymerase Chain Reaction) kit for detecting the expression level of HER2 genes and relates to a PCR kit. The real-time fluorescence quantitative PCR kit comprises PCR reaction liquid of the HER2 genes, PCR reaction liquid of ACTB genes, a Taq enzyme system, a control product and a packaging object; and the adopted primer is an annular primer, 3-8 self-designed basic groups are at the 5' end, and can be combined with the sequence at the 3' end under proper conditions so as to form double chains. The real-time fluorescence quantitative PCR kit has the advantages that the non-specific amplification products especially primer dimer generated by nonspecific amplification products especially the primer can be effectively prevented from being formed, the specificity is increased; and a relative-quantitative RT-PCR method is utilized for detecting the expression level of the HER2 genes of a patient with the breast cancer, adopts a 2-delta deltaCt value method for quantifying the detected result, and can be used for diagnosis of early stage and transferring of the breast cancer and assisting multiple fields such as clinicalmedicine selection and prognosis.

An immobilized metal affinity chromatography (IMAC) method for separating and / or purifying compounds containing a non-shielded purine or pyrimidine moiety or group such as nucleic acid, presumably through interaction with the abundant aromatic nitrogen atoms in the purine or pyrimidine moiety. The method can also be used to purify compounds containing purine or pyrimidine moieties where the purine and pyrimidine moieties are shielded from interaction with the column matrix from compounds containing a non-shielded purine or pyrimidine moiety or group. Thus, double-stranded plasmid and genomic DNA, which has no low binding affinity can be easily separated from RNA and / or oligonucleotides which bind strongly to metal-charged chelating matrices. IMAC columns clarify plasmid DNA from bacterial alkaline lysates, purify a ribozyme, and remove primers and other contaminants from PCR reactions. The metal ion affinity of yeast RNA decreases in the order: copper (II), nickel (II), zinc (II), and cobalt (II).

Methods for increasing multiplex level by externalization of a passive reference in polymerase chain reactions (PCR) are provided. An exemplary method comprises providing a first mastermix including a passive fluorescence dye in at least a first well of a plate; providing a second mastermix including an active fluorescence dye in at least a second well of the plate; wherein the passive fluorescence dye and the active fluorescence dye emit a same spectrum and an intensity of the spectrum is adapted to be measured; and wherein the first mastermix is devoid of an active fluorescence dye emitting the same spectrum and the second mastermix is devoid of the passive fluorescence dye emitting the same spectrum. Numerous other aspects are provided.

The invention relates to a multiplex-PCR three-round amplification method, a primer and a kit thereof and application of the method in establishment of next-generation sequencing platform libraries. In the first two rounds of a PCR, a specific primer with the low concentration is consumed as much as possible, so that the differences of different amplicons are reduced and the homogeneity of multiplex-PCR products is improved; when the third round of the PCR is performed, adapter primers carrying different bar code sequences are added to mark different templates, the different adapter primers carry same universal amplification primers, therefore, it is guaranteed that the amplification efficiency of the different templates is consistent, and the differences of different template amplification products are reduced. Accordingly, not only is the problem that the homogeneity of ordinary multiplex-PCR amplification is poor solved, but also establishment of the sequencing libraries can be quickly and conveniently completed.

The present invention discloses a high-flux nucleic acid analysis apparatus based on a microfluidic chip. The high-flux nucleic acid analysis apparatus comprises a microfluidic chip, an extraction unit, a heating unit and a detection unit. According to the present invention, under the assisting of the extraction unit, nucleic acid in a cleaved sample is captured by using the AOM membrane in the microfluidic chip, the AOM membrane is washed by using a purification and washing reagent so as to purify the nucleic acid, the nucleic acid template is retained on the AOM membrane, and the heating unit supplies stable reaction temperatures for the upper end and the lower end of a reaction chamber to make the nucleic acid template on the AOM membrane participate into a convection PCR reaction so as to achieve the integration of the nucleic acid extraction and the nucleic acid amplification; the detection unit comprises an LED lamp and a camera, wherein the LED lamp excites the fluorescent dye in the reaction reagent, and the fluorescence signals of a plurality of reaction hole positions are simultaneous detected through the camera; and the system has advantages of low complexity, simple operation and short detection time, integrates the nucleic acid extraction, the nucleic acid amplification and the nucleic acid detection, and can improve the nucleic acid diagnosis and analysis efficiency.

The invention discloses a method and a device for gene sequencing of a plurality of mixed DNA (Deoxyribonucleic Acid) or RNA (Ribonucleic Acid) sequences, which is mainly characterized by adding random labels at two ends of each DNA or RNA template sequence before establishing a bank. The method comprises the following steps of: mixing a first forward primer containing the random label and a connector, a first reverse primer and the DNA sequences, performing multiple PCR (Polymerase Chain Reaction) amplification on two PCR circulations, and purifying the PCR product to obtain a first DNA product; mixing the first DNA product, a second forward primer containing a sample indexing sequence and the connector, and a second reverse primer, performing PCR reaction to obtain a second PCR product; purifying to obtain a second DNA product; and sequencing the second DNA product to obtain a sequencing result of each DNA sequence. According to the method disclosed by the invention, by introducing the random labels to each DNA molecule, the sequencing precision is improved, the error rate of the sequencing is obviously reduced, and the copy number of each DNA is precisely detected.

The invention provides a multiplex quantitative PCR (polymerase chain reaction detection kit for vibrio parahaemolyticus toxin gene and a detection method. The kit mainly comprises specific primers, probes and PCR reaction reagent, wherein the specific primers and the probes consist of specific primers and probes of vibrio parahaemolyticus thermostable direct hemolysin gene (tdh), thermolabile hemomysin gene (tlh), toxin expression regulating protein gene (toxR) and thermostable related hemolysin gene (trh). The invention provides the quick, sensitive and specific multiplex fluorescent quantitative PCR detection kit and the detection method aiming at the vibrio parahaemolyticus toxin gene, and provides basis for controlling food poisoning caused by the vibrio parahaemolyticus in time and early diagnosis of the food poisoning caused by the vibrio parahaemolyticus.

The invention provides a method for quickly and simply screening a CRISPR / Cas gene editing positive object. Compared with the prior art, the method disclosed by the invention has the characteristics that an Oligo DNA segment for constructing a knockout vector is used as a PCR (Polymerase Chain Reaction) primer for screening a target sample, and possible mutations on the DNA can be detected by using the feature that PCR can be efficiently initiated by completely matching a sequence at 3'-terminal of the primer with a template needed in the PCR reaction. By adoption of the method disclosed by the invention, experimental steps are reduced and design of extra PCR primers is not needed; the method is suitable for almost all experimental designs; special DNA endonuclease does not need to be purchased and a false positive result is hardly generated. Moreover, a sample for sequencing has higher qualification rate and qualified samples are hardly missed.