572 results about "Dna amplification" patented technology

This disclosure describe three related novel methods for Recombinase-Polymerase Amplification (RPA) of a target DNA that exploit the properties of the bacterial RecA and related proteins, to invade double-stranded DNA with single stranded homologous DNA permitting sequence specific priming of DNA polymerase reactions. The disclosed methods has the advantage of not requiring thermocycling or thermophilic enzymes. Further, the improved processivity of the disclosed methods allow amplification of DNA up to hundreds of megabases in length.

The present invention relates to the field of fluid dynamics. More specifically, this invention relates to methods and apparatus for conducting densely packed, independent chemical reactions in parallel in a substantially two-dimensional array. Accordingly, this invention also focuses on the use of this array for applications such as DNA sequencing, most preferably pyrosequencing, and DNA amplification.

A method is provided for adding a terminal sequence tag to nucleic acid molecules for use in RNA or DNA amplification. The method involves contacting with a mixture of oligonucleotides, each having a sequence tag template, a random sequence and a blocked 3′ terminus, under conditions such that, the random sequence anneals with the nucleic acid molecules and the nucleic acid molecules are extended using the sequence tag template as template. For synthesis of RNA from DNA molecules having terminal sequence tags, the method includes forming DNA templates having a double stranded promoter sequence and synthesizing RNA from the DNA templates. For amplification of sequences from DNA molecules having terminal sequence tags, the method includes forming DNA templates by extension of one primer having a sequence that is complementary to the terminal sequence tag and another primer having a sequence that is derived form one of the DNA molecules.

The invention provides an apparatus that can be used in methods of preparing, amplifying, detecting, and / or optionally selecting for further analysis the genomic material from an organism for the rapid detection and / or classification of an organism in a sample (e.g., screening for, identifying, quantifying, and / or optionally further analyzing, e.g., sequencing, the genomic material of the organism). The invention further provides methods of using the apparatus, e.g., in combination with novel SGP primers for improved use in waveform-profiling methods of DNA amplification. It is an object of the invention to provide an apparatus for fully automated analysis of genomic material, and multiple methods of using the apparatus that are beneficial to society, e.g., the apparatus may be used in methods of screening for, identifying, quantifying, and / or selecting genomic material for further analysis (e.g., sequencing) in relation to monitoring a source for the presence of contaminating organisms.

A method of isolating genomic or RNA-derived duplex fragments which are unique to one of two fragment mixtures. The fragments in positive-source and negative-source mixtures are separately equipped with end linkers, and each mixture is amplified by successive primed-strand replications, using a single primer which is homologous to the associated linker. The second-source linker is biotinylated, and the fragments in this mixture are hybridized in molar excess with the fragments in the positive-source mixture. DNA species which are not hybridized with the biotinylated species, i.e., species that are unique to the positive-source mixture, are isolated after removal of hybridized species by affinity chromatography. Also disclosed is a method of amplifying a mixture of DNA fragments by repeated linker / primer replication.

We describe a method for interrogating the content and primary structure of DNA by microarray analyses and to provide comprehensive genetic screening and diagnostics prior to embryo transfer within an IVF setting. We will accomplish this by the following claims: 1) an optimized embryo grading system, 2) a less invasive embryo biopsy with reduced cellular contamination, 3) an optimized DNA amplification protocol for single cells, 4) identify aneuploidy and structural chromosome abnormalities using microarrays, 5) identifying sub-telomeric chromosome rearrangements, 6) a modified DNA fingerprinting protocol, 7) determine imprinting and epigenetic changes in developing embryos, 8) performing genome-wide scans to clarify / diagnose multi-factorial genetic disease and to determine genotype / haplotype patterns that may predict future disease, 9) determining single gene disorders with or without a known DNA mutation, 10) determining mtDNA mutations and / or the combination of mtDNA and genomic (nuclear) DNA aberrations that cause genetic disease.

The present invention provides an apparatus and method for performing heat-exchanging reactions on an electro wetting-based micro fluidic device. The apparatus provides one or multiple thermal contacts to an electro wetting-based device, where each thermal contact controls the part of the electro wetting-based device it communicates with to a designed temperature. The electrowetting-based device can be used to create, merge and mix liquids in the format of droplets and transport them to different temperature zones on the micro fluidic device. The apparatus and methods of the invention can be used for heat-exchanging chemical processes such as polymerase chain reaction (PCR) and other DNA reactions, such as ligase chain reactions, for DNA amplification and synthesis, and for real-time PCR.

A multilayered microfluidic DNA analysis system includes a cell lysis chamber, a DNA separation chamber, a DNA amplification chamber, and a DNA detection system. The multilayered microfluidic DNA analysis system is provided as a substantially monolithic structure formed from a plurality of green-sheet layers sintered together. The substantially monolithic structure has defined therein a means for heating the DNA amplification chamber and a means for cooling the DNA amplification chamber. The means for heating and means for cooling operate to cycle the temperature of the DNA amplification chamber as required for performing a DNA amplification process, such as PCR.

A thermal cycling method and device is disclosed. The device comprises a sample chamber whose temperature can be rapidly and accurately modulated over a range of temperatures needed to carry out a number of biological procedures, such as the DNA polymerase chain reaction. Biological samples are placed in glass micro capillary tubes and then located inside the sample chamber. A programmable controller regulates the temperature of the sample inside the sample chamber. Monitoring of the DNA amplification is monitored by fluorescence once per cycle or many times per cycle. The present invention provides that fluorescence monitoring of PCR is a powerful tool for DNA quantification.

The present invention provides for amplification of one or more polynucleotides by multi-staged linear amplifications using one or more RNA polymerases. At each stage RNA transcripts are accumulated at a linear rate, so that multiple stages provide for faster than linear transcript accumulation. In one aspect, the invention provides for polynucleotide amplification by ligating hairpin adaptors to an end of polynucleotides wherein the hairpin adaptors each contain a promoter sequence oriented so that transcription proceeds in the direction of the loop of the hairpin adaptor. Upon transcription through such loop region and to the complementary strand a replicate is made of the promoter sequence as well as the polynucleotide, thereby permitting exponential amplification upon reverse transcription, second strand synthesis, and repetition of the above cycle. Preferably such amplification is carried out under isothermal reaction conditions.

A method for parallel sequencing target RNA from samples from multiple sources while maintaining source identification is provided. The method includes providing samples of RNA comprising target RNA from two or more sources; labeling, at the 3′ end, the RNA from the two or more sources with a first nucleic acid adaptor that comprises a nucleic acid sequence that differentiates between the RNA from the two or more sources; reverse transcribing the two or more sources to create a single stranded DNA comprising the nucleic acid sequence that differentiates between the RNA from the two or more sources; amplifying the single stranded DNA to create DNA amplification products that comprise the nucleic acid sequence that differentiates between the RNA from the two or more sources; sequencing the DNA amplification products thereby parallel sequencing target RNA from samples from multiple sources while maintaining source identification.

A method of modulation of synthesis capacity on and cleavage properties of synthetic oligomers from solid support is described. The method utilizes linker molecules attached to a solid surface and co-coupling agents that have similar reactivities to the coupling compounds with the surface functional groups. The preferred linker molecules provide an increased density of polymers and more resistance to cleavage from the support surface. The method is particularly useful for synthesis of oligonucleotides, oligonucleotides microarrays, peptides, and peptide microarrays. The stable linkers are also coupled to anchor molecules for synthesis of DNA oligonucleotides using on support purification, eliminating time-consuming chromatography and metal cation presence. Oligonucleotides thus obtained can be directly used for mass analysis, DNA amplification and ligation, hybridization, and many other applications.

The present invention features novel, diverse, hybrid and engineered recombinase enzymes, and the utility of such proteins with associated recombination factors for carrying out DNA amplification assays. The present invention also features different recombinase ‘systems’ having distinct biochemical activities in DNA amplification assays, and differing requirements for loading factors, single-stranded DNA binding proteins (SSBs), and the quantity of crowding agent employed.

The present invention relates to a novel method for the amplification of DNA, this method being particularly useful for the amplification of the DNA or the whole genome of a single cell, chromosomes or fragments thereof. Described is also the use of the method in DNA analysis for medical, forensic, diagnostic or scientific purposes, like comparative genomic hybridization (CGH)-, fluorescence in situ hybridization (FISH)-, polymerase chain reaction (PCR)-, single strand conformation polymorphism (SSCP)-, DNA sequence-, "loss of heterozygosity" (LOH)-, fingerprint- and / or restriction fragment length polymorphism (RFLP)-analysis.

A thermal cycling method and device is disclosed. The device comprises a sample chamber whose temperature can be rapidly and accurately modulated over a range of temperatures needed to carry out a number of biological procedures, such as the DNA polymerase chain reaction. Biological samples are placed in containers each comprising a reservoir and a reaction portion, wherein the reaction portion has a small volume. The small volume reaction portion permits the rapid and accurate temperature modulation. With an optically transmissible reaction portion, DNA amplification may be monitored by fluorescence during PCR.

A thermal cycling method and device is disclosed. The device comprises a sample chamber whose temperature can be rapidly and accurately modulated over a range of temperatures needed to carry out a number of biological procedures, such as the DNA polymerase chain reaction. Biological samples are placed in containers each comprising a reservoir and a reaction portion, wherein the reaction portion has a small volume. The small volume reaction portion permits the rapid and accurate temperature modulation. With an optically transmissible reaction portion, DNA amplification may be monitored by fluorescence during PCR.

A convenient and effective method for amplifying a nucleic acid sequence characterized by effecting a DNA synthesis reaction in the presence of chimeric oligonucleotide primers; a method for supplying a large amount of DNA amplification fragments; an effective method for amplifying a nucleic acid sequence by combining the above method with another nucleic acid sequence amplification method; a method for detecting a nucleic acid sequence for detecting or quantitating a microorganism such as a virus, a bacterium, a fungus or a yeast; and a method for detecting a DNA amplification fragment obtained by the above method in situ.

Methods are provided for adding a terminal sequence tag to nucleic acid molecules for use in RNA or DNA amplification. The tag introduced may be used as a primer binding site for subsequent amplification of the DNA molecule and / or sequencing of the DNA molecule and therefore provides means for identification and cloning of the 5′-end or the complete sequence of mRNAs.

DNA amplification and hybridization are successively carried out in a reaction system containing primers for the DNA amplification and hybridization probes, followed by detecting the hybrid in the reaction solution by affinity chromatography, wherein at least one of the primers to be used in the DNA amplification is labeled with a first labeling agent so that the amplified DNA will be labeled with the first labeling agent, a hybridization probe is labeled with a second labeling agent and contained in a reaction solution for effecting the DNA amplification, the base sequence of the hybridization probe is designed not to inhibit the DNA amplification, and a hybrid is detected by affinity chromatography with the use of the first and second labeling agents.

The present invention relates to methods and apparatuses for conducting densely packed, independent chemical reactions in parallel in fluid-permeable arrays. Accordingly, this invention also focuses on the use of such arrays for applications such as DNA sequencing, most preferably pyrophosphate sequencing, and DNA amplification.

The portable micro PCR chip system prepared by MEMS or micro-model technique in bio-micro EMC system field comprises some trap-type reaction ponds for DNA amplification connected to liquid inlet / outlet by flow passages, an integrated micro-heater and sensor for amplifying DNA by the temperature cycle control system, a micro-pump and micro-valve on pond inlet to lead the DNA initiator and ponds insulation, and maybe a fluorescence excitation source, a detector and a fluorescence signal detection and analysis system for on-line detection on PCR product. This chip system provides a high-flux and fast DNA amplification means with low cost and well reliability.

The present invention relates generally to the use of terminal-phosphate-labeled nucleotides having three or more phosphates as substrates for nucleic acid polymerases and their use in DNA amplification. The labels employed are chemiluminescent, fluorescent, electrochemical and chromogenic moieties as well as mass tags and include those that are directly detectable, detectable after enzyme activation or feed into other processes to generate a different signal. The signal generated from the attached dyes may also be used to quantify the amount of amplification. Further provided are stabilizers that enhance the stability of terminal-phosphate labeled nucleoside polyphosphates in aqueous solutions and are useful for reducing non-enzymatic hydrolysis of these nucleotides, hence decrease background.

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 present disclosure is a cassette for PCR amplification and detection of DNA amplicons, the cassette having a cassette pipette which may be moved vertically and horizontally within the cassette, at least one reagent chamber, and at least one detector, such as a microarray, for the detection of DNA amplified during the PCR amplification steps.

The invention provides a microRNA quantitative detection analytic method by utilizing isothermal amplification to synthesize a fluorescent nano silver cluster probe. The method is as below: a DNA amplification template containing three kinds of functional sequences is designed: a sequence binding with a target microRNA, a nicking endonuclease enzyme sequence and a DNA complementary sequence for synthesis of fluorescent nano cluster; when the target microRNA and the DNA amplification template are combined, isothermal amplification reaction and specific enzyme reaction of nicking endonuclease are induced to obtain a large number of single-stranded DNA products; the DNA sequence for synthesis of fluorescent nano silver cluster and a silver nitrate solution are employed to prepare the fluorescent nano silver cluster probe under the reduction of sodium borohydride; fluorescence signal of the reaction system are determined, and a fluorescence change value is calculated; the value is compared with a standard working curve to calculate the concentration of the target microRNA. The method has the advantages of high sensitivity, strong specificity, wide linear detection range, low background signal and simple operation, and can be widely applied to microRNA detection of biological samples such as tissue, blood or cells.

A convenient and effective method for amplifying a nucleic acid sequence characterized by effecting a DNA synthesis reaction in the presence of chimeric oligonucleotide primers; a method for supplying a large amount of DNA amplification fragments; an effective method for amplifying a nucleic acid sequence by combining the above method with another nucleic acid sequence amplification method; a method for detecting a nucleic acid sequence for detecting or quantitating a microorganism such as a virus, a bacterium, a fungus or a yeast; and a method for detecting a DNA amplification fragment obtained by the above method in situ.

The present invention relates to a method of performing a chemical reaction, in particular a small-scale chemical reaction. The method involves the use of two (or more) phases which, when formed into an emulsion, have the characteristic of being subject to “collapse” under certain physical or chemical conditions such that the discontinuous phase dispersed in the emulsion becomes a substantially continuous phase—the chemical reaction taking place in the newly-formed continuous phase.