Abstract: Sample preparation processes for in situ RNA or DNA analysis, methods and compositions therefor are provided. Processes provided herein allow DNA or RNA analysis to be carried out in the same tube or on an aliquot of the prepared sample without centrifugation or extraction. The preparation process can be carried out at room temperature in as little as seven minutes and is amenable to high throughput processing using manual or robotic platforms.

Abstract: The invention provides an assay method for detection and/or quantification of a plurality of nucleic acid or protein targets in a sample. In the method probes are used to associate a detectable tag sequence with each of the selected targets present in the sample. Probes or primers sufficient to identify at least 25, and preferably at least 500, different targets are used. The method involves segregating aliquots of the sample from each other and detecting the tag sequences in each aliquot.

Abstract: Compositions and methods for amplifying selected polynucleotides, including DNA and RNA, particularly in multiplex amplification reactions using common primers amplification. Generally, methods of the invention employ multiple steps such as template-specific hybridization, a linear amplification, partial degradation of nucleic acid, and ligation. At the end of the process the sequences of selected polynucleotides are flanked by the common sequences which can be used for exponential amplification using common primers. In some aspects the polynucleotides are associated with a barcode and the presence of the barcode is detected to measure the amount of the polynucleotide.

Abstract: The present invention is referred to two human nucleic 5 acids that comprise sequences encoding two new isoforms of the human somatostatin receptor type 5 originated by alternative splicing, named sst5B and sst5C, with possible involvement in tumor processes. In addition, the invention is referred to oligonucleotide pairs allowing 10 their differential detection in several tissues using the PCR technique.

Abstract: The embodiments herein provide a dipstick nano-biosensor for diagnosing Plasmodium vivax and Plasmodium falciparum. The dipstick biosensor comprises a backing plate coated with a cellulose membrane, nitrocellulose membrane and fibreglass. Gold nanoparticles coated with antidigoxigenin are immobilized on the dipstick along with probes comprising strptavidin, texas red, biotin and fluorescein. The dipstick biosensor has three regions comprising a wicking pad, conjugate pad having two control lines and two test lines and an absorbent pad. The first Control line comprises nitrocellulose membrane coated with antifluorescein. The second control line comprises nitrocellulose membrane coated with anti anti-sheep. The first test line comprises nitrocellulose membrane coated with streptavidin conjugated to biotin. The second test line comprises nitrocellulose membrane coated with anti texas red. The two test lines help to confirm the diagnostic results.

Abstract: The present invention is in the field of nucleic acid amplification, and in particular in transcription-based amplification, providing improvements thereof. Specifically, the present invention provides primers, and methods for using them, that improve transcription-based amplification reactions, in particular multiplex reactions.

Abstract: Methods are provided for diagnosing in a subject a condition, such as a carcinoma, sarcoma or leukemia, associated with hypermethylation of genes by isolating the genes from tissue containing as few as 50 to 1000 tumor cells. Using quantitative multiplex methylation specific PCR (QM-MSP), multiple genes can be quantitatively evaluated from samples usually yielding sufficient DNA for analysis of only 1 or 2 genes. DNA sequences isolated from the sample are simultaneously co-amplified in an initial multiplex round of PCR, and the methylation status of individual hypermethylation-prone gene promoter sequences is then determined separately or in multiplex using a real time PCR round that is methylation status-specific. Within genes of the panel, the level of promoter hypermethylation as well as the incidence of promoter hypermethylation can be determined and the level of genes in the panel can be scored cumulatively. The QM-MSP method is adaptable for high throughput automated technology.

Abstract: The present invention provides methods of: identifying pathogens in biological samples from humans and animals, resolving a plurality of etiologic agents present in samples obtained from humans and animals, determining detailed genetic information about such pathogens or etiologic agents, and rapid detection and identification of bioagents from environmental, clinical or other samples.

Abstract: A device for amplifying target nucleic acid in a sample can include a planar fluidic assembly including a transparent substrate, a porous material layer on a surface of the transparent substrate, and a cover over the porous material layer and sealingly affixed to the substrate. The cover may be spaced from the porous material layer and a flow channel defined between the porous material layer and the cover. The flow channel may have a uniform cross-section from a first end to a second end.

Abstract: The invention relates to prognostic markers and prognostic signatures, and compositions and methods for determining the prognosis of cancer in a patient, particularly for melanoma. Specifically, the invention relates to the use of genetic and protein markers for the prediction of the risk of progression of a cancer, such as melanoma, based on markers and signatures of markers. In various aspects, the invention provides methods, compositions, kits, and devices based on prognostic cancer markers, specifically melanoma prognostic markers, to aid in the prognosis and treatment of cancer.

Abstract: The present invention is directed to method for analyzing multiple nucleic acid molecules of interest comprising in the steps of (i) providing a plurality of beads, characterized in that each bead comprises at least two sequence specific amplification primers, further characterized in that at least one of the primers is bound to the bead via a cleavable linker, (ii) capturing the nucleic acid molecules of interest from a sample, (iii) clonally isolating the plurality of beads, (iv) cleaving the at least one primer, (v) clonally amplifying the nucleic acid thereby creating multiple amplification products, and (vi) analyzing the amplification products.

Abstract: The invention relates to a method for the high throughput discovery, detection and genotyping of one or more genetic markers in one or more samples, comprising the steps of restriction endonuclease digest of DNA, adaptor-ligation, optional pre-amplification, selective amplification, pooling of the amplified products, sequencing the libraries with sufficient redundancy, clustering followed by identification of the genetic markers within the library and/or between libraries and determination of (co-)dominant genotypes of the genetic markers.

Abstract: Provided herein is a method for identifying a genetic variant that is associated with development of a condition of interest (e.g., Alzheimer's disease), and genetic variants so identified. Methods of treatment with an active agent (e.g., with a particular active agent and/or at an earlier age) is also provided, upon detecting a genetic variant described herein. In some embodiments, the genetic variant is a deletion/insertion polymorphism (DIP) of the TOMM40 gene. Kits for determining if a subject is at increased risk of developing late onset Alzheimer's disease is also provided. Kits for determining if a subject is responsive to treatment for a condition of interest with an active agent are further provided.

Abstract: The present invention provides methods for universally detecting citrus viroids in plant material such as germplasm. In particular embodiments, the invention enables the determination of citrus viroid infection and plant resistance. Accordingly, the present method provides methods for improved universal detection of any citrus viroid.

Abstract: Described herein are methods, compositions and kits directed to the detection of gene dysregulations such as those arising from gene fusions and/or chromosomal abnormalities, e.g., translocations, insertions, inversions and deletions. Samples containing dysregulated gene(s) of interest may show independent expression patterns for the 5? and 3? regions of the gene. The methods, compositions and kits are useful for detecting mutations that cause the differential expression of a 5? portion of a target gene relative to the 3? region of the target gene.

Abstract: A two-step multiplex amplification reaction includes a first step which truncates the standard initial multiplex amplification round to “boost” the sample copy number by only a 100-1000 fold increase in the target. Following the first step the product is divided into optimized secondary single amplification reactions, each containing one of the primer sets that were used previously in the first or multiplexed booster step. The booster step can occur using an aqueous target nucleic acid or using a solid phase archived nucleic acid. In particular, nucleic acid sequences that uniquely identify E. Coli were identified using the multiplex amplification method.

Abstract: A method for in vitro diagnosis or prognosis of testicular cancer in a biological sample from a patient suspected of suffering from testicular cancer, having a step of detecting the presence or absence of methylation of CpG dinucleotides in at least one genomic DNA target sequence of the sample, the target sequence being selected from at least one of the sequences identified in SEQ ID NOS: 1 to 7 or from at least one sequence which exhibits at least 99% identity with one of the sequences identified in SEQ ID NOS: 1 to 7 and the sequences complementary thereto; to the DNA sequences and to the use thereof as a testicular cancer marker.

Abstract: The inventors have developed a rapid and sensitive fluorescence-based assay to quantify dNTPs. This assay relies on the principle that incorporation of a limiting dNTP is required for primer-extension and polymerase-mediated 5-3? exonuclease hydrolysis of a quenched fluorophore-labeled probe resulting in fluorescence. The concentration of limiting dNTPs is directly proportional to the fluorescence generated. This assay has important applications in research that investigates the influence of pathological conditions or pharmacological agents on dNTP biosynthesis and regulation.

Abstract: Disclosed are mutant DNA polymerases having increased 3?-mismatch discrimination relative to a corresponding, unmodified polymerase. The mutant polymerases are useful in a variety of disclosed primer extension methods. Also disclosed are related compositions, including recombinant nucleic acids, vectors, and host cells, which are useful, e.g., for production of the mutant DNA polymerases.

Abstract: A method for replicating and amplifying a target nucleic acid sequence is described. A method of the invention involves the formation of a recombination intermediate without the prior denaturing of a nucleic acid duplex through the use of a recombination factor. The recombination intermediate is treated with a high fidelity polymerase to permit the replication and amplification of the target nucleic acid sequence. In preferred embodiments, the polymerase comprises a polymerase holoenzyme. In further preferred embodiments, the recombination factor is bacteriophage T4 UvsX protein or homologs from other species, and the polymerase holoenzyme comprises a polymerase enzyme, a clamp protein and a clamp loader protein, derived from viral, bacteriophage, prokaryotic, archaebacterial, or eukaryotic systems.

Abstract: Disclosed are methods for synthesizing and/or assembling at least one polynucleotide product having a predefined sequence from a plurality of different oligonucleotides. In exemplary embodiments, the methods involve synthesis and/or amplification of different oligonucleotides immobilized on a solid support, release of synthesized/amplified oligonucleotides in solution to form droplets, recognition and removal of error-containing oligonucleotides, moving or combining two droplets to allow hybridization and/or ligation between two different oligonucleotides, and further chain extension reaction following hybridization and/or ligation to hierarchically generate desired length of polynucleotide products.

Abstract: The present invention provides a novel method for identifying an olfactory receptor included in one olfactory cell. In the present invention, amplified is the cDNA derived from the mRNA of the one olfactory cell by a PCR method using a forward primer represented by SEQ ID: 01 and a reverse primer represented by SEQ ID: 02. Subsequently, determined is whether or not a gene sequence of the amplified cDNA is identical to one gene sequence included in gene sequences coding for olfactory receptors included in the mouse olfactory receptor group A. Finally, determined is that, if the gene sequence of the cDNA is identical to the one gene sequence in the previous step, the olfactory receptor included in the one olfactory cell is the olfactory receptor corresponding to the one gene sequence which is identical to the gene sequence of the cDNA in the previous step.

Abstract: Method for the detection of a target sequence comprising ligating two probes when annealed adjacent to the target sequence, hybridization of a compound primer to the ligated probes and after elongation of the compound primer, amplifying the elongated compound primer from primers annealing to primer binding sites provided in the compound primer and one of the probes to produce detectably amplicons.

Abstract: The present invention relates to a linker or population of linkers that include an oligonucleotide fixed portion and an oligonucleotide variable portion represented by formula (N)n, wherein N is A, C, G, T or U, or their derivatives, and n is an integer equal to or higher than 1. A linker-polynucleotide or a population of linker-polynucleotides of the invention may be constituted by said linker or population of linkers and a target first strand polynucleotide bound to said linker. The invention also encompasses a method of preparing said linker or population of linkers and a method of preparing a linker-polynucleotide using said linker or population of linkers. The linkers or polynucleotide-linkers of the invention can be used in a method of preparing a cDNA library.

Abstract: A process includes providing a mixture that includes a recombinase, a single-strand binding protein, and one or more oligonucleotides; and detecting particles in the reaction mixture.

Abstract: This invention relates to the use of tumor-derived or associated extracellular ribonucleic acid (RNA) found circulating in blood plasma or serum fraction for the detection, monitoring, or evaluation of cancer or premalignant conditions. Specifically, this invention enables the extraction of circulating RNA from plasma or serum and utilizes nucleic acid amplification assays for the identification, detection, inference, monitoring, or evaluation of any neoplasm, benign, premalignant, or malignant, in humans or other animals, which might be associated with that RNA. Further, this invention allows the qualitative or quantitative detection of tumor-derived or associated extracellular RNA circulating in the plasma or serum of humans or animals with or without any prior knowledge of the presence of cancer or premalignant tissue.

Abstract: The present invention provides methods for selectively enriching a biological sample for short nucleic acids, such as fetal DNA in a maternal sample or apoptic DNA in a biological sample from a cancer patient and for subsequently analyzing the short nucleic acids for genotype, mutation, and/or aneuploidy.

Abstract: Disclosed are mutant DNA polymerases having increased 3?-mismatch discrimination relative to a corresponding, unmodified polymerase. The mutant polymerases are useful in a variety of disclosed primer extension methods. Also disclosed are related compositions, including recombinant nucleic acids, vectors, and host cells, which are useful, e.g., for production of the mutant DNA polymerases.

Abstract: The present invention provides a biological substance-immobilized gel which comprises a gel containing 2%-7% by mass of N,N-dimethylacrylamide and a biological substance immobilized on and/or in the gel.

Abstract: Methods of producing substrates having selected active chemical regions by employing elements of the substrates in assisting the localization of active chemical groups in desired regions of the substrate. The methods may include optical, chemical and/or mechanical processes for the deposition, removal, activation and/or deactivation of chemical groups in selected regions of the substrate to provide selective active regions of the substrate.

Abstract: The present invention provides methods for rapid forensic analysis of mitochondrial DNA and methods for characterizing heteroplasmy of mitochondrial DNA, which can be used to assess the progression of mitochondrial diseases.

Abstract: The present invention relates to a method for identifying a target nucleotide sequence. This method involves forming a ligation product on a target nucleotide sequence in a ligation detection reaction mixture, amplifying the ligation product to form an amplified ligation product in a polymerase chain reaction (PCR) mixture, detecting the amplified ligation product, and identifying the target nucleotide sequence. Such coupling of the ligase detection reaction and the polymerase chain reaction permits multiplex detection of nucleic acid sequence difference.

Abstract: This invention relates to a rapid method for detection and characterization of STEC bacteria based on the presence of nucleic acid sequences, in particular, to a PCR-based method for detection, and to oligonucleotide molecules and reagents and kits useful therefore. This method is preferably employed to detect STEC bacteria in a food or water sample, such as a beef enrichment. The present invention further relates to isolated polynucleotides, replication compositions, kits, and reagent tablets for carrying out the method of the present invention.

Abstract: It is an object to provide a method of suitably analyzing the amount of gene expression of a single-cell. A method of detecting a nucleic acid comprising a step of sampling a single-cell from a sample containing at least a single-cell, a cell lysis step of lysing cell membrane of the sampled single-cell and extracting nucleic acids from the cell, a DNase treatment step of degrading DNA of the extracted nucleic acids with DNase, a step of hybridizing mRNA of the total RNA contained in the single-cell with oligo (dT) fixed onto a carrier, a step of performing reverse transcription of the mRNA hybridized with the oligo (dT) to fix cDNA derived from the single-cell onto the carrier, thereby preparing a single-cell derived cDNA library fixed onto a carrier, and a step of amplifying cDNA fixed onto the carrier and simultaneously detecting an amplification amount of the cDNA.

Abstract: Systems and methods for synthesizing long-chain nucleic acids molecules are disclosed. The systems and methods described in this application use a Ligation- Purification-Amplification (“LPA”) technique. The LPA technique requires first producing nucleotide sequences with the length of at least 500 bp-1 kbp by assembling smaller oligonucleotide fragments (30 bp-200 bp). The assembled nucleotide sequences are then purified and amplified. This technology can be used to achieve parallel amplification of three or more nucleotide sequences at the same time. In embodiments of the invention, a solid-phase ligation-purification method is adopted, in which nucleic acid molecules are fixed on the surface of beads or other solid particles in order to rapidly purify products obtained from the ligation reaction.

Abstract: Disclosed are mutant DNA polymerases having increased 3?-mismatch discrimination relative to a corresponding, unmodified polymerase. The mutant polymerases are useful in a variety of disclosed primer extension methods. Also disclosed are related compositions, including recombinant nucleic acids, vectors, and host cells, which are useful, e.g., for production of the mutant DNA polymerases.

Abstract: A method for producing a single stranded DNA (ssDNA) molecule of a defined length and sequence is disclosed. This method enables the preparation of, inter alia, probes of greater length than can be chemically synthesized. The method starts with a double stranded molecule, such as genomic, double stranded DNA (dsDNA) from any organism. A fragment of the starting molecule (dsDNA) is amplified by specific primers engineered to introduce cleavage sites on either side of the desired sequence. Cleavage steps on the amplified, engineered fragment are combined with a phosphate removal step, thereby creating a construct that can be digested with an exonuclease without damage to the desired ssDNA. Probes, which hybridize with large gaps between the ends of the probes, are also disclosed.

Abstract: Improved compositions, methods, apparatus, and kits for high-throughput nucleic acid amplification, detection and sequencing are disclosed. A nucleic acid cluster having an identifiable center is produced by generating on a solid support an immobilized nucleic acid complement from a template, one of which comprises a detectable label; and amplifying the complement and the template to obtain a nucleic acid cluster on the support, the cluster having a substantially central location marked by the detectable label and a surrounding region comprising immobilized copies. Also disclosed are nucleotide sequence determination in nucleic acid clusters so produced, center position annotation in the clusters, assignment of sequence information to overlapping clusters, and related compositions and methods.

Abstract: Disclosed are methods of multiplexed analysis of oligonucleotides in a sample, including: methods of probe and target “engineering”, as well as methods of assay signal analysis relating to the modulation of the probe-target affinity constant, K by a variety of factors including the elastic properties of target strands and layers of immobilized (“grafted”) probes; and assay methodologies relating to: the tuning of assay signal intensities including dynamic range compression and on-chip signal amplification; the combination of hybridization-mediated and elongation-mediated detection for the quantitative determination of abundance of messages displaying a high degree of sequence similarity, including, for example, the simultaneous determination of the relative expression levels, and identification of the specific class of, untranslated AU-rich subsequences located near the 3? terminus of mRNA; and a new method of subtractive differential gene expression analysis which requires only a single color label.

Abstract: There is a need for improved methods for determining the diagnosis and prognosis of patients with conditions, including autoimmune disease and cancer. Provided herein are methods for using DNA sequencing to identify personalized biomarkers in patients with autoimmune disease and other conditions. Identified biomarkers can be used to determine the disease state for a subject with an autoimmune disease or other condition.

Abstract: Embodiments of the disclosure relate to isolated nucleic acid sequences, methods of use thereof, and workflows for detection of several Listeria species in a sample, particularly in a food or environmental sample. Embodiments of the disclosure may also be used to detect one or more species or strains of Listeria from each other, for example L. grayi may be detected independently of other Listeria spp. Some embodiments also describe a duplexed assay that can detect L. monocytogenes, L. innocua, L. welshimeri, L. seelgeri, L. marthii (formerly incertae-sedis), L. ivanovii, and L. grayi. Kits for detection of Listeria are also described. In some embodiments, methods and kits of the disclosure may comprise a TAQMAN® assay. In some embodiments, 0.2-2 cfu of Listeria spp. are detected using the compositions, methods and kits after a 24-28 hour enrichment period.

Abstract: Disclosed are mutant DNA polymerases having increased 3?-mismatch discrimination relative to a corresponding, unmodified polymerase. The mutant polymerases are useful in a variety of disclosed primer extension methods. Also disclosed are related compositions, including recombinant nucleic acids, vectors, and host cells, which are useful, e.g., for production of the mutant DNA polymerases.

Abstract: Disclosed are mutant DNA polymerases having increased 3?-mismatch discrimination relative to a corresponding, unmodified polymerase. The mutant polymerases are useful in a variety of disclosed primer extension methods. Also disclosed are related compositions, including recombinant nucleic acids, vectors, and host cells, which are useful, e.g., for production of the mutant DNA polymerases.

Abstract: Provided herein is a method for detecting the presence or absence of at least one of Clostridium botulinum toxin gene A, B, E, and F in a biological sample by means of PCR amplification using toxin specific primers and labeled probes in connection with real time or delayed detection. Also provided are specific primer and probe sequences, a diagnostic method and a kit comprising primers and probes for detection of toxin genes A, B, E, or F in a biological sample.

Abstract: Oligonucleotides useful for determining the presence of Trichomonas vaginalis in a test sample.

Abstract: Provided are methods of enrichment and detection of target nucleic acids during target amplification in the presence of excess amounts of highly homologous sequences, said methods having substantial diagnostic utility (e.g., cancer diagnostics). Provided are amplification reaction mixtures having at least one cleavage-directing oligonucleotide, the respective binding sites of which, for the target and homologous sequences, include one or more nucleotide positions differing in sequence between the target homologous sequences.

Abstract: Fabrication of a microfluidic multi-temperature reaction device (MMR) and the design and fabrication of the equipment to drive various molecular biological methods on the device are provided. The device can be applicable, for example, to nucleic acid (DNA, RNA, cDNA, etc) amplification, cell lysis, reverse transcription and other enzymatic temperature sensitive and also temperature cycling reactions.

Abstract: Microfluidic devices, and methods for their use are described. The microfluidic devices include articles formed from a thermoplastic composition comprising a poly(aliphatic ester)-polycarbonate comprising soft block ester units, derived from monomers comprising an alpha, omega C6-20 aliphatic dicarboxylic acid or derivative thereof, a dihydroxyaromatic compound, and a carbonate source.

Abstract: A marking apparatus (1) for marking an item (12). The apparatus comprises: means to receive the item; a nucleic acid marker; means to release a marking fluid (8); and a distribution mechanism (11) coupled to the nucleic acid marker and the means to release the marking fluid. The means to release the marking fluid can be activated to release the marking fluid such that the distribution mechanism disperses a mixture of the nucleic acid marker and the marking fluid onto the item.

Abstract: The invention provides a method for reducing stutter in the amplification of a microsatellite comprising the steps of providing a sample comprising a microsatellite having a G+C content of 50% or less; contacting the sample with at least one enzyme having nucleic acid polymerase activity; and incubating the sample with the enzyme for a sufficient amount of time and under conditions sufficient to amplify the microsatellite; wherein the incubation is performed in the presence of an amount of betaine, sorbitol or mixtures thereof, effective to reduce stutter relative to the amount of stutter observed in the absence of betaine and/or sorbitol. The invention also provides compositions containing betaine and/or sorbitol, kits for amplifying microsatellites having a G+C content of 50% or less, and methods of using all of the foregoing.