Abstract: Amplification-based methods and kits for rapidly producing siRNA expression cassettes are provided. Also provided are methods for expressing amplified siRNA expression cassettes in cells.

Abstract: The application relates generally to methods useful for the selective amplification of one or more target nucleic acid or fragments thereof, as well as compositions and kits comprising said amplification reaction mixtures. More specifically, the application relates to a composite primer that comprises a 5? promoter portion and a 3? target-recognition portion which is complementary to the 3? end portion of a target polynucleotide sequence; and optionally, a means for identifying the 5? end portion of the target polynucleotide sequence. The amplification reaction mixture comprises at least one handle-stem-loop structure which comprises a 5? single-stranded handle comprising the promoter portion and a double-stranded stem comprising at least one pair of self-folding segments hybridized to each other, and optionally, a single-stranded loop comprising the sequence between the pair of self-folding segments.

Abstract: The present invention is directed to compositions and methods useful for the amplification of nucleic acid molecules by reverse transcriptase-polymerase chain reaction (RT-PCR). Specifically, the invention provides compositions and methods for the amplification of nucleic acid molecules in a simplified one- or two-step RT-PCR procedure using combinations of reverse transcriptase and thermostable DNA polymerase enzymes in conjunction with sulfur-containing molecules or acetate-containing molecules (or combinations of such sulfur-containing molecules and acetate-containing molecules), and optionally bovine serum albumin. The invention thus facilitates the rapid and efficient amplification of nucleic acid molecules and the detection and quantitation of RNA molecules. The invention also is useful in the rapid production and amplification of cDNAs which may be used for a variety of industrial, medical and forensic purposes.

Abstract: A method of amplifying a target nucleic acid with reduced amplification bias and a method of determining a relative amount of a target nucleic acid in a sample.

Abstract: The present invention provides improved methods for the amplification of nucleic acid molecules. Methods for amplifying target polynucleotides, including mRNA, using oligonucleotides, DNA and RNA polymerases are provided. The invention further provides compositions and kits for practicing the methods, as well as methods which use the amplification products.

Abstract: The present invention provides methods for the amplification of nucleic acid molecules. Methods for amplifying target polynucleotides, including mRNA, using oligonucleotides, DNA and RNA polymerases are provided. The invention further provides compositions and kits for practicing the methods, as well as methods which use the amplification products.

Abstract: The invention relates to methods for the detection of a specific sequence of RNA in a cell or tissue sample. The invention also relates to methods to enzymatically manipulate the RNA in a crude cell lysate in a number of applications.

Abstract: An assay for detection of short sequences of RNA in a synthetic or clinically isolated sample is presented herein. Particular reference is made to detecting RNA based pathogens, such as H5 influenza.

Abstract: There is disclosed an improved high-throughput and quantitative process for determining methylation patterns in genomic DNA samples based on amplifying modified nucleic acid, and detecting methylated nucleic acid based on amplification-dependent displacement of specifically annealed hybridization probes. Specifically, the inventive process provides for treating genomic DNA samples with sodium bisulfite to create methylation-dependent sequence differences, followed by detection with fluorescence-based quantitative PCR techniques. The process is particularly well suited for the rapid analysis of a large number of nucleic acid samples, such as those from collections of tumor tissues.

Abstract: New styles of hepatitis C virus (HCV), referred to as HCV-3 and HCV-4, have been identified and sequenced. Antigenic regions of HCV-2, HCV-3 and HCV-4 polypeptides have been identified. Immunoassays for HCV and antibodies thereto are described, which allow more complete screening of blood samples for HCV, and allow HCV genotyping.

Abstract: Compositions and methods of use are disclosed for clonally amplifying target polynucleotide sequences in solution and attaching the amplicons to a surface by activation of a masked binding moiety. In an embodiment, the amplicons comprise the masked binding moiety and the surface comprises a binding partner of the binding moiety. Upon activation of the binding moiety, the amplicons bind to the binding partner on the surface. In a non-limiting example, the masked binding moiety is caged biotin or caged fluorescein, while the corresponding binding partner is avidin or an anti-fluorescein antibody.

Abstract: A method for producing a monolayer of molecules on a surface comprises: loading a stamp with seed molecules; transferring seed molecules from the stamp to the surface; and, amplifying the seed molecules via an amplifying reaction to produce the monolayer. The method permit generation of complete monolayers from incomplete or sparse monolayers initially printed on the surface.

Abstract: The present invention relates to a method for exponential amplification of RNA using a primer independent RNA-dependent RNA polymerase (RdRp) wherein reactants are premixed cycle and then transferred into the reaction chamber in which the steps of polymerisation of the complementary strand and separation of the resulting double-stranded RNA occur. The invention also relates to a RNA reactor for carrying out the exponential RNA amplification.

Abstract: The present invention relates to a method for exponential amplification of RNA in vitro by using a thermostable RNA-dependent RNA polymerase (RdRp) of a sapovirus or norovirus.

Abstract: A method for preparing aRNA to be used for gene expression analysis from an RNA sample extracted from a tissue or cell(s) fixed with a fixative includes an amplification step of the RNA sample by reverse transcription and in vitro transcription, the ratio of aminoallyl uridine 5?-triphosphate (AA-UTP) in a nucleotide reagent used in the in vitro transcription is not less than 5 mol % and less than 25 mol % with respect to the total of uridine 5?-triphosphate (UTP) and AA-UTP.

Abstract: The present invention relates to a disposable device (100) for amplifying at least one target nucleic acid present in a liquid and biological sample of interest, which consists of a solid body (2), at least one fluid channel (3) connecting an inlet (4), via which all or part of the sample of interest can be drawn up and/or discharged, and an outlet (5), which is itself connected to a means for the drawing up/discharging of the said sample of interest, the fluid channel (3) further comprising from the inlet (4) to the outlet (5): a first compartment (8) containing all or part of the thermostable constituents, a means (15) for mixing the constituents with the sample of interest, a second compartment (9) containing all or part of the non-thermostable constituents, and in addition, at least one zone intended for heating the said sample of interest (6) mixed with the said amplification constituents in order to allow the amplification of the target nucleic acid.

Abstract: The present invention relates to a biomarker and a composition for diagnosis of preeclampsia. In accordance with one aspect of the present invention, there is provided a biomarker for diagnosis of preeclampsia using an enzyme selected from the group consisting of placental chondroitin 4-O-sulfotransferase 1 (C4ST), chondroitin 6-sulfotransferase (C6S), heparan sulfate 6-O-sulfotransferase 1 (HS6S), and dermatan/chondroitin sulfate 2-sulfotransferase (CS-2OST), or uronic acid-2-sulfate (UA2S).

Abstract: Disclosed is a single stranded primer-promoter-selector construct comprising (in 3? to 5? orientation) a primer subsequence annealing to the target, a T7 or other promoter subsequence (the template strand), and a selector subsequence. The primer can be extended by template mediated elongation, including reverse transcription, or ligation to another oligonucleotide. The promoter sequence is oriented to direct the in-vitro transcription (IVT) opposite to that of primer extension, where the selector subsequence serves as a template for IVT. The selector is associated with the target subsequence of interest and it, and the amplified product are unique subsequences, dissimilar to other sequence present in the sample. The construct's is useful for determination of the presence and relative abundance of designated subsequences in the sample, multiplex gene expression analysis, multiplex allele counting, determination of polymorphic/mutation site, and loss of heterozygosity.

Abstract: The invention provides methods for isothermal amplification of RNA. The methods are particularly suitable for amplifying a plurality of RNA species in a sample. The methods employ a composite primer, a second primer and strand displacement to generate multiple copies of DNA products comprising sequences complementary to an RNA sequence of interest. In another aspect, the methods employ a single primer (which is a composite primer) and strand displacement to generate multiple copies of DNA products comprising sequences complementary to an RNA sequence of interest. In some embodiments, a transcription step is included to generate multiple copies of sense RNA of an RNA sequence of interest. The methods are useful for preparation of nucleic acid libraries and substrates for analysis of gene expression of cells in biological samples. The invention also provides compositions and kits for practicing the amplification methods, as well as methods which use the amplification products.

Abstract: A method and apparatus converts host cells of a first type into cells of a second type when the host cells are placed in intimate contact with donor cells of the second type. Under predetermined conditions there is transport of a sufficient number of mRNA molecules from the donor cells into the host cells to reprogram the host cells into the second type. The host and donor cells may be subjected to while in intimate contact to a transporting force that enables the mRNA molecules of the donor cells to penetrate an outer membrane wall of host cells without damaging the membrane wall. The transporting force may include an electric field, a magnetic field, or a combined electric field and magnetic field.

Abstract: The present invention provides methods and compositions for amplifying and sorting adapter tagged nucleic acid fragments using selective primer extension. Immortalized pooled polynucleotide samples and method of producing the same are also provided.

Abstract: The present invention provides systems and methods for generating clonal root lines, clonal root cell lines, clonal plant cell lines, and clonal plants and for expressing gene products in such cell lines and plants. In some embodiments, a viral vector containing a polynucleotide of interest operably linked to a promoter is introduced into a plant or portion thereof to generate clonal root lines, clonal root cell lines, clonal plant cell lines, and clonal plants. According to certain inventive methods, a viral vector containing a polynucleotide of interest operably linked to a promoter is introduced into cells of a plant cell line that is maintained in culture to generate clonal plant cell lines and clonal plants. The invention provides clonal root lines, clonal root cell lines, clonal plant cell lines, and clonal plants generated using inventive methods.

Abstract: Methods and compositions for making and using pre-adenylated oligonucleotide sequences are provided.

Abstract: A method for amplifying oligonucleotide in vitro by polymerase-endonuclease chain reaction (PECR) which utilizes a single-stranded DNA probe containing repeat sequences, extends a target oligonucleotide by a thermostable DNA polymerase, cleaves extended products with a thermostable endonuclease, and amplifies target oligonucleotide by thermocycling. In PECR, a specific oligonucleotide is exponentially amplified using one single probe instead of a pair of primers, and the reaction is precisely controlled by thermal cycles whose parameters are flexibly adjustable according to length, sequence, melting temperature and initial amount of the target oligonucleotide. Amplification speed depends totally on initial amount of target oligonucleotide present in the reaction system. The method can be used to amplify specific small nucleic acids, such as oligonucleotides and microRNAs, and further conduct quantitative analysis.

Abstract: Materials and methods are provided for producing aptamer therapeutics having modified nucleotide triphosphates incorporated into their sequence.

Abstract: Compositions, methods and kits for detecting Group B streptococci. Particularly described are oligonucleotides that are useful as amplification primers and hybridization probes for detecting very low levels of Group B streptococci nucleic acids.

Abstract: Methods and kits are provided for performing multiple rounds of sense RNA synthesis. The sense RNA molecules can be used in various research and diagnostic applications, such as gene expression studies involving nucleic acid microarrays.

Abstract: The present invention relates to a multiwell plate for amplification and a lid with a foil for sealing the multiwell plate, wherein two positions of said lid on said plate exist, one position for storage, one position for sealing of the foil to the plate. The invention also relates to a method for.

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 presently claimed invention provides for novel methods and kits for reducing the complexity of a nucleic acid sample by providing non-gel based methods for amplification of a subset of the sequences in a sample. In a preferred embodiment, amplification of a subset can be accomplished by digesting a sample with two or more restriction enzymes and ligating adaptors to the fragments so that only a subset of the fragments can be amplified. The invention further provides for analysis of the above amplified sample by hybridization to an array, which may be specifically designed to interrogate the desired fragments for particular characteristics, such as, for example, the presence or absence of a polymorphism.

Abstract: Embodiments of the invention are directed to compositions and methods that use non-extendable oligonucleotides to enhance or improve synthesis or amplification of nucleic acids.

Abstract: Disclosed are compositions and methods useful for labeling and detection of analytes. The compositions generally are associations of three components: reporter binding agents, amplification target circles, and DNA polymerase. The compositions are assembled prior to their use in a rolling circle amplification reaction and can be stored and transported prior to use without substantial loss of activity. The reporter binding agents generally are composed of a specific binding molecule and a rolling circle replication primer. The specific binding molecule can be specific for a target molecule. The rolling circle replication primer has sequence complementary to the amplification target circle. The DNA polymerase can interact with the rolling circle replication primer and amplification target circle. For use as a general reagent, the specific binding molecule is not bound to the target molecule until the composition is used in an assay.

Abstract: The present teachings provide methods, compositions, and kits for reverse transcribing and amplifying small nucleic acids such as micro RNAs. High levels of multiplexing are provided by the use of a zip-coded stem-loop reverse transcription primer, along with a PCR-based pre-amplification reaction that comprises a zip-coded forward primer. Detector probes in downstream decoding PCRs can take advantage of the zip-code introduced by the stem-loop reverse transcription primer. In some embodiments, further amplification is achieved by cycling the reverse transcription reaction. The present teachings also provide compositions and kits useful for performing the reverse transcription and amplification reactions described herein.

Abstract: The present invention provides a method of predicting the risk of a patient for developing adverse drug reactions, particularly SJS or TEN. It was discovered that an HLA-B allele, HLA-B* 1502, is associated with SJS/TEN that is induced by a variety of drugs. The correlation with HLA-B* 1502 is most significant for carbamazepine-induced SJS/TEN, wherein all the patients tested have the HLA-B* 1502 allele. In addition, another HLA-B allele, HLA-B*5801, is particularly associated with SJS/TEN induced by allopurinol. Milder cutaneous reactions, such as maculopapular rash, erythema multiforme (EM), urticaria, and fixed drug eruption, are particularly associated with a third allele, HLA-B *4601. For any of the alleles, genetic markers (e.g., HLA markers, microsatellite, or single nucleotide polymorphism markers) located between DRB1 and HLA-A region of the specific HLA-B haplotype can also be used for the test.

Abstract: The present teachings provide methods, compositions, and kits for quantifying target, polynucleotides. In some embodiments, a reverse stem-loop ligation probe is ligated to the 3? end of a target polynucleotide, using a ligase that can ligate the 3? end of RNA to the 5? end of DNA using a DNA template, such as T4 DNA ligase. Following digestion to form an elongated target polynucleotide with a liberated end, a reverse transcription reaction can be performed, followed by a PCR. In some embodiments, the methods of the present teachings can discriminate between polymorphic polynucleotides that vary by as little as one nucleotide.

Abstract: Methods are disclosed for rapid, reliable and simple isolation of RNA, DNA and proteins from formalin-fixed paraffin-embedded tissue samples. RNA purified in this manner can be used to monitor gene expression levels. The tissue sample can be a tumor or other pathological tissue.

Abstract: Methods are disclosed for rapid, reliable and simple isolation of RNA from formalin-fixed paraffin-embedded tissue samples. RNA purified in this manner can be used for quantitative measurement gene expression levels. The tissue sample can be a tumor or other pathological tissue.

Abstract: Compositions and methods of use are disclosed for clonally amplifying target polynucleotide sequences in solution and attaching the amplicons to a surface by activation of a masked binding moiety. In an embodiment, the amplicons comprise the masked binding moiety and the surface comprises a binding partner of the binding moiety. Upon activation of the binding moiety, the amplicons bind to the binding partner on the surface. In a non-limiting example, the masked binding moiety is caged biotin or caged fluorescein, while the corresponding binding partner is avidin or an anti-fluorescein antibody.

Abstract: A method for prognostic or diagnostic assessment of a gastrointestinal-related disorder, such as ulcerative colitis, in a subject correlates the presence, absence, and/or magnitude of a gene in a sample with a reference standard to determine the presence and/or severity of the disorder, and/or the response to treatment for the disorder. The method enables identification of the effectiveness of candidate therapies.

Abstract: Provided is a simple and highly sensitive nucleic acid amplification method including hybridizing two types of oligonucleotide probes with a target gene and ligating the oligonucleotide probes with DNA ligase and amplifying the resultant single-stranded oligonucleotide in accordance with LAMP.

Abstract: The present invention is directed to a method for performing an RT-PCR for amplifying a target RNA including the steps of (i) cultivation of a population of adherent cells in a cell culture vessel (ii) lysis of the population of adherent cells which is supposed to contain the target RNA in the sample vessel with a lysis buffer comprising between 0.05 M and 1 M of a chaotropic agent (iii) adding reagents to the sample vessel which are necessary to perform a reverse transcription reaction such that the the chaotropic agent is present in a concentration of about 10 to 60 mM in the sample vessel, and reverse transcribing the target RNA and (iv) amplifying the first strand cDNA by means of subjecting the sample to multiple cycles of a thermocycling protocol.

Abstract: The present application discloses a diagnostic method and a kit for prognosis assessment of colorectal cancer (CRC) and a novel tumor suppressor gene to be used for diagnosis of colorectal cancer (CRC), the method comprising the steps of: (a) identifying recurrently altered regions (RAR) on a chromosome; and (b) detecting genomic alterations in the RAR. The present method makes it possible to perform early diagnosis as well as prognosis assessment for various cancers and tumors including colorectal cancer (CRC).

Abstract: Described herein are methods for increasing the annealing specificity of an amplification reaction using Iso-base Amplification Primers (“IAPs”). IAPs containing an iso-region are capable of regulating sequence-specific annealing thereby enhancing primer-template hybridization for sequence-specific amplification of nucleotides.

Abstract: This invention relates to a composition, kit, or DNA chip comprising polynucleotides and antibodies as probes for detecting, determining, or predicting the presence or metastasis of esophageal cancer, and to a method for detecting, determining, or predicting the presence or metastasis of esophageal cancer using the same.

Abstract: The present invention provides methods for the amplification of nucleic acid molecules. Methods for amplifying target polynucleotides, including mRNA, using oligonucleotides, DNA and RNA polymerases are provided. The invention further provides compositions and kits for practicing the methods, as well as methods which use the amplification products.

Abstract: A simple, rapid, inexpensive, and promising commercial biomarker assay method for multiple diseases is described herein. The present invention detects miRNA-based biomarkers in human stool specimens. The method of the present invention amplifies miRNA directly from stool specimens without any prior miRNA extraction. Differential expression of specific microRNAs in stool of colorectal cancer CRC and adenoma patients suggest fecal microRNAs as a novel potential biomarker for colorectal neoplasia detection. The method of the present invention has diagnostic, prognostic, and therapeutic relevance for gastroenterological cancers/colorectal cancer and as well as further acquired or hereditary GI diseases.

Abstract: Methods are disclosed for rapid, reliable and simple isolation of RNA from formalin-fixed paraffin-embedded tissue samples. RNA purified in this manner can be used to monitor gene expression levels. The tissue sample can be a tumor or other pathological tissue.

Abstract: The present invention provides methods for synthesis and therapeutic use of DNA and RNA oligonucleotides and analogs. RNA oligonucleotides are synthesized using a small, circular DNA template which lacks an RNA polymerase promoter sequence. The RNA synthesis is performed by combining a circular single-stranded oligonucleotide template with an effective RNA polymerase and at least two types of ribonucleotide triphosphate to form an RNA oligonucleotide multimer comprising multiple copies of the desired RNA oligonucleotide sequence. Preferably, the RNA oligonucleotide multimer is cleaved to produce RNA oligonucleotides having well-defined ends. Preferred RNA oligonucleotide multimers contain ribozymes capable of both cis (autolytic) and trans cleavage.

Abstract: The present invention relates to a method for enzymatically synthesizing chemically modified RNA by using RNA-dependent RNA polymerases (RdRp), especially RdRps from viruses of the Caliciviridae family. The method of the present invention is particularly useful for preparing RNA molecules of increased stability especially with respect to RNA degradation, for example for in vivo applications. Further subject matter of the present invention relates to a kit for carrying out the enzymatic synthesis of the chemically modified RNA.

Abstract: The present methods are exemplified by a process in which maternal blood containing fetal DNA is diluted to a nominal value of approximately 0.5 genome equivalent of DNA per reaction sample. Digital PCR is then be used to detect aneuploidy, such as the trisomy that causes Down Syndrome. Since aneuploidies do not present a mutational change in sequence, and are merely a change in the number of chromosomes, it has not been possible to detect them in a fetus without resorting to invasive techniques such as amniocentesis or chorionic villi sampling. Digital amplification allows the detection of aneuploidy using massively parallel amplification and detection methods, examining, e.g., 10,000 genome equivalents.