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: The present invention generally relates to a devices, systems, and methods for amplifying nucleic acids in flowing droplets. In certain embodiments, the invention provides a device for amplifying nucleic acids including at least one channel through which sample droplets including nucleic acids flow, in which the nucleic acids in the droplets are optically detectable while the droplets are flowing through the channel, and a plurality of temperature zones in thermal contact with the channel, in which the zones are located at different locations along the channel and the zones are separated from each other.

Abstract: The present invention comprises novel methods, compositions and kits that use N4 vRNAP deletion mutants to detect and quantify analytes comprising one or multiple target nucleic acid sequences, including target sequences that differ by as little as one nucleotide or non-nucleic acid analytes, by detecting a target sequence tag that is joined to an analyte-binding substance. The method consists of an annealing process, a DNA ligation process, an optional DNA polymerase extension process, a transcription process, and, optionally, a detection process.

Abstract: Methods and kits are provided for diagnosing, monitoring, or predicting the conditions of pre-eclampsia, fetal chromosomal aneuploidy, and pre-term labor in a pregnant woman, as well as for detecting pregnancy in a woman, by quantitatively measuring in the maternal blood the amount of one or more mRNA species encoding human chorionic gonadotropin ? subunit (hCG-?), human placental lactogen (hPL), human corticotropin releasing hormone (hCRH), KiSS-1 metastasis-suppressor (KISS1), tissue factor pathway inhibitor 2 (TPFI2), placenta-specific 1 (PLAC1), or glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and comparing the amount of the mRNA species with a standard control.

Abstract: This invention relates generally to a method for quantifying the number of occurrences of a specific nucleic acid sequence within a nucleic acid sample in order to circumvent the shortcomings of the methods currently available and to provide reliable quantification of a specific nucleic acid sequence within a nucleic acid sample. The present invention provides a method of assessing an amount of a known target nucleic acid sequence in a sample comprising co-amplifying said target nucleic acid sequence and a known amount of a known control nucleic acid sequence to produce respective target and control amplicons, wherein said control nucleic acid sequence is different than said target nucleic acid sequence; and determining relative amounts of said respective amplicons by determining relative quantities of a primer extension reaction using each of said respective amplicons as a template.

Abstract: The present invention relates in part to methods for suppressing the innate immune response of a cell to transfection with an exogenous nucleic acid, to methods for increasing expression of a protein encoded by an exogenous nucleic acid by repeated delivery of the exogenous nucleic acid to a cell, and to methods of changing the phenotype of a cell by differentiating, transdifferentiating or dedifferentiating cells by repeatedly delivering one or more nucleic acids that encode defined proteins. A method is provided for extended transient transfection by repeated delivery of an in vitro-transcribed RNA (“ivT-RNA”) to a cell to achieve a high and sustained level of expression of a protein encoded by an ivT-RNA transcripts.

Abstract: A novel use of a template-dependent polymerase. The novel use is effected by employing the template-dependent polymerase for incorporating at least one oligonucleotide triphosphate onto a nascent oligonucleotide-3?-OH in a template-dependent manner.

Abstract: The invention relates to a method for amplification of a target RNA sequence, wherein the first primer comprises a hybridizing sequence of 7 to 14 nucleotides, which is capable of binding to a first segment of the target RNA sequence, a transcription enhancing sequence, and an anchor which is capable of binding to a second segment of the target RNA sequence, and/or wherein the second primer comprises a hybridizing sequence of 7 to 14 nucleotides, an amplification enhancing sequence and an anchor which is capable of binding to a second segment of the first single stranded cDNA. The invention further relates to primers for the amplification of target RNA sequences and to a kit comprising one or more of the primers.

Abstract: The present invention provides improved methods of attenuating gene expression through the phenomenon of RNA interference. The invention provides methods of synthesis of double stranded RNAs (dsRNAs) of increased potency for use as small interfering RNA (siRNA). Surprisingly and unexpectedly, siRNAs made by the methods of the invention are significantly more potent than previously available siRNAs.

Abstract: The present teachings provide methods, compositions, and kits for performing primer extension reactions on at least two target polynucleotides in the same reaction mixture. In some embodiments, a reverse transcription reaction is performed on a first target polynucleotide with a hot start primer comprising a self-complementary stem and a loop, and extension products form at high temperatures but extension products form less so at low temperatures since the self-complementary stem of the hot start primer prevents hybridization of the target specific region to the target. However, non-hot start primers with free target specific regions can hybridize to their corresponding targets at the low temperature and extension can happen at the low temperature.

Abstract: The present invention is directed to a method for performing an RT-PCR for amplifying a target RNA comprising the steps of a) lysis of a cellular sample which is supposed to contain the target RNA with a lysis buffer comprising between 0.2 M and 1 M guanidine thiocyanate, b) diluting the sample to an extend such that guanidine thiocyanate is present in a concentration of about 30 to 50 mM, c) reverse transcribing in the presence of a mixture of first strand cDNA synthesis primers, the mixture consisting of oligo dT primers and random primers, and d) subjecting the sample to multiple cycles of a thermocycling protocol and monitoring amplification of the first strand cDNA in real time.

Abstract: The invention relates generally to the fields of making biological unit lysates or admixtures of body fluids and of RNA analysis. More specifically, it relates to direct methods for the detection of a specific sequence of RNA in a biological unit, for example a virus, cell or tissue sample, or a body fluid, for example saliva, sputum, blood plasma, etc. More generally, the invention may be used to enzymatically manipulate and protect the RNA in lysate or bodily fluids for a number of applications.

Abstract: The present invention is generally related to compositions, kits and methods for labeling nucleic acid molecules using reverse transcriptases, preferably multi-subunit reverse transcriptases such as ASLV reverse transcriptases. Specifically, the invention relates to methods, kits and compositions for to fluorescently labeling nucleic acid molecules during nucleic acid synthesis. The labeled nucleic acid molecules produced in accordance with the invention are particularly suited as labeled probes for nucleic acid detection and diagnostics.

Abstract: The present teachings are generally directed to methods for normalizing at least one species of small nucleic acid that is present in a population of small nucleic acid species, wherein the relative concentration of at least one small nucleic acid species is substantially greater than the relative concentration of at least one other small nucleic acid species in the population. At least one small nucleic acid species is normalized using a multiplicity of primers comprising degenerate sequences. In some embodiments, a small nucleic acid species is identified by inserting at least part of an extension product from a normalized population into a vector and subsequently sequencing the insert. In some embodiments, a small nucleic acid species is identified by determining the sequence of at least part of an extension product.

Abstract: The present invention relates to HCV variants, particularly variants that are resistant to a protease inhibitors such as VX-950. Also provided are methods and compositions related to the HCV variants. Further provided are methods of isolating, identifying, and characterizing multiple viral variants from a patient.

Abstract: The present invention relates to a method for the diagnosis and/or the follow up of the evolution of cancer, which includes the analysis and quantification of over expressed and amplified genes in the plasma/serum of cancer patients or persons suspected to harbor cancer. This is achieved by analyzing together the amount of DNA and RNA of certain genes in the plasma/serum of cancer patients that are the reflection of a gene amplification and/or a gene over expression in comparison to healthy controls.

Abstract: The present invention provides a method of covalently joining a DNA strand to an RNA strand comprising (a) forming a topoisomerase-DNA intermediate by incubating a DNA cleavage substrate comprising a topoisomerase cleavage site with a topoisomerase specific for that site, wherein the topoisomerase-DNA intermediate has one or more 5? single-strand tails; and (b) adding to the topoisomerase-DNA intermediate an acceptor RNA strand complementary to the 5? single-strand tail under conditions permitting a ligation of the covalently bound DNA strand of the topoisomerase-DNA intermediate to the RNA acceptor strand and dissociation of the topoisomerase, thereby covalently joining the DNA strand to the RNA strand. The present invention also provides a method of tagging a 5? end of an RNA molecule. The present invention further provides a DNA-RNA molecule which has been joined in vitro by the use of a topoisomerase. The present invention also provides a method of tagging a 5? end of an mRNA.

Abstract: A method of diagnosing a disease that includes obtaining experimental data on gene selections. The gene selection functions to characterize a cancer when the expression of that gene selection is compared to the identical selection from a noncancerous cell or a different type of cancer cell. The invention also includes a method of targeting at least one product of a gene that includes administration of a therapeutic agent. The invention also includes the use of a gene selection for diagnosing a cancer.

Abstract: The present invention concerns preparation of DNA molecules, such as a library, using a stem-loop oligonucleotide. In particular embodiments, the invention employs a single reaction mixture and conditions. In particular, at least part of the inverted palindrome is removed during the preparation of the molecules to facilitate amplification of the molecules. Thus, in specific embodiments, the DNA molecules are suitable for amplification and are not hindered by the presence of the palindrome.

Abstract: The present invention relates to methods of treating viral disease using mutagenic nucleoside analogs. In particular, the invention provides isoguanosine nucleosides and derivatives thereof as well a method of increasing the mutation rate of a virus such as bovine viral diarrhea virus (BVDV) and hepatitis C virus (HCV).

Abstract: This invention provides expression vectors for a ribonucleic acid (RNA) molecule comprising a double-stranded region of random sequence, sets and libraries of same, methods of generating same, and methods for identifying an RNA therapeutic or RNA molecule that has an ability to affect a biological parameter, for identifying a drug target for a disease or disorder of interest, and for identifying a variant of an RNA molecule that has an altered ability to affect a biological parameter of interest.

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: 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 invention provides methods for amplification of polynucleotide sequences using primers containing single-stranded RNA. The methods employ use of an enzyme capable of cleaving single-stranded RNA, such as RNase I, to degrade a first RNA-containing primer prior to addition of a second RNA-containing primer. The invention also provides compositions and kits for practicing the amplification methods, as well as methods which use the amplification products.

Abstract: The present invention relates to a method for isothermal amplification of nucleic acids and a method for detecting nucleic acids, which comprises characterized in simultaneous isothermal amplification of nucleic acids and a signal probe to a method for isothermal amplification of target nucleic acids using an external primer set and RNA/DNA hybrid primer set, and a method for detecting amplification products by amplifying nucleic acids and a signal probe simultaneously using an external primer set, RNA-DNA hybrid primer set and DNA-RNA-DNA hybrid probe.

Abstract: This invention relates to a method for selecting and/or for qualitative and/or quantitative detection of predetermined biological cells from or in a sample containing biological cells, the sample being mixed with a predetermined combination of at least two antibodies and/or antibody derivatives, which bind preferentially with their binding sites to different epitopes of the cells to be selected or detected, and/or with at least one biospecific antibody and/or antibody derivative, which binds preferentially with its two binding sites to different epitopes of the cells to be selected or detected, which are separated from the sample with cells marked with at least one of the antibodies and/or antibody derivatives, and the separated cells being tested with a predetermined combination of at least two molecular-biological detection reagents, the at least two detection reagents reacting preferentially with at least one component of the cells to be selected or detected.

Abstract: The invention relates to methods and devices for analyzing single molecules, i.e. nucleic acids. Such single molecules may be derived from natural samples, such as cells, tissues, soil, air and water without separating or enriching individual components. In certain aspects of the invention, the methods and devices are useful in performing nucleic acid sequence analysis by probe hybridization.

Abstract: Methods for making cDNA molecules, for amplification of RNA by PCR and for preparation of cDNA libraries are provided. Kits for making cDNA molecules also are provided. Compositions are also provided comprising mixtures of reagents, including reverse transcriptases, buffers, cofactors and other components, suitable for immediate use in conversion of RNA into cDNA and RT PCR without dilution or addition of further components. These compositions are useful, alone or in the form of kits, for cDNA synthesis or nucleic acid amplification (e.g., by the Polymerase Chain Reaction) or for any procedure utilizing reverse transcriptases in a variety of research, medical, diagnostic, forensic and agricultural applications.

Abstract: This invention provides compositions and methods for HCV typing, e.g., genotyping and/or subtyping. The compositions and methods of the invention can be used to assign an HCV isolate to one of at least five HCV genotypes (for example, selected from genotypes 1, 2, 3, 4, 5 or 6), or assign an HCV isolate to one of at least six subtypes (for example, selected from subtypes 1a/b/c, 2a/c, 2b, 3a, 4a, 5a or 6a), where the methods of the invention use only a single typing probe to make the HCV type assignment.

Abstract: Overexpression of the gene, BAALC, in biological samples from a patient is prognostic for tumor aggressiveness and unfavorable patient outcome. The present invention provides polynucleotide primers and probes for assaying for overexpression of BAALC transcripts. Kits containing the primers and probes are also provided. Also provided are antibodies for assaying for overexpression of BAALC proteins as well as peptide immunogens for producing the anti-BAALC antibodies. The present invention also provides methods for characterizing acute myelogenous leukemia, chronic myelogenous leukemia and prostate cancer in a patient, base on detection of BAALC overexpression.

Abstract: In one aspect the present invention provides methods of synthesizing a preparation of nucleic acid molecules, the methods comprising the steps of: (a) utilizing an RNA template to enzymatically synthesize a first DNA molecule that is complementary to at least 50 contiguous bases of the RNA template; (b) utilizing the first DNA molecule as a template to enzymatically synthesize a second DNA molecule, thereby forming a double-stranded DNA molecule wherein the first DNA molecule is hybridized to the second DNA molecule; (c) utilizing the first or second DNA molecule of the double-stranded DNA molecule as a template to enzymatically synthesize a first RNA molecule that is complementary to either the first DNA molecule or to the second DNA molecule; and (d) utilizing the first RNA molecule as a template to enzymatically synthesize a third DNA molecule that is complementary to the first RNA molecule.

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: This invention relates to new polypeptides which exhibit kinase activity or, more specifically, which show phosphoinositide (PI) 3-kinase activity. Such polypeptides are involved in pathways responsible for cellular growth and differentiation. An isolated polypeptide which possesses PI3-kinase activity when produced by recombinant production in insect cells is disclosed.

Abstract: The invention relates to a method for amplification of a target RNA sequence, wherein the first primer comprises a hybridizing sequence of 7 to 14 nucleotides, which is capable of binding to a first segment of the target RNA sequence, a transcription enhancing sequence, and an anchor which is capable of binding to a second segment of the target RNA sequence, and/or wherein the second primer comprises a hybridizing sequence of 7 to 14 nucleotides, an amplification enhancing sequence and an anchor which is capable of binding to a second segment of the first single stranded cDNA. The invention further relates to primers for the amplification of target RNA sequences and to a kit comprising one or more of the primers.

Abstract: A method and system for amplifying non-coding RNA, microRNA, and small polynucleotide sequences through the generation of a pool of signature sequences to the target sequences. The target sequences can be amplified through DNA synthesis, RNA synthesis, or the combination of DNA and RNA synthesis. The amplification of signature sequences provides an efficient and reproducible means to determine the presence or absence of the target miRNAs, analyze the quantities of the said target miRNAs, and miRNA profiling. The present invention may also be used for screening for unknown non-coding RNAs, including novel miRNAs.

Abstract: Materials and methods for incorporating adenosine derivatives into the 5? end of transcribed RNA are disclosed. Adenosine derivatives include naturally occurring compounds such as Coenzyme A, NAD, and FAD, as well as various non-naturally occurring compounds. The derivatives can be used to impart desirable properties to the RNA such as fluorescence, the ability to bind to receptors or ligands, and improved catalytic activity. The transcribed RNAs can be used in a variety of applications including nucleic acid detection, designed or random generation of catalytic RNAs, antisense applications, and in the study of RNA structure and function.

Abstract: The present invention provides methods for detecting Helicobacter pylori (H. pylori) DNA and/or fragments thereof in blood. The first method involves extracting DNA from a blood sample, preferably plasma, by amplifying the DNA using a polymerase chain reaction (PCR) or a ligase chain reaction (LCR) method, and detecting a target DNA sequence in the amplified DNA. The preferred target DNA sequence comprises a Mr26 or a 16S rRNA gene or fragments thereof specific to H. pylori. The second method involves extracting DNA from a blood sample, preferably serum, by hybridizing the extracted DNA with a radioisotope or fluorescence labeled H. pylori DNA probe.

Abstract: The invention relates to a new method for sequence-specific identification, separation and quantitative measurement of nucleic acid fragments. The invention is based on the use of restriction endonucleases that have degenerate bases in their recognition or cleavage sequence. The method has broad applications, including DNA fingerprinting, differential display of mRNA, mutation and polymorphism identification, diagnosis and drug screening.

Abstract: The present invention relates to methods and products for the evaluation of HIV treatment. The methods are based on evaluating molecular events at the HIV integrase resulting in altered therapeutic efficacy of the investigated compounds. The methods rely on providing an integrase gene and evaluating either through genotyping or phenotyping the integrase gene. The present invention relates to the fields of diagnostics, drug screening, pharmacogenetics and drug development.

Abstract: The present invention describes an in situ reverse transcriptase PCR method in which the background fluorescence is greatly reduced as compared to traditional in situ PCR.

Abstract: The present invention relates to improved reverse transcription methods using a modified thermostable DNA polymerases, particularly in a magnesium ion buffer. These methods are particularly useful in combined reverse-transcription/amplification reactions.

Abstract: The present invention relates to universal-tagged oligonucleotide primers, and to methods of using the primers for amplifying the genome.

Abstract: The invention concerns a method for the diagnosis and/or the follow up of the evolution of cancer comprising the analysis of the RNA components of the telomerase enzyme present in the plasma or serum of the blood.

Abstract: The present invention relates to a method for predicting whether or not transplant recipients are likely to reject tissue allografts. It is based, at least in part, on the discovery that, based on analysis of three immunologic factors, cardiac transplant recipients could be classified into risk categories for progression to high-grade rejection. The present invention, by enabling a determination of the risk for high-risk rejection in a transplant patient, reduces unnecessary diagnostic and therapeutic procedures in low risk patients and clinical intervention in patients who would most benefit.

Abstract: The present invention relates to prognostic methods which are useful in medicine, particularly cancer chemotherapy. The object of the invention to provide a method for assessing ERCC1 expression levels in fixed or fixed and paraffin embedded tissues and determine a platinum-based chemotherapy by examination of the amount of ERCC1 mRNA in a patient's tumor cells and comparing it to a predetermined threshold expression level. More specifically, the invention provides to oligonucleotide primer pair ERCC1 and methods comprising their use for detecting levels of ERCC1 mRNA.

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: The present invention relates to oligonucleotides for use in amplifying and detecting HIV nucleic acid in a sample.

Abstract: This invention provides for a novel amplification procedure for nucleic acid. The method uses a wild type or mutant RNA polymerase designed to transcribe both deoxyribonucleotides and ribonucleotides.

Abstract: The invention provides a nucleotide sequence comprising at least a portion of the nucleotide sequence of FIG. 10A, FIG. 6B or FIG. 10A or FIG. 10B; nucleotides which hybridise to the nucleotide sequences of FIG. 6A, FIG. 6B or FIG. 10A or FIG. 10B; nucleotides which are degenerate to the nucleotide sequences of FIG. 6A, FIG. 6B or FIG. 10A or FIG. 10B; all of which nucleotides encode a polypeptide having transglutaminase activity.

Abstract: An oligonucleotide useful for detection of an RNA derived from HIV-1 consisting of at least 10 consecutive bases in any of SEQ ID NOS:1, 2, 4 to 10 and 13 to 17, which can bind to a specific site of the RNA.