Abstract: This invention relates to a method for producing a zinc finger nucleic acid binding protein comprising preparing a zinc finger protein according design rules, varying the protein at one or more positions, and selecting variants which bind to a target nucleic acid sequence by polysome display.

Abstract: The present invention provides a method for the in vitro selection of signaling aptamers comprising the steps of synthesizing a DNA pool, the DNA having a random insert of nucleotides of a specific skewed mole ratio; amplifying the DNA pool; transcribing an RNA pool from the amplified DNA using a fluorescently labeled nucleotide; applying the fluorescently labeled RNA pool to an affinity column to remove the high-affinity fluorescent RNA molecules from the fluorescently labeled RNA pool; obtaining a cDNA pool from the high-affinity fluorescent RNA molecules; repeating the amplification and selection steps on the fluorescent RNA molecules and cloning the fluorescent RNA molecules to yield signaling aptamers. Signaling aptamers comprising DNA molecules are also selected for. Also provided is a signaling aptamer that transduces the conformational change upon binding a ligand to a change in fluorescence intensity of the signaling aptamer.

Abstract: A medium is disclosed for embedding and preserving single cells or a cell tissue for an extended period of time at a temperature not exceeding 0° C. in a state suitable for DNA and/or RNA amplification, comprising an aqueous solution of at least one water-soluble cellulose derivative and, optionally, an osmotic pressure stabilizing agent. A method for preserving single cells or a cell tissue for an extended period of time at a temperature not exceeding 0° C. in a state suitable for DNA and/or RNA amplification using the above medium is also disclosed.

Abstract: The invention relates to methods for the qualitative and quantitative determination of differentially expressed mRNA molecules. Said methods are used especially to determine all possible mRNA molecules present in a cell or a tissue, and to compare them with other cells or tissues, with other conditions (stages of disease or development), or with stages of treatment for these conditions. The method provided for in the invention therefore makes it possible, for example, to establish a comprehensive map of the different mRNA molecules present in a defined mRNA population and subsequently to use the preferably digital information obtained in this way in data base analyses.

Abstract: Embodiments of the present invention are directed to the detection of fetal or maternal RNA in a blood sample from a pregnant subject, and may involve subjecting the sample to a test for fetal or maternal analysis indicative of a fetal or maternal condition or characteristics. For instance, the RNA analysis may involve the assessment of the gene expression pattern of an unborn fetus by analyzing a blood sample from the mother. The prenatal monitoring technology allows, for the first time, the detection of genes which are expressed by the fetus, just by analysis of a sample of maternal blood. In specific embodiments, the prenatal monitoring technology is based on the discovery of circulating RNA of fetal origin in the plasma of pregnant women. In general, the detection method performed on a maternal serum or plasma sample from a pregnant female comprises detecting the presence of RNA of fetal or maternal original in the sample.

Abstract: The present invention provides a method for covalently joining a DNA strand to an RNA strand using a topoisomerase enzyme. This invention also provides a method for obtaining a cDNA corresponding to a gene using a topoisomerase enzyme.

Abstract: The present invention relates to a process for the preparation of full-length complementary DNA (cDNA).

Abstract: The invention relates to a novel method for analyzing the composition of an mRNA sample and analyzing differential gene expression using two differently labeled primers, and to the use of the method. In the method for analyzing an RNA sample, a) a first primer, which is, where appropriate, labeled with a first dye, is used to prepare the first strand of a complementary DNA sample or cDNA sample from an RNA sample, b) a second primer, which is preferably labeled with a second dye, is used to prepare the second strand of this cDNA sample, c) the first primer, which is labeled with a first dye, and the second primer, which is labeled with a second dye, are used to amplify the cDNA sample, and d) the composition of the amplified, labeled cDNA sample is analyzed.

Abstract: Disclosed are compositions and a method for amplification of nucleic acid sequences of interest. The method is based on stand displacement replication of the nucleic acid sequences of interest by multiple primers. In one preferred form of the method, referred to as multiple strand displacement amplification, two sets of primers are used, a right set and a left set. The primers in the right set are complementary to one strand of the nucleic acid molecule to be amplified and the primers in the left set are complementary to the opposite strand. The 5′ end of primers in both sets are distal to the nucleic acid sequence of interest when the primers have hybridized to the nucleic acid sequence molecule to be amplified. Amplification proceeds by replication initiated at each primer and continuing through the nucleic acid sequence of interest. A key feature of this method is the displacement of intervening primers during replication by the polymerase.

Abstract: The present invention relates to a method for the determination of the presence and amount of DNA in a sample. The method is based on the use of a nucleic acid template dependent enzyme in combination with a random primer to generate an enzymatic product which incorporates a binding species and a detectable species covalently linked.

Abstract: The presently claimed invention provides methods and kits for amplifying a target sequence from within a nucleic acid population. The presently claimed invention provides selection probes which are complementary to at least a portion of said target sequence and mechanisms for adding a probe sequence to the 3′ end of a target sequence that is hybridized to a selection probe. The added 3′ probe sequence and a probe sequence added at the 5′ end of the target by adaptor ligation allow for selective amplification of the target sequence.

Abstract: The disclosed nucleic acid primer sets, used in combination with quantitative amplification (PCR) of tissue cDNA, can indicate the presence of specific proteases in a tissue sample. The detected proteases are themselves specifically overexpressed in certain cancers, and their presence may serve for early detection of associated ovarian and other malignancies, and for the design of interactive therapies for cancer treatment. More specifically, the present invention relates to the uses of stratum corneum chymotrytic enzyme as a marker for ovarian tumor cells.

Abstract: Methods of manipulation of nucleic acid, in particular amplification by means of the polymerase chain reaction (PCR), including use of oligonucleotides and combinations and kits comprising such oligonucleotides, also methods comprising use of nested PCR, allowing for improved results in methods wherein large numbers of nucleic acid fragments are manipulated by means of PCR and electrophoresis. Oligonucleotides are provided for use a size standards in electrophoresis, and internal controls allowing for calculation of relative amounts of material present. Improved results can be achieved in methods of profiling mRNA transcribed in a system under investigation.

Abstract: A method for generating and amplifying closed circular DNA having a specific sequence in vitro in a cell-free system is disclosed. Prior to the invention of this method, closed circular DNA could only be amplified in vivo in appropriate host cells. The essence of the method is the inclusion of a thermostable DNA ligase in a PCR reaction. This procedure is referred to as ligation-during-amplification (LDA), in which the fully extended DNA strands are ligated by the DNA ligase and used as templates for subsequent amplification. Closed circular DNA having a specific sequence can be selectively amplified exponentially by the use of two sequence-specific primers in the LDA reaction. In addition, one or more site-specific mutations can be introduced into a closed circular DNA by the use of one or more mutagenic primers in the LDA reaction. Various thermostable DNA polymerases and thermostable ligases can be used for LDA amplification.

Abstract: A process for generating multiple linear complements of a single strand, circular nucleic acid template containing at least one cleavage site is described. The process consists of combining the single strand, circular nucleic acid template with polynucleotide primers under conditions sufficient for hybridization; extending the polynucleotide primer more than once around the circle to generate a complementary displacement of more than one continguous complement of the single strand, circular nucleic acid template. Also described is a process of synthesizing novel single strand, circular nucleic acids between 30 an 2200 nucleotides. The process consist of synthesizing a linear polynucleotide; combining the linear polynucleotide with a complementary linking oligonucleotide under conditions sufficient for hybridization; and ligating the linear polynucleotide pto produce a single strand, circular nucleic acid.

Abstract: This invention relates to methods of detecting extracellular hTR RNA and hTERT RNA in blood, plasma, serum, and other bodily fluids. The invention thereby provides an aid for the detection, diagnosis, monitoring, treatment, or evaluation of neoplastic disease.

Abstract: The present invention pertains to methods, reagents, compositions, kits, and instruments for use in simultaneously amplifying multiple targets. In particular, this invention is based on the discovery of a two-step multiplex amplification reaction wherein the first step 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.

Abstract: The proportional amplification of nucleic acids can increase the amount of nucleic acids while preserving the relative abundance of the individual nucleic acid species, or portions thereof, in the original sample. A proportionally amplified nucleic acid preparation may be analyzed in a gene expression monitoring system, preferably involving a nucleic acid probe array.

Abstract: An oligonucleotide for detection or amplification of a gene selected from the group consisting of Vibrio parahaemolyticus thermostable direct hemolysin-related hemolysin genes (trh1 and trh2) and Vibrio parahaemolyticus thermostable direct hemolysin gene (tdh2) or RNA derived therefrom is provided. Further, method for detecting trh1, trh2 or tdh2 using said oligonucleotide is provided.

Abstract: cDNA including the 5′-terminal sequence of full-length mRNA with a cap structure is synthesized from a mRNA sample containing the full-length mRNA with the cap structure and non-full-length mRNA without any cap structure in mixture. At the first step, the phosphate group at 5′-terminus of the non-full-length mRNA in the mRNA sample is removed. At the second step, the cap structure at the 5′-terminus of the full-length mRNA in the mRNA sample is removed. At the third step, an oligoribonucleotide is ligated to the phosphate group at 5′-terminus of mRNA generated through the first and second steps. At the fourth step, mRNA with the oligoribonucleotide ligated at the 5′-terminus thereof at the third step is subjected to a reverse transcriptase process using a short-chain oligonucleotide capable of being annealed to an intermediate sequence within the mRNA as primer, to synthesize a first-strand cDNA.

Abstract: This invention provides methods for constructing a normalized cDNA library, constructing a low copy gene library, preparing a probe from a biological sample and a kit for constructing a normalized cDNA library.

Abstract: A method for assaying a target nucleic acid, comprising: providing an RNA amplification system comprising producing a double-stranded DNA using, as a template, a target RNA containing a specific nucleotide sequence in a sample, said double-stranded DNA having a promoter sequence and being capable of transcribing an RNA comprising the specific nucleotide sequence or a sequence complementary to the specific nucleotide sequence, producing an RNA transcription product comprising the specific nucleotide sequence or a sequence complementary to the specific nucleotide sequence in the presence of an RNA polymerase, and producing the double-stranded DNA using the RNA transcription product as a template, in the presence of a probe labeled with an intercalating fluorochrome having a sequence complementary to the RNA transcription product; measuring the fluorescence intensity in the RNA amplification system with time; calculating a time when the fluorescence intensity satisfies a prescribed criterion based on the measure

Abstract: The present invention provides new methods of synthesizing cDNAs, methods of verifying full-length cDNAs, methods of producing cDNA libraries enriched for full-length inserts, and the like.

Abstract: The invention relates to a substantially pure thermostable DNA polymerase from Thermotoga (Tne and Tma) and mutants thereof. The Tne DNA polymerase has a molecular weight of about 100 kilodaltons and is more thermostable than Taq DNA polymerase. The mutant DNA polymerase has at least one mutation selected from the group consisting of (1) a first mutation that substantially reduces or eliminates 3′→5′ exonuclease activity of said DNA polymerase; (2) a second mutation that substantially reduces or eliminates 5′→3′ exonuclease activity of said DNA polymerase; (3) a third mutation in the O helix of said DNA polymerase resulting in said DNA polymerase becoming non-discriminating against dideoxynucleotides. The present invention also relates to the cloning and expression of the wild type or mutant DNA polymerases in E. coli, to DNA molecules containing the cloned gene, and to host cells which express said genes.

Abstract: Methods and compositions for synthesizing cDNA in vivo are disclosed, wherein a synthetic polynucleotide molecule which anneals in vivo to an RNA template molecule is utilized as a primer for reverse transcriptase in vivo.

Abstract: A kit is provided for preferentially amplifying target RNA in a sample of tester RNA relative to non-target RNA in the sample, the kit driver sequences complementary to the non-target tester RNA under conditions where the driver sequences hybridize to the non-target RNA, a nucleic acid primer capable of hybridizing to the target RNA under conditions suitable for extension of the nucleic acid primer; and a promoter template capable of hybridizing to a DNA that is complementary to the target RNA under conditions suitable for extension of the complementary DNA such that a functional double promoter is formed.

Abstract: The even length proportional amplification of nucleic acids can increase the amount of nucleic acids while preserving the relative abundance of the individual nucleic acid species, or portions thereof, in the original sample. An even length proportionally amplified nucleic acid preparation may be analyzed in a gene expression monitoring system, preferably involving a nucleic acid probe array.

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: Recombinant retroviruses carrying a vector construct capable of preventing, inhibiting, stabilizing or reversing infectious, cancerous or auto-immune diseases are disclosed. More specifically, the recombinant retroviruses of the present invention are useful for (a) stimulating a specific immune response to an antigen or a pathogenic antigen; (b) inhibiting a function of a pathogenic agent, such as a virus; and (c) inhibiting the interaction of an agent with a host cell receptor. In addition, eucaryotic cells infected with, and pharmaceutical compositions containing such a recombinant retrovirus are disclosed. Various methods for producing recombinant retroviruses having unique characteristics, and methods for producing transgenic packaging animals or insects are also disclosed.

Abstract: A process is provided for labeling with signal amplification a ribonucleic acid (RNA), comprising fragmenting the RNA to form RNA fragments, fixing a first ligand to a terminal phosphate located at least one of the 3′ end and the 5′ end of each of a plurality of the RNA fragments, the terminal phosphate having been released during the fragmentation, and binding a plurality of labeling agents to the first ligand on each of a plurality of the fragments.

Abstract: The present invention describes the identification, isolation and cloning of two human presenilin genes, PS-1 and PS-2, mutations in which lead to Familial Alzheimer's Disease. Also identified are presenilin homologue genes in mice, C. elegans and D. melanogaster. Transcripts and products of these genes are useful in detecting and diagnosing Alzheimer's disease, developing therapeutics for treatment of Alzheimer's disease, as well as the isolation and manufacture of the protein and the constructions of transgenic animals expressing the mutant genes.

Abstract: An object of the present invention is to establish a method for amplifying an RNA in a biologically-derived sample by preparing a reaction solution in which a nucleic acid synthesis is not inhibited even in the presence of various biologically-derived impurities and as well as to enable a convenient, rapid and highly sensitive analysis of the RNA in the sample. In a method of the present invention, a reaction solution having a pH higher than that of an ordinarily employed reaction solution and/or containing a polyamine and/or a sulfated polysaccharide.

Abstract: A process for detecting live microbiological contaminants in a food product sample, according to which the presence of mRNA coding for the synthesis of a protein involved in the synthesis of proteins by the contaminants is detected in the sample.

Abstract: A method and apparatus for the manipulation of colloidal particulates and biomolecules at the interface between an insulating electrode such as silicon oxide and an electrolyte solution. Light-controlled electrokinetic assembly of particles near surfaces relies on the combination of three functional elements: the AC electric field-induced assembly of planar aggregates; the patterning of the electrolyte/silicon oxide/silicon interface to exert spatial control over the assembly process; and the real-time control of the assembly process via external illumination. The present invention provides a set of fundamental operations enabling interactive control over the creation and placement of planar arrays of several types of particles and biomolecules and the manipulation of array shape and size. The present invention enables sample preparation and handling for diagnostic assays and biochemical analysis in an array format, and the functional integration of these operations.

Abstract: The method provided by the present invention sets forth a novel combination of methods and principles which allows for the rapid and accurate isolation and identification of a large number of differentially expressed mRNAs.

Abstract: The present invention relates to a variant form of the receptor for the obese gene product. In particular, the invention relates to methods of detecting this receptor variant in cells and tissues of obese individuals. In addition, it relates to methods of inhibiting or down-regulating expression of this variant in cells to augment their responsiveness to weight regulation by leptin as well as methods of using compounds to directly activate signal transduction pathways associated with this ligand-receptor system.

Abstract: A method for selectively detecting the presence of C. parvum organisms in a sample. A method for selectively detecting the presence of C. parvum organisms and for detecting the presence of G. lamblia organisms, simultaneously, in a sample. A method for selectively detecting viable C. parvum organisms in a sample potentially containing viable C. parvum organisms. A method for selectively detecting viable C. parvum organisms and for detecting viable G. lamblia organisms, simultaneously. A method for selectively detecting infectious C. parvum organisms in a sample, and in another embodiment, additionally comprising detecting viable G. lamblia organisms in the sample, simultaneously. Kit for use in performing these methods.

Abstract: The present invention concerns a process for the amplification of nucleic acids, wherein the tailing of the nucleic acid to be amplified is effected by extending the nucleic acid with ribonucleotides at its 3′ end with the aid of terminal transferase. Further, a kit for the amplification of a nucleic acid, which includes at least one ribonucleotide and terminal transferase, is provided.

Abstract: The invention provides methods and compositions for amplifying RNA sequences by (a) hybridizing to a target RNA a first primer comprising a 3′ target RNA hybridizing sequence and a first 5′ defined amplifyable sequence; (b) extending the first primer with a reverse transcriptase to form a first cDNA strand; (c) hybridizing to the first cDNA strand a second primer comprising a 3′ random cDNA hybridizing sequence and a second 5′ defined amplifyable sequence; (d) extending the second primer with a DNA polymerase to form a second cDNA strand; and (e) amplifying the second cDNA strand with a third primer comprising the first 5′ defined amplifyable sequence.

Abstract: The present invention concerns new compositions and methods for the detection of pathological events. It more specifically concerns methods for the detection in vitro of the presence of a pathology or a pathological event in a subject, comprising taking a sample of blood cells from the subject and determining, in this sample, the presence of blood cells presenting a physiological state characteristic of the pathology. The invention also concerns the tools, kits and compositions for the implementation of such methods, as well as their uses in the field of human and animal health, or in experimental research for example.

Abstract: A method of detecting binding interactions and target molecules, such as proteins, protein fragments, recombinant proteins, recombinant protein fragments, extracellular matrix proteins, ligands, carbohydrates, steroids, hormones, drugs, drug candidates, immunoglobulins and receptors of eukaryotic, prokaryotic or viral origin, by mediated electrochemistry using labels that react with transition metal mediator complexes in a detectable catalytic redox reaction. These labels are attached directly to binders, target molecules, surrogate target molecules, or to affinity ligands capable of binding to the target or to surrogate target molecules capable of competing with the target for binding to another binder. The labels can be naturally present (endogenous) in the binder, target or affinity ligand, or constructed by the covalent attachment of the label to the binder, target, affinity ligand or surrogate target (exogenous).

Abstract: Disclosed is a method for producing double-stranded DNA comprising treating double-stranded DNA having a homopolymer part or parts at one or both ends with a restriction enzyme to partly or fully eliminate at least one of the homopolymer part or parts. The restriction enzyme is capable of cleaving double-stranded DNA at a cleavage site separate from a recognition site therefor. Disclosed is a method for determining a nucleotide sequence of double-stranded DNA utilizing one or both strands of the double-stranded DNA as a template, wherein the double-stranded DNA used as the template is double-stranded DNA prepared by the above method of the present invention.

Abstract: The invention relates to a novel method for analyzing the composition of an mRNA sample and analyzing differential gene expression using two differently labeled primers, and to the use of the method. In the method for analyzing an RNA sample, a) a first primer, which is, where appropriate, labeled with a first dye, is used to prepare the first strand of a complementary DNA sample or cDNA sample from an RNA sample, b) a second primer, which is preferably labeled with a second dye, is used to prepare the second strand of this cDNA sample, c) the first primer, which is labeled with a first dye, and the second primer, which is labeled with a second dye, are used to amplify the cDNA sample, and d) the composition of the amplified, labeled cDNA sample is analyzed.

Abstract: The invention concerns stable aqueous solutions containing one or several nucleoside triphosphates wherein the respective solution has a pH value of more than 7.5 and contains no additional substances with a stabilizing effect. The nucleoside triphosphate solutions are used in particular for DNA synthesizing reactions such as e.g. RT-PCR, cycle sequencing, random priming and nick translation. One of the most important applications of such solutions containing deoxynucleoside triphosphates (d-NTP) is their use in the polymerase chain reaction (PCR).

Abstract: This invention relates to the use of tumor-derived or associated extracellular ribonucleic acid (RNA) found circulating in the plasma or serum fraction of blood for the detection, monitoring, or evaluation of cancer or premalignant conditions. Extracellular RNA may circulate as non-bound RNA, protein-bound RNA, lipid-RNA complexes, lipoprotein (proteolipid)—RNA complexes, protein-RNA complexes including within or in association with ribonucleoprotein complexes, nucleosomes, or within apoptotic bodies. Any intracellular RNA found in plasma or serum can additionally be detected by this invention. 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.

Abstract: Methods and compositions are provided for producing full-length cDNA libraries. In the subject methods, full length first strand cDNAs are isolated using a fusion protein of an eIF-4E domain and an eIF-4G domain separated by a flexible linker. Also provided is the novel fusion protein employed in the subject methods, as well as nucleic acids encoding, and host cells capable of expressing, the same. Finally, kits for use in practicing the subject methods are provided. The subject invention finds use in a variety of applications in which full-length CDNA libraries are employed.

Abstract: The present invention concerns a method for generating multiple double stranded nucleic acids by (a) elongating a primer molecule comprising a nucleobase sequence B′ by using one or more nucleotide(s) and a target nucleic acid T that acts as a template for the elongation of said primer such that the elongation product E formed is capable of acting as a template for the elongation of a further primer molecule containing the nucleobase sequence B′; (b) separating the target nucleic acid T from said elongation product E; (c) using said elongation product E as a template for the elongation of a further primer molecule yielding an elongation product E′; and (d) repeating the steps of elongating primer molecules and of separating said elongation products a sufficient number of times to achieve the desired amount of double stranded nucleic acid.

Abstract: A method for detecting reverse transcriptase (RT), utilizing RT-catalyzed generation of cDNA complementary to an RNA template and detecting cDNA in a DNA hybridization reaction, is provided. Also provided is a method for detecting and/or quantitating drug resistance of reverse transcriptase in a sample and a method for evaluating ability of a compound to inhibit reverse transcriptase activity.

Abstract: This invention relates to a methodology for assessing the sensitivity of an HIV-1 sample to zidovudine and to diagnostic assays for use in such assessment.

Abstract: The present invention relates to methods of monitoring, via polymerase chain reaction, the clinical progression of human immunodeficiency virus infection and its response to antiretroviral therapy. According to the invention, polymerase chain reaction assays may be used to predict immunological decline and to identify, at an early stage, patients whose infection has become resistant to a particular antiretroviral drug regimen.