Abstract: Ligase detection reaction is utilized to distinguish minority template in the presence of an excess of normal template with a thermostable ligase. This process can be carried out with a mutant ligase, thermostable ligase, or a modified oligonucleotide probe. This procedure is particularly useful for the detection of cancer-associated mutations. It has the advantage of providing a quantitative measure of the amount or ratio of minority template.

Abstract: Methods, employing a nucleotide integrase, for cleaving single-stranded RNA substrates, single-stranded DNA substrates, and double-stranded DNA substrates at specific sites and for inserting a nucleic acid molecule into the cleaved substrate are provided. One method uses a nucleotide integrase to cleave one strand of a double-stranded DNA substrate. The method comprises the steps of: providing an isolated nucleotide integrase comprising a group II intron RNA having two hybridizing sequences for hybridizing with two intron RNA binding sequences on the top strand of the DNA substrate, and a group II-intron encoded protein which binds to a first sequence element of the substrate; and reacting the nucleotide integrase with the double-stranded DNA substrate to permit the nucleotide integrase to cleave the top strand of the DNA substrate and to insert the group II intron RNA into the cleavage site.

Abstract: This invention is directed to a plus strand RNA viral vector for transformation of a host organism with a foreign RNA, and expression of said foreign RNA. The foreign RNA is inserted into an infective RNA viral segment containing cis-acting viral replication elements, and allowed to infect the host organism. The RNA vector is modified to obtain infectivity by including an intervening sequence between the cap and the 5′ end. The modified RNA is able to tolerate the exogeneous RNA segment without disrupting the replication of the modified RNA, in the absence of a trans-acting viral replication element in a single component plant virus host cell.

Abstract: This invention is directed to a plus strand RNA viral vector for transformation of a host organism with a foreign RNA, and expression of said foreign RNA. The foreign RNA is inserted into an infective RNA viral segment containing cis-acting viral replication elements, and allowed to infect the host organism. The RNA vector is modified to obtain infectivity by not incorporating a cap at the 5′ end of the genome. The modified RNA is able to tolerate the exogenous RNA segment without disrupting the replication of the modified RNA, in the absence of a trans-acting viral replication element in a single component plant virus host cell.

Abstract: A method for sequencing a target DNA fragment in which along with amplification of the target DNA fragment, nucleic acid transcripts are generated using an RNA polymerase and the amplified target DNA fragments are used as templates in the presence of terminators for nucleic acid transcription reaction and the generated nucleic acid transcripts are analyzed, characterized in that the amplification of target DNA fragments and the generation of nucleic acid transcripts are carried out at a constant temperature is disclosed. The amplification of target DNA fragments and the generation of nucleic acid transcripts can be carried out around the room temperature. A DNA sequencing method using a novel method in which without using a thermo-resistant RNA polymerase, the amplification of target DNA fragments and generation of nucleic acid transcript can be carried out simultaneously in parallel is provided.

Abstract: The present invention is directed to products and methods for storing, separating or analyzing a sample of genetic material. The invention is particularly suited for the separation of a selected genetic material from a sample containing genetic material from multiple sources.

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: Methods for screening a patient for pancreatic disease are disclosed and are based upon detection of a mutation in the gene encoding insulin promoter factor-1 (IPF-1) which is linked to diabetes mellitus and pancreatic agenesis.

Abstract: A method for detecting gene expression in cells by reverse transcribing mRNA molecules into cDNA, cutting the cDNA with at least one restriction endonuclease, adding adaptor sequences to the cDNA fragments and selectively amplifying a subset of the cDNA by a polymerase chain reaction (PCR) to present a two-dimensional display of the DNA fragments or for cloning the DNA fragments into a vector is disclosed. In one embodiment, cDNA corresponding to the 3′ end of the mRNA is amplified and displayed or cloned, whereas in another embodiment, cDNA corresponding to the entire mRNA molecule is amplified and displayed or cloned.

Abstract: The invention relates to a method for enzyme stabilization. A method for improved reverse transcription at high temperatures is provided, wherein a thermostable heat shock protein (HSPs) stabilizes a reverse transcriptase, as well as reduces the RNase H activity of said reverse transcriptase. The present invention thus relates to a stabilizing agent, that prevents thermal denaturing and enhances thermostability of a reverse transcriptase. The invention further relates to a method of producing a polypeptide complex consisting of a Chaperonin and a Moloney murine leukemia virus (MMVL) reverse transcriptase, characterized by having enhanced thermostability as well as reduced RNase H activity, compared to a (MMVL) reverse transcriptase alone. The invention further relates to a kit for the preparation of cDNA from mRNA, comprising either both stabilizing agent and reverse transcriptase or the polypeptide complex of the invention.

Abstract: This invention is directed to methods for the quantitative measurement of specific gene expression levels in biological samples. In one embodiment, methods for the quantitative monitoring of gene expression without either co-amplification of an added template or use of an endogenous constitutive transcript are provided. The former involves a duplex amplification reaction in which a single set of primers is used to amplify both genomic DNA and expressed mRNA from the same gene sequence. These primers are targeted for sequences flanking the splice junction and intron sequences for the mRNA and DNA respectively. By their use, any suitable nucleic acid amplification technology yields mRNA and DNA amplimers which are distinguishable by length and sequence heterogeneity.

Abstract: This invention provides a method of detecting cancer metastatic cells in a subject, comprising: a) obtaining a nucleic acid sample from the subject's blood; b) amplifying nucleic acid encoding the product of prostate carcinoma tumor antigen gene-1; and c) detecting the presence of nucleic acid encoding the product of the prostate carcinoma tumor antigen gene-1, thereby detecting cancer metastatic cells in a subject. This invention also provides the above-described methods, wherein the method of amplification is PCR. This invention further provides the above-described methods, wherein the primers are 5′-AAGCTGACGCCTCATTTGCA-3′ SEQ ID NO: 1 and 5′-AACCACCAATGGAACTGGGT-3′ SEQ ID NO: 2. This invention also provides the above-described methods, wherein the primers are 5′-AATGGCTTCTGTGATACT-3′ SEQ ID NO: 3 and 5′-GGCTATAAGTGTTGCTGC-3′SEQ ID NO: 4.

Abstract: This invention relates to improved methods for sequencing and genotyping nucleic acid in a single molecule configuration. The method involves single molecule detection of fluorescent labeled PPi moieties released from NTPs as a polymerase extension product is created.

Abstract: The present invention is directed to a method for identifying and/or cloning within a biological sample alternatively spliced nucleic acid regions ocurring between two physiological conditions, comprising hybridizing RNA derived from a test condition with cDNA derived from the standard condition and further identifying and/or cloning nucleic acids corresponding to alternative forms of splicing.

Abstract: A method and apparatus for the manipulation of colloidal particles 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: Isolated nucleic acid molecules encoding Thyroid Receptor-Associated Proteins (TRAPS) are provided. TRAPS are members of protein complexes that bind to nuclear hormone receptors in a ligand-dependent manner so that the receptor, upon activation by a corresponding hormone, regulates the transcription of a particular gene. Also provided are methods of replicating and expressing such isolated nucleic acid molecules, pharmaceutical compositions comprising TRAPS, and methods of modulating gene expression via administration of therapeutically effective amounts of such pharmaceutical compositions.

Abstract: The present invention is directed to methods, compositions, kits and apparatus to identify and detect the presence or absence of target analytes. The embodiments of the present invention have utility in identification of protein and measurement of its levels in specimens and samples, as well as the design of test kits and apparatus for implementing such methods.

Abstract: Hepatocyte culturing system, primer sets and an analytical method for selectively detecting and quantitatively assessing the levels of mRNA expression of the major isoenzymes of cytochrome P450 (CYP450 1A1, 1A2, 2B1/2, 2C11, 2E1, 3A1, 3A2 and 4A1), fatty acyl-CoA oxidase (FACO) and select Phase II conjugating enzymes (UDPGT, GST and ST) in the rat using specific 5′ and 3′ oligonucleotide primers and reverse transcriptase-polymerase chain reaction. The method closely reproduces the expression obtained from rat liver tissue following treatment with the same enzyme inducers. Constitutive and inducible expression was maintained by resuspending, culturing and then overlaying adult rat hepatocytes with an extracellular matrix such as Matrigel®.

Abstract: For the genus-specific or/and species-specific detection of bacteria in a sample liquid, bacterial RNA is hybridized with a primer which is complementary to a genus-specific or species-specific region of the RNA of particular bacteria or to a highly conserved region of the RNA of bacteria in general, but which is not complementary at its 3′ end to the RNA of bacteria of another genus or species, the primer is elongated in the presence of a suitable polymerase and the four deoxyribonucleotides, if desired, with a concurrent or subsequent labelling of the elongation product and an elongation product formed is hybridized with a genus-specific or species-specific oligonucleotide after denaturation and the hybridization is detected by means of the oligonucleotide label.

Abstract: A high throughput virus in vitro infectivity assay method comprising growing cells in a multi-well format, infecting the cells with intact virion incubated with a test agent, and measuring expression of at least one viral nucleic acid sequence in the cells. The method also preferably comprises incubating intact virion without test agent to define a control. The cells are preferably human keratinocyte cells grown in monolayers. The viral nucleic acid sequence will generally comprise viral mRNA. In one preferred embodiment, the intact virion comprise Human Papilloma Virus, and more preferably Human Papilloma Virus-11. Measuring expression is generally carried out by releasing the viral mRNA from the cells by lysis, amplifying the mRNA as CDNA via RT-PCR, and detecting amplicons with specific probes. Cell lysis may be carried out by heating or by treatment with detergent.

Abstract: The present invention provides a fast, simple and specific method for generating a complete full-length cDNA library from single cells. The first reverse transcription of intracellular mRNAs with an oligo(dT)n-promoter primer introduces a recognition site for following transcription of newly reverse-transcribed cDNAs. The poly-nucleotide tailing of above cDNAs in addition to aforementioned promoter region further forms binding templates for specific PCR amplification. After repeating the reverse transcription, transcription, reverse transcription and PCR procedure, we can multiply a single copy of mRNA to two billion folds by calculation based upon the comparison between the amount of a synthesized cDNA library and that of theoretically presumed mRNAs within a cell (0.1 pg). In conjunction with a cell fixation and permeabilization step, the complete full-length cDNA library can be directly generated from few single cells without mRNA degradation.

Abstract: This invention provides methods for the isolation of complete representation of cellular messenger ribonucleic acid from wax-embedded tissue samples. Expression of genes of interest can thus be fortuitously determined and cDNA probes can be readily developed.

Abstract: The present invention concerns a method for evaluating the metastatic tendency of tumor cells by determining the level of expression (mRNA level) of the gene coding for the thrombin receptor (ThR) or by determining the level of the thrombin receptor present on the membranes or within the tumor cells. A high level of either of the above indicates a high metastatic tendency, a low level indicates a low metastatic tendency and an intermediate level indicates a moderate metastatic tendency. The present invention further concerns a kit for use in the above method.