Abstract: Methods are provided for the replication and amplification of RNA sequences by thermoactive DNA polymerases. In a preferred embodiment, high temperature reverse transcription is coupled to nucleic acid amplification in a one tube, one enzyme procedure using a thermostable DNA polymerase. Methods for eliminating carry over contamination of amplifications due to prior reverse transcription reactions are also provided. Reagents particularly suited for the methods of the present invention are provided.

Abstract: The present invention provides primers for the polymerase chain reaction amplification of a nucleic acid sequence encompassing the polymorphic regions of the second, third, and fourth exons of the HLA Class I B gene (HLA-B). The polymorphic regions of the second, third, and fourth exons of the HLA-B gene contain sufficient variability so that each allele is uniquely identified by the nucleic acid sequence within these regions. The primers and amplification methods of the present invention enable genotyping at the HLA-B locus using samples from a variety of sources and for detecting allelic variants not distinguishable by serological methods. The HLA-B DNA genotyping can be used for tissue typing, determining individual identity, and identifying disease susceptible individuals.

Abstract: Methods are provided for detecting carcinoma metastases in selected body tissues and fluids. These methods offer greater than 1,000-fold enhanced sensitivity compared to prior standard diagnostic methods. In one embodiment of the invention, target carcinoma associated nucleic acid sequences are identified for detecting minimal residual disease in lung carcinomas. The methods utilize nucleic acid amplification techniques, preferably, the polymerase chain reaction.

Abstract: A process for determining the genotype of an individual with respect to the alleles at the HLA DP locus involves obtaining a sample of nucleic acid from the individual, and hybridizing the nucleic acids with a panel of probes specific for variant segments of DPalpha and DPbeta genes. Because the variation between DPbeta alleles is highly dispersed throughout the second exon of the DPbeta gene, the discovery of many different DPbeta alleles makes the process far more discriminating and informative than cellular, RFLP, or serological methods. The process can also be carried out on amplified nucleic acid produced by the polymerase chain reaction using primers specific for the second exon of the DPalpha and DPbeta genes. HLA DP DNA typing methods are useful in the prevention of graft rejection and host versus graft disease, in determining susceptibility to autoimmune diseases, in providing evidence concerning the derivation from an individual of forensic samples, and in paternity testing.

Abstract: Improvements to the in situ polymerase chain reaction (PCR), a process of in vitro enzymatic amplification of specific nucleic acid sequences within the cells where they originate, can be achieved by changing the way that the enzymatic reaction is started. Reaction initiation is delayed until the start of PCR thermal cycling, either by withholding a subset of PCR reagents from the cellular preparation until the preparation has been heated to 50.degree. C. to 80.degree. C., immediately before thermal cycling is begun, or by adding to the PCR reagents a single-stranded DNA binding protein which blocks reaction at temperatures below about 50.degree. C. If the in situ PCR is performed on cellular preparations already attached to a microscope slide, thermal cycling also is facilitated by use of a thermal cycler sample block or compartment designed optimally to hold the microscope slide and any vapor barrier covering the slide.

Abstract: The presence of human papillomavirus (HPV) in a sample can be detected and the HPV typed by a method that involves the amplification of HPV DNA sequences by the polymerase chain reaction (PCR). The primers used in the method are consensus primers that can be used to amplify a particular region of the genome of any HPV. The presence of HPV in a sample is indicated by the formation of amplified DNA. The HPV nucleic acid is detected by consensus probes that may be short oligonucleotide probes or long generic probes. The HPV is typed by the use of type-specific DNA probes specific for the amplified region of DNA.

Abstract: Oligonucleotides primers can be used to amplify and detect Hepatitis C virus nucleic acids in a process that involves reverse transcription of the viral genomic RNA to create cDNA and the subsequent amplification of the cDNA by the polymerase chain reaction. Oligonucleotide probes can be used to detect the presence of amplified DNA by hybridization. The invention provides improved methods, compositions, and kits for amplifying and detecting U.S., Japan, and HCV-C prototype nucleic acids.

Abstract: The present invention is directed to methods for controlling the light emission of an oligonucleotide labeled with a light-emitting label that are useful in nucleic acid detection assays. A reaction that results in the cleavage of single-stranded oligonucleotide probes labeled with a light-emitting label is carried out in the presence of a DNA binding compound that interacts with the label to modify the light emission of the label. The methods utilize the change in light emission of the labeled probe that results from degradation of the probe. The methods are applicable in general to assays that utilize a reaction that results in cleavage of oligonucleotide probes, and, in particular, to homogeneous amplification/detection assays wherein hybridized probe is cleaved concomitant with palmer extension. A homogeneous amplification/detection assay is provided which allows the simultaneous detection of the accumulation of amplified target and the sequence-specific detection of the target sequence.

Abstract: This invention provides for superior nucleic acid primers for amplification of select target regions of the genome of the genus Legionella. The invention facilitates detection of pathogenic and nonpathogenic forms of this genus. The invention further provides for processes for using the primers in template dependent nucleic acid polymerase extension reactions to amplify select target regions. Kits for the use of these primers are also provided. This invention further provides for methods of controlling the intensity of visual signal for detection of duplex formation in nucleic acid hybridization assays under high stringent conditions. This method involves the blending of different capture probes onto a solid support.

Abstract: Recombinant DNA sequences encoding the DNA polymerase activity of Pyrodictium species can be used to construct recombinant vectors and transformed host cells for production of the activity. Pyrodictium enzymes for catalyzing 3'.fwdarw.5' exonuclease activity, i.e., proofreading enzymes, are also provided. The Pyrodictium enzymes are useful in DNA amplification procedures and are not irreversibly inactivated by exposure to 100.degree. C. in a polymerase chain reaction.

Abstract: A process of detecting a target nucleic acid using labeled oligonucleotides which uses the 5' to 3' nuclease activity of a nucleic acid polymerase to cleave annealed labeled oligonucleotide from hybridized duplexes and thus releasing labeled oligonucleotide fragments for detection. This process is easily incorporated into a PCR amplification assay.

Abstract: The present invention provides a method for determining the amount of a target acid segment in a sample by polymerase chain reaction. The method involves the simultaneous amplification or the target nucleic acid segment and an internal standard nucleic acid segment. The amount of amplified DNA from each segment is determined and compared to standard curves to determine the amount of the target nucleic acid segment present in the sample prior to amplification. The method is especially preferred for determining the quantity of a specific mRNA species in a biological sample. Additionally, an internal standard is provided useful for quantitation of multiple mRNA species.

Abstract: Single or multiple nucleotide variations in nucleic acid sequence can be detected in nucleic acids by a process whereby the sample suspected of containing the relevant nucleic acid is repeatedly treated with primers, nucleotide triphosphates, and an agent for polymerization of the triphosphates and then denatured, in a process which amplifies the sequence containing the nucleotide variation if it is present. In one embodiment, the sample is spotted on a membrane and treated with a labeled sequence-specific oligonucleotide probe. Hybridization of the probe to the sample is detected by the label on the probe.

Abstract: The present invention relates to thermostable DNA polymerases which have been mutated such that a lesser amount of 5' to 3' exonuclease activity is exhibited from that which is exhibited by the native enzyme.

Abstract: The present invention provides methods and reagents for the estimation of the quantity of human DNA contained in a sample. Immobilized sample DNA is hybridized to a biotinylated oligonucleotide probe that hybridizes to a human genomic or mitochondria DNA sequence. The subsequent binding of streptavidin-horseradish peroxidase to the bound probe allows for chemiluminescent detection using a luminol-based reagent and X-ray film. In addition, the present invention provides methods and reagents to assess the quality of DNA contained in a sample. The sample is first size fractionated by agarose gel electrophoresis, and then immobilized, hybridized to a biotinylated oligonucleotide probe, and detected using the chemiluminescent method as used in the quantity estimation methods of the present invention.

Abstract: A purified thermostable enzyme is derived from the eubacterium Thermus species Z05. The enzyme has DNA polymerase, activity reverse transcriptase activity, and optionally 5'.fwdarw.3' exonuclease activity. The enzyme can be native or recombinant, and may be used with primers and nucleoside triphosphates in a temperature-cycling chain reaction where at least one nucleic acid sequence is amplified in quantity from an existing sequence.

Abstract: The present invention provides methods for tagging and tracing materials using nucleic acids as taggants. The process of tagging involves altering a substance in a manner that allows for the subsequent identification of the substance by detecting the alteration. The alteration disclosed herein involves nucleic acids.

Abstract: Primers for amplification of specific nucleic acid sequences of the second and third exon of HLA Class I A gene and probes for identifying polymorphic sequences contained in the amplified DNA can be used in processes for typing homozygous or heterozygous samples from a variety of sources and for detecting allelic variants not distinguishable by serological methods. This HLA-A DNA typing system can be used in a forward or reverse dot-blot format that is simple and rapid to perform, produces detectable signals in minutes, and can be used for tissue typing, determining individual identity, and identifying disease susceptible individuals.

Abstract: The presence of human papillomavirus (HPV) in a sample can be detected and the HPV typed by a method that involves the amplification of HPV DNA sequences by the polymerase chain reaction (PCR). The primers used in the method are consensus primers that can be used to amplify a particular region of the genome of any HPV. The presence of HPV in a sample is indicated by the formation of amplified DNA. The HPV nucleic acid is detected by consensus probes that may be short oligonucleotide probes or long genetic probes. The HPV is typed by the use of type-specific DNA probes specific for the amplified region of DNA.

Abstract: A purified thermostable enzyme is derived from the eubacterium Thermotoga maritima. The enzyme has a molecular weight as determined by gel electrophoresis of about 97 kilodaltons and DNA polymerase I activity. The enzyme can be produced from native or recombinant host cells and can be used with primers and nucleoside triphosphates in a temperature-cycling chain reaction where at least one nucleic acid sequence is amplified in quantity from an existing sequence.