Abstract: Improved methods for amplifying nucleic acids can reduce non-specific amplification and minimize the effects of contamination of nucleic acid amplification reaction assays due to amplified product from previous amplifications. The methods involve the introduction of unconventional nucleotide bags into the amplification reaction products and treating the products by enzymatic (e.g., glycosylases) and/or physical-chemical means to render the product incapable of acting as a template for subsequent amplifications.

Abstract: Improvements to the polymerase chain reaction (PCR), a process for in vitro enzymatic amplification of specific nucleic acid sequences, can be achieved by changing the way that PCR reagents are mixed and the enzymatic reaction is started and by the replacement of mineral oil, commonly used as a vapor barrier to minimize solvent evaporation, by a grease or wax. The use of such mixtures allows for the delay of reagent mixing until the first heating step of a PCR amplification, thereby reducing the enzymatic generation of nonspecific products which occurs when a complete mixture of PCR reagents, with or without test sample, stands at room temperature or below. These mixtures increase the shelf-life of PCR reagents and increase protection of the laboratory environment against contamination by PCR product.

Abstract: Methods are provided for the replication and amplification of RNA sequences by thermoactive DNA polymerases. Reverse transcription of RNA is catalyzed by, for example, 94 kDa Taq, 62 kDa Taq, and recombinant Tth DNA polymerase. Reverse transcription is coupled to PCR amplification in a one enzyme procedure using a thermostable polymerase.

Abstract: A purified thermostable enzyme is derived from the bacterium Thermus species sps17. The enzyme has DNA polymerase activity, reverse transcriptase activity, and optionally, 5'.fwdarw.3' exonuclease activity. The enzyme can be native or recombinant, and can be used with selected 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 a method for determining the relative amount of a nucleic acid segment in a sample by the polymerase chain reaction. The method involves the simultaneous amplification of the nucleic acid segment and a second nucleic acid segment present in the sample. The amount of amplified DNA from each segment is determined and compared to standard curves to determine the amount of the nucleic acid segment present in the sample before amplification expressed as a ratio of first segment to second segment. The method is especially preferred for determining the viral load, or copies of virus genome/host cell, in a sample of cells from an individual infected with a virus.

Abstract: The presence or absence of a nucleic acid sequence associated with AIDS in a sample containing one or more nucleic acids and suspected of containing such sequence can be detected by amplifying the sequence using primers to form extension products as templates and detecting the amplified product if it is present. This may be accomplished by adding a labeled hybridization probe to the amplified product either free in solution or after immobilization on a solid support. Exemplary primers and probes for amplifying and detecting AIDS virus are provided.

Abstract: A purified thermostable enzyme is derived from the eubacterium Thermotoga maritima. The enzyme has a molecular weight 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 temperaturecycling chain reaction where at least one nucleic acid sequence is amplified in quantity from an existing sequence.

Abstract: A purified thermostable enzyme is obtained that has unique characteristics. Preferably the enzyme is isolated from the Thermus aquaticus species and has a molecular weight of about 86,000-95,000 daltons. The thermostable enzyme may be native or recombinant and may be used in a temperature-cycling chain reaction wherein at least one nucleic acid sequence is amplified in quantity from an existing sequence with the aid of selected primers and nucleotide triphosphates. The enzyme is preferably stored in a buffer containing non-ionic detergents that lends stability to the enzyme.

Abstract: Methods are provided for enhanced specificity and sensitivity of nucleic acid amplification. The methods are simplified nested amplification procedures wherein both inner and outer primer pairs are present in the amplification reaction mixture. According to the methods, the thermocycling profile, as well as the sequences, length, and concentration of amplification primers, is modified to regulate which primers are annealed and extended on the target during any particular amplification cycle. The methods described are particularly suitable in PCR amplifications and have numerous applications in molecular biology, medical diagnostics, and forensics.

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: 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.

Abstract: Processes and kits therefor for detection of water-borne pathogens and indicator organisms in water samples by recovering cells of the pathogens or indicator organisms from a water sample, lysing the cells to recover undegraded DNA, amplifying a target gene sequence of a target gene present in cells of the pathogens or indicator organisms by polymerase chain reaction amplification and detecting the presence of amplified target gene sequence to determine the presence or absence of pathogens or indicator organism in the test sample.

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 is typed by the use of type-specific DNA probes specific for the amplified region of DNA.

Abstract: Methods and reagents for determining an individual's genotype at the .sup.G .gamma.-globin locus with respect to a set of three alleles using sequence-specific oligonucleotide probes enable one to type samples from a variety of sources, including samples comprising RNA or cDNA templates, and can be applied to nucleic acids in which a target region spanning the polymorphism has first been amplified. This three-allele system facilitates typing tissue for determining individual identity and has application in the field of forensic science.

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 of 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: 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 is typed by the use of type-specific DNA probes specific for the amplified region of DNA.

Abstract: The presence or absence of a nucleic acid sequence associated with one or more related viruses in a sample containing one or more nucleic acids and suspected of containing such sequence can be detected by amplifying the sequence using primers to form extension products as templates and detecting the amplified product if it is present. This may be accomplished by adding a labeled hybridization probe to the amplified product either free in solution or after immobilization on a solid support. Preferably the virus constitutes AIDs viruses and hepadnaviruses.

Abstract: Structure-independent amplification of DNA by the polymerase chain reaction can be achieved by incorporation of 7-deaza-2'-deoxyguanosine-5'-triphosphate into the amplified DNA.

Abstract: HLA typing based on restriction length polymorphism is carried out by: digesting an individual's DNA with a restriction endonuclease that produces a polymorphic digestion pattern with HLA DNA; subjecting the digest to genomic blotting using a labeled DNA hybridization probe that hybridizes to an HLA DNA sequence involved in the polymorphism; and comparing the resulting genomic blotting pattern with a standard. This technique may be adapted to make paternity or transplant or transfusion compatibility determinations or to make disease association correlations to diagnose diseases or predict susceptibility to diseases. Locus specific cDNA hybridization probes, particularly probes for genes of Class II loci, for use in the typing procedure are described.

Abstract: Structure-independent amplification of DNA by the polymerase chain reaction can be achieved by incorporation of 7-deaza-2'-deoxyguanosine-5'-triphosphate into the amplified DNA.