Abstract: The present invention is generally related to compositions and methods for the reverse transcription of nucleic acid molecules, especially messenger RNA molecules. Specifically, the invention relates to compositions comprising mixtures of polypeptides having reverse transcriptase (RT) activity, and to methods of producing, amplifying or sequencing nucleic acid molecules (particularly cDNA molecules) using these compositions or polypeptides, particularly at temperatures above about 55° C. The invention also relates to nucleic acid molecules produced by these methods, to vectors and host cells comprising these nucleic acid molecules, and to the use of such nucleic acid molecules to produce desired polypeptides.

Abstract: The present invention is generally related to compositions and methods for the reverse transcription of nucleic acid molecules, especially messenger RNA molecules. Specifically, the invention relates to compositions comprising mixtures of polypeptides having reverse transcriptase (RT) activity, and to methods of producing, amplifying or sequencing nucleic acid molecules (particularly cDNA molecules) using these compositions or polypeptides, particularly at temperatures about about 55° C. The invention also relates to nucleic acid molecules produced by these methods, to vectors and host cells comprising these nucleic acid molecules, and to the use of such nucleic acid molecules to produce desired polypeptides.

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: 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: Compositions, systems, kits and methods relating to in vitro synthesis are provided. The system includes one or more extracts having reduced activity of an enzyme that catalyses hydrolysis of high energy phosphate bonds or hydrolysis or formation of phosphodiester bonds, an inhibitor that inhibits hydrolysis of high energy phosphate bonds or hydrolysis or formation of phosphodiester bonds, and/or at least two energy sources. The composition may include a nucleic acid template and one or more products of the nucleic acid template.

Abstract: The present invention provides methods for use in identifying, analyzing and typing polymorphic DNA fragments, particularly minisatellite, microsatellite or STR DNA fragments. In particular, the invention provides methods using DNA polymerases, more particularly thermostable DNA polymerases, and most particularly Thermotoga polymerases or mutants or derivatives thereof, whereby minisatellite, microsatellite or STRDNA molecules may be amplified and analyzed for polymorphisms. The invention also relates to polymerases having reduced, substantially reduced or eliminated ability to add non-template 3′ nucleotides to a synthesized nucleic acid molecule. In accordance with the invention, such reduction or elimination may be accomplished by modifying or mutating the desired polymerase.

Abstract: The invention relates to a substantially pure thermostable DNA polymerase from Thermotoga neapolitana (Tne). The Tne DNA polymerase has a molecular weight of about 100 kilodaltons and is more thermostable than Taq DNA polymerase. The present invention also relates to the cloning and expression of the Tne DNA polymerase in E. coli, to DNA molecules containing the cloned gene, and to host cells which express said genes. The Tne DNA polymerase of the invention may be used in well-known DNA sequencing and amplification reactions.

Abstract: The invention relates to a DNA and RNA polymerases which have increased fidelity (or reduced misincorporation rate). In particular, the invention relates to a method of making such polymerases by increasing or enhancing 3′-5′ exonuclease activity of a polymerase by, for example, substituting the 3′-5′ exonuclease domain of one polymerase with a 3′-5′ exonuclease domain with the desired activity from another polymerase. The invention also relates to DNA molecules containing the genes encoding the polymerases of the invention, to host cells containing such DNA molecules and to methods to make the polymerases using such host cells. The polymerases of the invention are particularly suited for nucleic acid synthesis, sequencing, amplification and cDNA synthesis.

Abstract: The present invention is generally related to compositions and methods for the reverse transcription of nucleic acid molecules, especially messenger RNA molecules. Specifically, the invention relates to compositions comprising mixtures of polypeptides having reverse transcriptase (RT) activity, and to methods of producing, amplifying or sequencing nucleic acid molecules (particularly cDNA molecules) using these compositions or polypeptides, particularly at temperatures above about 55° C. The invention also relates to nucleic acid molecules produced by these methods, to vectors and host cells comprising these nucleic acid molecules, and to the use of such nucleic acid molecules to produce desired polypeptides.

Abstract: The present invention provides methods for use in identifying, analyzing and typing polymorphic DNA fragments, particularly minisatellite, microsatellite or STR DNA fragments. In particular, the invention provides methods using DNA polymerases, more particularly thermostable DNA polymerases, and most particularly Thermotoga polymerases or mutants or derivatives thereof, whereby minisatellite, microsatellite or STR DNA molecules maybe amplified and analyzed for polymorphisms. The invention also relates to polymerases having reduced, substantially reduced or eliminated ability to add non-template 3′ nucleotides to a synthesized nucleic acid molecule. In accordance with the invention, such reduction or elimination may be accomplished by modifying or mutating the desired polymerase.

Abstract: The present invention is directed generally to methods facilitating the cloning of nucleic acid molecules. In particular, the invention relates to the use of polymerase inhibitors, including but not limited to anti-polymerase antibodies (such as anti-Taq antibodies) and fragments thereof, to inactivate residual polymerase activity remaining after the amplification (particularly via PCR) of a target nucleic acid molecule. The invention further provides compositions, particularly storage-stable compositions, comprising one or more components, such as one or more restriction endonucleases and one or more polymerase inhibitors, that are useful in cloning amplified or synthesized nucleic acid molecules by the above-described methods. The invention also relates to nucleic acid molecules produced by these methods, and to genetic constructs (such as vectors) and host cells comprising these nucleic acid molecules.

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'.fwdarw.5' exonuclease activity of said DNA polymerase; (2) a second mutation that substantially reduces or eliminates 5'.fwdarw.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: The invention relates to the cloning of a gene encoding rat liver ribonuclease inhibitor (RI), and its expression in a cellular host. In addition, the invention relates to the successful cloning of a gene encoding porcine liver RI, and its expression in a cellular host.

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'.fwdarw.5' exonuclease activity of said DNA polymerase; (2) a second mutation that substantially reduces or eliminates 5'.increment.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: The invention relates to a substantially pure thermostable DNA polymerase from Thermotoga neapolitana (Tne) 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 Tne DNA polymerase has at least one mutation selected from the group consisting of (1) a first mutation that substantially reduces or eliminates 3'.fwdarw.5' exonuclease activity of said DNA polymerase; (2) a second mutation that substantially reduces or eliminates 5'.fwdarw.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 Tne DNA polymerase in E. coli, to DNA molecules containing the cloned gene, and to host cells which express said genes.

Abstract: The invention relates to the cloning of a gene encoding rat liver RI, and its expression in a cellular host. In addition, the invention relates to the successful cloning of a gene encoding porcine liver RI, and its expression in a cellular host. The invention also relates to the cloning and expression of human liver RI, and the cloning and expression of chimeric mammalian RIs, particularly chimeric porcine/human liver RIs, which may be thermostable. The invention also relates to methods and kits for use in producing nucleic acid molecules and polypeptides using the RIs of the invention, and to nucleic acid molecules and polypeptides produced using these methods and kits.

Abstract: The invention relates to a substantially pure thermostable DNA polymerase from Thermotoga neapolitana (Tne). The Tne DNA polymerase has a molecular weight of about 100 kilodaltons and is more thermostable than Taq DNA polymerase. The present invention also relates to the cloning and expression of the Tne DNA polymerase in E. coli, to DNA molecules containing the cloned gene, and to host cells which express said genes. The Tne DNA polymerase of the invention may be used in well-known DNA sequencing and amplification reactions.

Abstract: The present invention discloses a recombinant DNA molecule having a structural gene encoding a processive, thioredoxin-independent DNA polymerase that is substantially reduced in processive 3'-to-5' DNA exonuclease activity, a promoter, and an origin of replication. A method for producing this enzyme is also disclosed, as is the protein produced by this process. This invention is exemplified by expression of exonuclease.sup.- T5 DNA polymerase in E. coli.

Abstract: The present invention discloses a recombinant DNA molecule having a structural gene encoding a processive, thioredoxin-independent DNA polymerase that is substantially reduced in processive 3'-to-5' DNA exonuclease activity, a promoter, and an origin of replication. A method for producing this enzyme is also disclosed, as is the protein produced by this process. This invention is exemplified by expression of exonuclease.sup.- T5 DNA polymerase in E. coli.

Abstract: The invention relates to a substantially pure thermostable DNA polymerase. Preferably, the DNA polymerase has a molecular weight of about 95 kilodaltons and is more thermostable than Taq DNA polymerase. The present invention also relates to cloning and expression of the DNA polymerase in E. coli, to DNA molecules containing the cloned gene, and to host cells which express said genes.