Abstract: The present invention discloses methods of using DNA polymerase fusions at high pH in PCR, DNA sequencing and mutagenesis protocols.

Abstract: The present invention discloses methods of using DNA polymerase fusions at high pH in PCR, DNA sequencing and mutagenesis protocols.

Abstract: The invention provides novel extracts, proteins, and complexes that improve the polymerization activity of nucleic acid polymerases. Included within the aspects of the invention are methods for identifying compositions with a polymerase enhancing activity, methods for purifying and using these compositions, and specific extracts, proteins, and complexes that function to enhance polymerase activity. As an example, specifically described is nucleotide and amino acid sequence information for Pyrococcus furiousus PEF (P45), which was used to produce a recombinant PEF.

Abstract: A system and method for fluorescence excitation and detection having distinct optical paths is disclosed. A system for detecting fluorescence comprises a light source that emits an excitation light into an illumination tube; a plurality of collection optics located around an aperture in the illumination tube for collecting fluorescence; and a detector for determining the amount of fluorescence. A method for detecting fluorescence comprises emitting an excitation light from a light source into an illumination tube; directing the excitation light to an excitation filter; illuminating a sample with the excitation light to generate an emission light; and detecting the optical characteristics of the emission light using a plurality of collection optics located around the illumination tube.

Abstract: This invention provides isolated polynucleotides that encode replication accessory factors. The invention also provides novel DNA replication accessory factors, which have been isolated and purified from the hyperthermophilic archaea Pyrococcus furiosus. The invention also provides various methods of enhancing a nucleic acid polymerase reaction comprising the addition of the replication accessory factors to the reaction. This invention further provides methods of synthesizing, amplifying, and mutagenizing nucleic acids of interest employing the replication accessory factors. This invention also provides kits comprising, at least one of the replication accessory factors. This invention also provides kits useful for various methods that comprise at least one replication accessory factor.

Abstract: The invention provides cells and methods of circularizing linear DNA molecules. The cell is an isolated Escherichia coli cell which transiently expresses the Cre recombinase protein from an integrated Cre recombinase gene, and which is at least transiently repressed for RecBCD activity. The cells are used in a method of circularizing a linear DNA molecule comprising at least two loxP sites. The DNA molecule is introduced into the cells, and the linear DNA molecule is joined at said loxp sites.

Abstract: The invention relates to compositions and methods for detection of nucleic acid sequences. The invention further relates to kit format of said compositions for detection of nucleic acid sequences. Oligonucleotide probes of the invention comprise a target binding sequence and a sequence at least partially complementary thereto, joined by an optional linker to form a hairpin structure in the absence of the target nucleic acid sequence. The probes of the invention comprise a pair of moieties that produce a detectable signal when the probe hybridizes to the target sequence.

Abstract: The invention relates to compositions for generating a signal indicative of the presence of a target nucleic acid in a sample, where the compositions include an RNA polymerase, a FEN nuclease, and a probe.

Abstract: The invention relates to an apparatus and methods for preventing condensation on the interior surfaces of sample tubes which are being exposed to temperature cycles, such as during a PCR amplification reaction. In particular, the invention relates to apparatus comprising a sample block comprising a plurality of sample wells for receiving sample tubes and heating elements disposed in the sample wells for heating at least a portion of the sides of sample tubes (e.g., at least the portion which forms the head space after a tube is filled with a PCR reaction mixture). In a preferred aspect, the sample block is part of a thermocycling device for performing PCR and the side-wall heater is used to enhance uniformity and speed of amplification reactions. For example, by decreasing or eliminating condensation, signal strength jumps in a real-time PCR assay can be minimized as can reaction non-homogeneity.

Abstract: This invention provides isolated polynucleotides that encode replication accessory factors. The invention also provides novel DNA replication accessory factors, which have been isolated and purified from the hyperthermophilic archaea Pyrococcus furiosus. The invention also provides various methods of enhancing a nucleic acid polymerase reaction comprising the addition of the replication accessory factors to the reaction. This invention further provides methods of synthesizing, amplifying, and mutagenizing nucleic acids of interest employing the replication accessory factors. This invention also provides kits comprising at least one of the replication accessory factors. This invention also provides kits useful for various methods that comprise at least one replication accessory factor.

Abstract: The invention features a novel isolated Family B DNA polymerase, a Thermococcus polymerase JDF-3, and mutant recombinant forms thereof. Mutant polymerases of the invention are deficient in 3? to 5? exonuclease activity and/or exhibit reduced discrimination against non-conventional nucleotides relative to the wild-type form of the polymerase.

Abstract: The invention relates to a method of generating a signal indicative of the presence of a target nucleic acid sequence in a sample, where the method includes forming a cleavage structure by incubating a sample containing a target nucleic acid sequence with a nucleic acid polymerase and cleaving the cleavage structure with a nuclease to generate a cleaved nucleic acid fragment. The invention also relates to methods of detecting or measuring a target nucleic acid sequence, where the method includes forming a cleavage structure by incubating a target nucleic acid sequence with a nucleic acid polymerase, cleaving the cleavage structure with a nuclease and detecting or measuring the release of a fragment.

Abstract: The invention relates to a method of generating a signal indicative of the presence of a target nucleic acid in a sample, where the method includes forming a cleavage structure by incubating a sample comprising a target nucleic acid with a probe having a secondary structure that changes upon binding of the probe to the target nucleic acid and further comprising a binding moiety. The invention also includes the steps of cleaving the cleavage structure with a nuclease to release a nucleic acid fragment to generate a signal, wherein generation of the signal is indicative of the presence of a target nucleic acid in a sample, and detecting and/or measuring the amount of the fragment captured by binding of a binding moiety to a capture element on a solid support.

Abstract: The invention relates to baseline subtraction algorithms developed to reduce tube-to-tube and cycle-to-cycle variabilities during real time PCR amplification. Particularly, the invention relates to algorithms for determining the threshold cycle for the first reliable detection of the amplified nucleic acid product. The invention also relates to computer programs comprising the algorithm and methods and/systems implementing the algorithm.

Abstract: The present invention relates to probes useful for the detection and measurement of target nucleic acids, as well as compositions and kits containing such probes. The probes of the present invention include an interactive pair of labels, as well as a hairpin sequence that does not hybridize to a target nucleic acid. According to the present invention, the probe generates a detectable signal indicative of a presence of a target nucleic acid. The detectable signal emitted by one of the pair of labels is substantially constant upon binding of the probe to target nucleic acid, and the detectable signal increases by at least 2 fold upon cleavage of the probe between the pair of labels.

Abstract: The present invention provides methods, kits and compositions for the detection of an analyte. The invention is particularly suited for the detection and quantification of analytes in solution. In the methods of the invention a complex is formed between two or more analyte specific probes (ASP) and an analyte. The reactive moieties of the probes interact upon the binding of the analyte specific probes to the analyte. The reactive moieties generate a nucleic acid cleavage product which is detected and indicative of the presence of the analyte.

Abstract: The invention relates to compositions, methods, and kits for nucleic acid replication, including polymerase chain reaction (PCR) and mutagenesis reactions. A buffer composition is provided which allows higher concentrations of DNA polymerase to be used, resulting in greater yield of amplified product and faster reaction kinetics.

Abstract: The invention relates to compositions and methods of generating a signal indicative of the presence of a target nucleic acid in a sample, where the method includes forming a cleavage structure by incubating a sample comprising a target nucleic acid with a probe. The invention also includes the steps of cleaving the cleavage structure with a nuclease to release a nucleic acid fragment to generate a signal, wherein generation of the signal is indicative of the presence of a target nucleic acid in a sample.

Abstract: The subject invention provides novel compositions containing a mixture of (a) an enzyme that possesses substantial 3?–5? exonuclease activity (b) a DNA polymerase with less 3?–5? exonuclease activity than the enzyme with substantial 3?–5? exonuclease activity. Preferably, the DNA polymerase for inclusion in the compositions are DNA polymerases that substantially lack 3?–5? exonuclease activity. A preferred embodiment of the invention is a composition comprising the Taq DNA polymerase (isolated from Thermus aquaticus) and the Pfu DNA polymerase (isolated from Pyrococcus furiosus). Another aspect of the invention is to provide methods for synthesizing polynucleotides, typically DNA, using compositions comprising an enzyme that possesses substantial 3?–5? exonuclease activity and a DNA polymerase with less 3?–5? exonuclease activity than the enzymes possessing substantial 3?–5? exonuclease activity, preferably a DNA polymerase that substantially lacks 3?–5? exonuclease activity.

Abstract: The invention relates to compositions and methods for generating a signal indicative of the presence of a target nucleic acid in a sample utilizing a cleavage resistant probe.