Abstract: The invention relates to a method of generating a signal indicative of the presence of a target nucleic acid sequence in a sample, comprising forming a complex by incubating a sample comprising a target nucleic acid sequence with a probe comprising a first and second subunit, and/or an upstream primer, and binding the probe to the target nucleic acid such that the first and second subunits dissociate to release the first subunit and generate a signal. In certain embodiments, the upstream primer is extended with a nucleic acid polymerase to displace at least a portion of the first subunit of the probe from the target nucleic acid strand and dissociate the first and second subunits to release the first subunit of the probe and generate a signal.

Abstract: The invention provides improved methods of introducing site-directed mutations into circular DNA molecules of interest by means of mutagenic primer pairs. The mutagenic primer pairs are also selected so as to be either completely complementary or partially complementary to each other, wherein the mutation site (or sites) is located within the region of complementarity. A mutagenic primer pair is annealed to opposite strands of a circular DNA molecule containing the DNA sequence to be mutagenized. After annealing, first and second mutagenized DNA strands, each incorporating a member of the mutagenic oligonucleotide primer pair is synthesized by a linear cyclic amplification reaction. After the linear cyclic amplification mediated synthesis step is completed, the reaction mixture is treated with a selection enzyme that digests the parental template strands. After the digesting step, a double-stranded circular DNA intermediate is formed.

Abstract: The subject invention provides for a method of selectively cloning homoduplex nucleic acid molecules, in particular, by using a strain of host cells that contains a conditionally expressed and/or conditionally active mismatch-recognizing enzyme, e.g., a temperature sensitive variant of the gene encoding the endonuclease VII from phage T4. Using this host strain, the invention features a novel cloning method that selects for PCR products that are devoid of PCR-generated mutations.

Abstract: The invention provides improved methods of introducing site-directed mutations into circular DNA molecules of interest by means of mutagenic primer pairs. The mutagenic primer pairs are also selected so as to be either completely complementary or partially complementary to each other, wherein the mutation site (or sites) is located within the region of complementarity. A mutagenic primer pair is annealed to opposite strands of a circular DNA molecule containing the DNA sequence to be mutagenized. After annealing, first and second mutagenized DNA strands, each incorporating a member of the mutagenic oligonucleotide primer pair is synthesized by a linear cyclic amplification reaction. After the linear cyclic amplification mediated synthesis step is completed, the reaction mixture is treated with a selection enzyme that digests the parental template strands. After the digesting step, a double-stranded circular DNA intermediate is formed.

Abstract: The invention relates to a method of generating a signal indicative of the presence of a target nucleic acid in a sample, as well as a method of detecting or measuring a target nucleic acid in a sample. The invention comprises 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 provides methods of covalently joining nucleic acid molecules and methods of molecular cloning. The methods provide either sequential or simultaneous ligation of flanking or vector nucleic acid molecules to nucleic acid insert molecules by topoisomerase and DNA ligase. The methods provide for directional and non-directional covalent joining and cloning of nucleic acid molecules.

Abstract: The detection of insertions and/or deletions in reiterated nucleotide sequences in tissues provides an identification of neoplastic changes that are associated with malignancy. The mutations are preferably detected by PCR based amplification of target sequences using selected primers, followed by standard analytic procedures. The detection of these mutations is useful as a diagnostic tool for cancer development and has direct application for cancer prognosis.

Abstract: The present invention provides a polynucleotides encoding mutants of green fluorescent protein from Renilla reniformis, including humanized sequences which permit enhanced expression of the encoded polypeptides in mammalian cells.

Abstract: The invention provided herein includes novel gram negative bacteria cells containing the Hte mutation. Other aspects of the invention include methods for rendering gram negative bacterial cells bearing the Hte region, such as E. coli cells competent for DNA transformation using any of a variety of competency inducing procedures. The competent cells of the subject invention may be frozen so as to provide for prolonged storage.

Abstract: Purified thermostable Pyrococcus furiosus DNA polymerase that migrates on a non-denaturing polyacrylamide gel faster than phosphorylase B and Taq polymerase and more slowly than bovine serum albumin and has an estimated molecular weight of 90,000–93,000 daltons when compared with a Taq polymerase standard assigned a molecular weight of 94,000 daltons.

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: An apparatus for thermally cycling samples of a biological material including a thermal block assembly including a plurality of sample holders for receiving samples of biological material; a heat sink thermally coupled to the thermal block assembly, the heat sink transferring heat away from the thermal block assembly to ambient air in contact with the heat sink; a first heat source thermally coupled to the thermal block assembly to provide heat to the thermal block assembly; and a second heat source thermally coupled to the first heat source and configured to provide heat to a portion of the first heat source. The arrangement of the heat sink, first heat source and second heat source can provide substantial temperature uniformity among the plurality of sample holders. The invention also includes a method for thermally cycling samples of biological material.

Abstract: Purified thermostable Pyrococcus furiosus DNA polymerase that migrates on a non-denaturing polyacrylamide gel faster than phosphorylase B and Taq polymerase and more slowly than bovine serum albumin and has an estimated molecular weight of 90,000-93,000 daltons when compared with a Taq polymerase standard assigned a molecular weight of 94,000 daltons.

Abstract: Purified thermostable Pyrococcus furiosus DNA polymerase that migrates on a non-denaturing polyacrylamide gel faster than phosphorylase B and Taq polymerase and more slowly than bovine serum albumin and has an estimated molecular weight of 90,000-93,000 daltons when compared with a Taq polymerase standard assigned a molecular weight of 94,000 daltons.

Abstract: The invention relates to polynucleotides for HSV detection and the use of these polypeptides in kits and methods for HSV detection.

Abstract: The present invention relates to a method for isolating from the immunological gene repertoire a gene coding for a receptor having the ability to bind a preselected ligand. Receptors produced by the gene isolated by the method, particularly catalytic receptors, are also contemplated.

Abstract: A method in which a temperature gradient is generated across a “gradient” block, and an apparatus comprising a block across which a temperature gradient can be generated. By setting up such a gradient, multiple reaction mixtures held in wells on the gradient block can be simultaneously run at temperatures which differ only slightly, thereby permitting an optimum temperature for the reaction to be quickly identified. In a preferred embodiment the gradient block is integrated into a thermal cycler used for nucleic acid amplification reactions.

Abstract: The invention relates to polynucleotides for HSV detection and the use of these polypeptides in kits and methods for HSV detection.

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 proteins or polypeptides that comprise intramolecular dimers of fluorescent protein monomers. More specifically, the invention relates to recombinant polypeptides comprising a monomer of a fluorescent polypeptide, a linker peptide, and a second monomer of that fluorescent polypeptide, where the monomers form an intramolecular dimer. The invention also relates to nucleic acids encoding Intramolecular Dimer Fluorescent Proteins (IDFPs) and vectors comprising such nucleic acids. The invention further relates to methods of making IDFPs and methods of using them. IDFPs are, useful in any application suited for fluorescent proteins and are particularly useful in applications in which more than one fluorescent protein sharing complementary dimerization interfaces is present in the same mixture or is expressed in the same cell, because IDFPs do not form heterodimers.