Abstract: Methods of using polymerase chain reactions to enrich a target sequence in a sample containing reference sequences and target sequences having high homology and amplifiable by the same primer pair are provided herein. In particular the methods provide a robust means to improve the fold enrichment of the target sequence and minimize reaction-to-reaction, well-to-well and run-to-run variations in the enrichment methods.

Abstract: The invention is based, at least in part, on the observation that the presence of particular biomarkers, e.g., particular mutations in any of the KCNQ1, KCNH2, SCN5A, KCNE1 and KCNE2 genes as identified in Tables 1-5 (and, in particular, those identified with an asterisk), is associated with Long QT Syndrome (LQTS).

Abstract: Methods and kits for sequencing a target DNA sequence in a sample having a related reference sequence are provided herein. In particular, kits and methods for sequencing cancer and cancer therapy associated mutations are described. Also provided are kits and methods for detecting mitochondrial mutations and for differentiating between closely related viral strains.

Abstract: The invention relates to methods for identifying the sequence of one or more variant nucleotides in a nucleic acid molecule. The method involves cleaving a double-stranded nucleic acid molecule containing a mismatch with a mismatch-specific endonuclease which cleaves on the 3? side of the mismatch, and preserving the integrity of the variant nucleotide by ligating a double-stranded linker with a 3?-overhang to said variant nucleotide. Because the variant nucleotide is immediately adjacent to the linker, PCR and/or sequence-by-synthesis analysis can be readily carried out.

Abstract: The invention relates to methods for identifying the sequence of one or more variant nucleotides in nucleic acid molecules. The method involves cleaving a double-stranded nucleic acid molecule containing a mismatch with a mismatch-specific endonuclease which cleaves on the 3? side of the mismatch, and preserving the integrity of the variant nucleotide by ligating a Double-Stranded Linker with a degenerate 3?-overhang to said variant nucleotide. Because the variant nucleotide is immediately adjacent to the linker, PCR and/or sequence-by-synthesis analysis can be readily carried out.

Abstract: The present invention relates to isolated nucleic acid sequences encoding CEL II endonuclease and vectors and host cells for producing a protein encoded thereby.

Abstract: In an extensive Matched Ion Polynucleotide Chromatography (MIPC) system and method, and the computer programs or software associated therewith, the system provides automated options for sample selection, mobile phase gradient selection and control, column and mobile phase temperature control, and fragment collection for a wide variety of MIPC separation processes. MIPC separation processes can be applied to effect size-based separation of DNA fragments, mutation detection, DNA fragment purification, PCR process monitoring and other novel processes. This invention is directed to the system and software which automates many of these procedures, facilitating use of the system to achieve complex separation methods. In one embodiment of the invention, a user specifies a size range of double stranded DNA fragment(s) in a mixture, the software calculates a solvent gradient to elute the fragment(s), and the system performs the chromatographic separation using the calculated gradient.

Abstract: Methods and devices based on capillary monolithic columns, preferably consisting of an underivatized poly(styrene-divinylbenzene) monolith, for separating a mixture of polynucleotides by ion pair-reverse phase-high performance chromatography (IP-RP-HPLC).

Abstract: In one aspect, the invention provides a method for separating a mixture of polynucleotides, such as DNA or RNA, including (a) applying the mixture to a polymeric separation medium having non-polar surfaces, wherein the surfaces are characterized by being substantially free from multivalent cations, such as metal ions, which are free to interfere with polynucleotide separation, and (b) eluting the mixture with a mobile phase containing organic solvent and counter ion agent. In the separation of single-stranded polynucleotides, improved separation is obtained at a temperature effective to fully denature secondary structure within the polynucleotides.

Abstract: Methods, compositions, and kits for separating heteroduplex and homoduplex DNA molecules in a test mixture by temperature-compression denaturing high performance liquid chromatography (tcDHPLC). The method includes use of nitrogen-containing additives in the mobile phase that allow detection of diverse heteroduplex molecules to be performed at the same pre-selected temperature. An example of a preferred additive is betaine. Standard mixtures of DNA fragments, such as mutation standards containing known heteroduplex and homoduplex molecules, can be used to select the concentration of additive and temperature. Compositions and kits including the mobile phase, mutation standards, PCR primers, separation media, and DNA polymerase are also provided.

Abstract: The present invention relates to the isolation and characterization of CEL I and CEL II endonuclease proteins. Methods and kits for identifying mismatches in double-stranded DNA are also provided.

Abstract: Substituted dihydrophthalazine sulfur containing compositions are provided which are active as non-NMDA ionotropic excitatory amino acid (EAA) receptor antagonists. The compositions are useful for treating disorders associated with excessive activation of the non-NMDA subtype of the ionotropic EAA receptor. The compounds further are useful as testing agents to identify and characterize other compounds for the treatment of these disorders. The compounds are useful therapeutically as sedatives or for the treatment of neuropsychopharmacological disorders such as stroke, ischemia and epilepsy. The compositions may be provided in combination with a suitable carrier for oral or parenteral administration. The compounds may be administered orally or parenterally for the treatment of a variety of disorders associated with non-NMDA EEA receptor function.

Abstract: Substituted benzodiazepine compositions are provided which are active as non-NMDA ionotropic excitatory amino acid (EAA) receptor antagonists. The compounds are generally 7- or 8-mono substituted 5H-2,3-benzodiazepines. The compositions are useful for treating disorders associated with excessive activation of the non-NMDA subtype of the ionotropic EAA receptor. The compounds further are useful as testing agents to identify and characterize other compounds for the treatment of these disorders. The compounds are useful therapeutically as sedatives or for the treatment of neurosychopharmacological disorders such as stroke, ischemia and epilepsy. The compositions may be provided in combination with a suitable carrier for oral or parenteral administration. The compounds may be administered orally or parenterally for the treatment of a variety of disorders associated with non-NMDA EEA receptor function.

Abstract: Covalently bound non-polar tags are used to increase the retention times of double stranded polynucleotides on Matched Ion Polynucleotide Chromatography (MIPC) columns. In doing so, separations of DNA mixture components is improved. Additionally, when the non-polar tags are fluorophores, detection limits are also greatly reduced. Strategically tagged primers are used in conduction with PCR to produce DNA fragments having specifically tagged strands. This improves mutation detection by MIPC in several ways. Separations are improved, detection sensitivity is enhanced, and non-stoichiometric addition of wild type DNA prior to hybridization is now possible since only tagged fragments will be observed with a fluorescence detector. Non-polar tags are also used as a novel alternative to G-C clamping during MIPC under partially denaturing conditions. Reversible DNA binding dyes, such as DNA intercalator dyes and DNA groove binding dyes, are used to reduce the detection limit of polynucleotides separated by MIPC.

Abstract: Methods, compositions, and kits for detecting mutations in the entire human mitochondrial genome. A preferred method includes amplifying mtDNA from a biological sample by polymerase chain reaction of total DNA using a plurality of pre-selected primer pairs to generate overlapping amplicons; cleaving the amplicons using restriction enzymes to produce fragments suitable for analysis by denaturing high performance liquid chromatography (DHPLC); denaturing and re-annealing the amplicons; and fragment analysis by DHPLC to detect the presence or absence of heteroduplex molecules.

Abstract: A system for separating an aqueous stream of mixed polynucleotides into a series of length-based polynucleotide fractions and collecting one or more of the length-based polynucleotide fractions into separate containers. The system comprises a separation column containing separation media for separating an aqueous stream of mixed polynucleotides into a series of length-based polynucleotide fractions; a container including one or more single-sample containers; an ejection chamber having a separated sample inlet for receiving the length-based polynucleotide fractions, a waste outlet for discharging uncollected sample, and a capillary-sized fraction outlet positioned to discharge a selected length-based polynucleotide fraction into a single-sample container. The system also includes means for effecting discharge of a selected length-based polynucleotide fraction into the separate container.

Abstract: Substituted dihydrophthalazine sulfur containing compositions are provided which are active as non-NMDA ionotropic excitatory amino acid (EAA) receptor antagonists. The compositions are useful for treating disorders associated with excessive activation of the non-NMDA subtype of the ionotropic EAA receptor. The compounds further are useful as testing agents to identify and characterize other compounds for the treatment of these disorders. The compounds are useful therapeutically as sedatives or for the treatment of neurosychopharmacological disorders such as stroke, ischemia and epilepsy. The compositions may be provided in combination with a suitable carrier for oral or parenteral administration. The compounds may be administered orally or parenterally for the treatment of a variety of disorders associated with non-NMDA EEA receptor function.

Abstract: Methods, systems, compositions and kits for improved detection of polynucleotides. In one aspect, there is provided a method for separating polynucleotides (such as DNA or RNA) using a liquid chromatographic separation device (such as a reverse phase column or an ion exchange column), contacting eluted polynucleotides with intercalating dye, and detecting (such as by fluorescence detection) dye bound to the eluted polynucleotides. The invention preferably uses a post-column reactor, such as a mixing tee, downstream of the separation column. Sensitivity of mutation detection by denaturing high performance liquid chromatography (DHPLC) is enhanced.

Abstract: Methods, compositions, and kits for separating heteroduplex and homoduplex DNA molecules in a test mixture by temperature-compression denaturing high performance liquid chromatography (tcDHPLC). The method includes use of nitrogen-containing additives in the mobile phase that allow detection of diverse heteroduplex molecules to be performed at the same pre-selected temperature. An example of a preferred additive is betaine. Standard mixtures of DNA fragments, such as mutation standards containing known heteroduplex and homoduplex molecules, can be used to select the concentration of additive and temperature. Compositions and kits including the mobile phase, mutation standards, PCR primers, separation media, and DNA polymerase are also provided.

Abstract: An improved separation column and method for separating a mixture of double stranded DNA fragments by Matched Ion Polynucleotide Chromatography. The cylindrical column has an ID greater than about 5 mm and contains polymer beads. The beads have an average diameter of 1 to 100 microns and are unsubstituted polymer beads or are polymer beads substituted with a hydrocarbon moiety having from 1 to 1,000,000 carbons. The preferred beads are characterized by being substantially free from multivalent cations which are free to bind with DNA. The improved column provides enhanced separation of DNA fragments with sizes ranging from about 100 to 20,000 base pairs. The column also provides enhanced separation of heteroduplex and homoduplex DNA molecules in a mutation detection procedure in which the chromatography is performed under conditions effecting partial denaturation of DNA at a site of mismatched base pairs.