Abstract: The present invention provides a method of performing hot start PCR reactions. The method is based on sequestration of magnesium ions in the form of a precipitate which renders a DNA polymerase inactive until the appropriate time in the PCR reaction when a certain temperature is reached and the magnesium ions are released from the precipitate. Also provided are kits comprising reagents and instructions for amplifying a target nucleic acid by the magnesium precipitate hot start.

Abstract: Methods are provided for evaluating the fidelity of a given set of polymerase chain reaction conditions. In the subject methods, a template polydeoxyribonucleotide is amplified under the to be evaluated polymerase chain reaction conditions, where the template polydeoxyribonucleotide includes a pseudo restriction endonuclease restriction site. The resultant amplified product population is then contacted with the corresponding restriction endonuclease and resultant cleavage products, if any, are detected. The fidelity of the polymerase chain reaction conditions is then derived from the detected cleavage products (or absence thereof). Also provided are kits for use in practicing the subject methods. The subject methods are suited for determining the fidelity of a given polymerase under PCR conditions, and are particularly suited for determining the fidelity of a thermostable polymerase under PCR conditions.

Abstract: The present invention provides a process for detecting and enumerating Campylobacter jejuni in an environmental sample. The present invention further provides a process which can distinguish antibiotic resistant strains of Campylobacter jejuni from wild-type strains, in particular, antibiotic-resistant strains resistant to high levels of ciprofloxacin. Both processes use PCR primers which flank a target sequence unique to Campylobacter jejuni in combination with one or more dual-labeled oligonucleotide probes complementary to the target sequence wherein the dual-labeled probes enable detection of PCR amplification by fluorescence detection means.

Abstract: A method for determining whether a DNA sequence is identical to a wild-type sequence is presented. A GC-rich DNA segment is attached to a DNA segment of interest. With the GC-clamp attached, the DNA segment of interest has two melting domains, a higher domain associated with the GC-clamp and a lower domain associated with the DNA segment of interest. The DNA segment of interest is labeled with a fluorescent label such as a double-stranded DNA binding dye and mixed with a denaturant. The mixture of denaturant and fluorescently labeled DNA is heated. Fluorescence is monitored to determine the melting point of the DNA segment of interest. The melting temperature of the DNA segment of interest is compared to the melting point of the wild-type sequence. A difference in melting temperatures of the DNA sequence and the wild-type sequence indicates an alteration in the DNA sequence.

Abstract: The present invention relates to novel human secreted proteins and isolated nucleic acids containing the coding regions of the genes encoding such proteins. Also provided are vectors, host cells, antibodies, and recombinant methods for producing human secreted proteins. The invention further relates to diagnostic and therapeutic methods useful for diagnosing and treating disorders related to these novel human secreted proteins.

Abstract: Polymerases from the Pol I family which are able to efficiently use ddNTPs have demonstrated a much improved performance when used to sequence DNA. A number of mutations have been made to the gene coding for the Pol II family DNA polymerase from the archaeon Pyrococcus furiosus with the aim of improving ddNTP utilisation. “Rational” alterations to amino acids likely to be near the dNTP binding site (based on sequence homologies and structural information) did not yield the desired level of selectivity for ddNTPs. However, alteration at four positions (Q472, A486, L490 and Y497) gave rise to variants which incorporated ddNTPs better than the wild type, allowing sequencing reactions to be carried out at lowered ddNTP:dNTP ratios. Wild type Pfu-Pol required a ddNTP:dNTP ratio of 30:1; values of 5:1 (Q472H), 1:3 (L490Y), 1:5 (A486Y) and 5:1 (Y497A) were found with the four mutants; A486Y representing a 150-fold improvement over the wild type.

Abstract: The present invention provides compositions and methods for performing an amplification reaction of nucleic acids with internal controls that test the integrity of all aspects of the amplification reaction.

Abstract: A nutrient medium for protein producing cells, characterized in that said medium consists essentially of the following components: a physiological saline containing Ca2+, K+, Mg2+ and Na+, an energy source, a pH buffer and methionine in an amount of 0.015-2.0 g/liter, and optionally antibiotics. The invention further relates to use of the nutrient medium for cells and human leukocytes.

Abstract: A method for sequencing a target DNA fragment in which along with amplification of the target DNA fragment, nucleic acid transcripts are generated using an RNA polymerase and the amplified target DNA fragments are used as templates in the presence of terminators for nucleic acid transcription reaction and the generated nucleic acid transcripts are analyzed, characterized in that the amplification of target DNA fragments and the generation of nucleic acid transcripts are carried out at a constant temperature is disclosed. The amplification of target DNA fragments and the generation of nucleic acid transcripts can be carried out around the room temperature. A DNA sequencing method using a novel method in which without using a thermo-resistant RNA polymerase, the amplification of target DNA fragments and generation of nucleic acid transcript can be carried out simultaneously in parallel is provided.

Abstract: A method for detecting the presence of a target nucleic acid sequence in a sample is provided. The method comprises subjecting the sample to an amplification reaction to obtain an amplification product where the target nucleic acid sequence is present using a set of nucleotides, at least one of which is fluorescently labelled. The amplification product is contacted with a probe under conditions in which the probe will hybridise to the target sequence. The probe comprises a reactive molecule which is capable of absorbing fluorescence energy from or donating fluorescent energy to the fluorescent labelled nucleotide. The fluorescence of the sample is monitored and related to the presence of the target nucleic acid sequence. The method can be used to quantitate the amount of target nucleic acid in the sample as well as to determine sequence characteristics. Kits for effecting the method are also provided.

Abstract: The present invention provides an alternative PCR amplification which does not draw upon the use of thermostable DNA polymerases. It provides means for the controlled manipulation of denaturing conditions which do not demand the use of high denaturing temperature. More particularly, it provides means for the controlled oscillation of divalent metal ions, preferably of divalent metal ions such as Cu2+, Zn2+, Mn2+ and Cd2+, which are known to destabilize the DNA helix and thereby decrease the melting temperature of the DNA helix. The invention also provides methods for the automatization of this process. For instance, by means of cathodic reduction of the divalent metal species the concentration can be decreased to levels that allows for reannealing of separated sands with the primers; while oxidation of deposited metals or oxidation of monovalent metal ions, can restore the initially high concentration that allows for separation of both strands that make up the DNA helix.

Abstract: The invention relates to a method for enzyme stabilization. A method for improved reverse transcription at high temperatures is provided, wherein a thermostable heat shock protein (HSPs) stabilizes a reverse transcriptase, as well as reduces the RNase H activity of said reverse transcriptase. The present invention thus relates to a stabilizing agent, that prevents thermal denaturing and enhances thermostability of a reverse transcriptase. The invention further relates to a method of producing a polypeptide complex consisting of a Chaperonin and a Moloney murine leukemia virus (MMVL) reverse transcriptase, characterized by having enhanced thermostability as well as reduced RNase H activity, compared to a (MMVL) reverse transcriptase alone. The invention further relates to a kit for the preparation of cDNA from mRNA, comprising either both stabilizing agent and reverse transcriptase or the polypeptide complex of the invention.

Abstract: Polynucleotide probes and accessory helper oligonucleotides useful for detecting the subset of High (G+C) subset of Gram-positive bacteria known as the “Actinomycetes.” The hybridization probes are highly specific for the Actinomycetes and do not cross-hybridize with the rRNA or rDNA of numerous other bacterial and fungal species.

Abstract: The present invention provides a method that is useful to predict an individual's increased risk of developing Alzheimer's disease. The method comprises the steps of: (a) collecting a biological sample from an individual; and (b) analyzing the biological sample for the presence of IL-1A 2,2 (−889) genotype, IL-1B 2,2 (+3953) genotype, or a combination of IL-1A 2,2 (−889) and IL-1B 2,2 (+3953) genotypes, the presence of any of the genotypes indicating that the individual is at risk of developing Alzheimer's disease.

Abstract: The present invention relates, in general, to cancer prognosis and, in particular, to a method of assessing the prognosis of a patent using the M6P/IGF-II receptor.