Abstract: Methods for determining quantities of nucleic acid sequences in samples include the steps of amplifying a plurality of known quantities of a nucleic acid sequence in respective calibration samples and an unknown quantity of a nucleic acid sequence in a test sample, in parallel, during a time interval. These samples may be amplified using an isothermal amplification method such as Strand Displacement Amplification (SDA), or a thermal cycling amplification method such as Polymerase Chain Reaction (PCR), for example. Indicia of the quantities of a nucleic acid sequence being amplified in the calibration and test samples are then measured using conventional techniques, at measurement points in the time interval. Steps are then performed to determine for a first potential cutoff level, a corresponding first set of time points in the time interval at which the indicia of the quantities of a nucleic acid sequence being amplified in each of the calibration samples equal the first cutoff level.

Abstract: A process for lysing Mycobacteria to liberate nucleic acids such as DNA and RNA comprises heating the Mycobacteria in an aqueous solution to lyse the Mycobacteria and release the nucleic acids, with the aqueous solution containing a chelating agent such as EDTA or EGTA in an amount effective to inhibit degradation of the released nucleic acids. Examples of Mycobacteria which can be lysed include Mycobacterium fortuitum and Mycobacterium tuberculosis. After lysis, the nucleic acid is preferably then amplified and detected. Kits useful for carrying out the present invention are also disclosed.

Abstract: Single-stranded signal primers are modified by linkage to two dyes which form a donor/acceptor dye pair. The two dyes are positioned in sufficiently close spatial proximity on the signal primer that the fluorescence of the first dye is quenched by the second dye. The signal primer may further comprise a restriction endonuclease recognition site (RERS) between the two dyes. As the signal primer is initially single-stranded and remains single-stranded in the absence of target, the restriction endonuclease recognition site is not cleavable or nickable by the restriction endonuclease. In the presence of target, however, signal primer and the restriction endonuclease recognition site are rendered double-stranded and cleavable or nickable by the restriction endonuclease. Cleavage or nicking separates the two dyes and a change in fluorescence due to decreased quenching is detected as an indication of the presence of the target sequence or of target sequence amplification.

Abstract: Methods employing internal oligonucleotide standards in isothermal nucleic acid amplification reactions to determine the efficacy of the amplification reaction and to quantify pre-amplification target levels.

Abstract: Cell culture substrates comprising dried films of native fibrillar collagen produced by a method in which collagen fibers are hydrolyzed in acid, solubilized, and reformed as gels on porous surfaces under non-physiologic salt conditions to produce large fibers with the striations characteristic of collagen fibers found in vivo. The gels are collapsed onto the porous surfaces by drawing the interfibril fluid out of the gel through the underside of the porous surface and then dried to form films. Dried collagen films made in this manner retain native fibrillar collagen structure and excellent diffusion characteristics. Native fibrillar collagen films produced according to the methods of the invention are useful as cell culture substrates. They have particularly advantageous properties for growth and differentiation of epithelial cells. This effect is synergistically enhanced by addition of butyric acid as a differentiation inducing agent.

Abstract: Amplification primers useful in assays for species-specific detection of a target sequence in the cryptic plasmid of C. trachomatis are described. The primers of the invention amplify a target in the region of nucleotides 2219-2366 of the cryptic plasmid sequence, and the target binding sequences disclosed may be adapted for use in amplification primers for a variety of amplification reactions.

Abstract: Primers and methods for adapter-mediated multiplex amplification of the IS6110 insertion element of the Mycobacterium tuberculosis (M.tb) complex and a 16S rDNA target common to essentially all mycobacteria are described. In certain embodiments, the primers are optimized for efficient multiplex amplification in thermophilic SDA. The multiplex Strand Displacement Amplification methods of the invention are capable, in a single amplification reaction, of simultaneously identifying M. tuberculosis and providing a screen for substantially all of the clinically relevant species of mycobacteria. Also disclosed are internal control sequences designed for coamplification with the two targets, allowing assessment of amplification efficiency and/or quantitation of the targets.

Abstract: Fluorescence polarization methods for detection of nucleic acid amplification at thermophilic temperatures employ a fluorescently labeled oligonucleotide signal primer which is converted from single- to double-stranded form in a target amplification-dependent manner. This conformational change is accompanied by an increase in fluorescence polarization values. The decrease in FP typically observed for the duplex at elevated temperatures is overcome by double-stranded DNA binding proteins which are believed to stabilize the double-stranded structure by reducing the single-strandedness normally associated with higher temperatures. The inventive methods provide a closed, homogeneous system for amplification and detection of amplification in real-time or at an endpoint.

Abstract: A process for lysing Mycobacteria to liberate nucleic acids such as DNA and RNA comprises heating the Mycobacteria in an aqueous solution to lyse the Mycobacteria and release the nucleic acids, with the aqueous solution containing a chelating agent such as EDTA or EGTA in an amount effective to inhibit degradation of the released nucleic acids. Examples of Mycobacteria which can be lysed include Mycobacterium fortuitum and Mycobacterium tuberculosis. After lysis, the nucleic acid is preferably then amplified and detected. Kits useful for carrying out the present invention are also disclosed.

Abstract: Cell culture substrates made of dried films of native fibrillar collagen produced by a method in which collagen fibers are hydrolyzed in acid, solubilized, and reformed as gels on porous surfaces under non-physiologic salt conditions to produce large fibers with the striations characteristic of collagen fibers found in vivo. The gels are collapsed onto the porous surfaces by drawing the interfibril fluid out of the gel through the underside of the porous surface and then dried to form films. Dried collagen films made in this manner retain native fibrillar collagen structure and excellent diffusion characteristics. Native fibrillar collagen films produced according to the methods of the invention are useful as cell culture substrates. They have particularly advantageous properties for growth and differentiation of epithelial cells. This effect is synergistically enhanced by addition of butyric acid as a differentiation inducing agent.

Abstract: Thermophilic Strand Displacement Amplification (tSDA) for amplification of nucleic acid target sequences in situ in cells in suspension, on slides or in tissues is described. Excellent specimen morphology is preserved, and either DNA targets, RNA targets, or both may be selectively amplified. In situ amplification by tSDA is compatible with immunochemical techniques, so that both amplification of target sequences and immunological staining can be perfonned on the same specimen.

Abstract: A newly-identified M. kansasii DNA sequence is described and designated KATS1. This sequence is highly M. kansasii-specific in nucleic acid hybridization assays and is useful for detecting both typical and atypical strains. KATS1 and segments of the KATS1 sequence are useful for species detection of M. kansasii in hybridization or nucleic acid amplification assays with 100% specificity and no cross-reactivity to non-M. kansasii species.

Abstract: Strand Displacement Amplification methods (thermophilic SDA) which can be performed over a broad temperature range (37.degree. C. to 70.degree. C.). The preferred temperature range for thermophilic SDA is 50.degree. C. to 70.degree. C. It has been found that certain thermophilic restriction endonucleases are capable of nicking the hemimodified restriction endonuclease recognition/cleavage site as required by SDA and dissociating from the site. It has further been found that certain thermophilic polymerases are capable of extending from the nick while displacing the downstream strand. Thermophilic SDA, because of reaction temperatures higher than previously possible with conventional SDA enzyme systems, has improved specificity and efficiency, reduced nonspecific background amplification, and potentially improved yields of amplification products.

Abstract: Cell culture substrates comprising dried films of native fibrillar collagen produced by a method in which collagen fibers are hydrolyzed in acid, solubilized, and reformed as gels on porous surfaces under non-physiologic salt conditions to produce large fibers with the striations characteristic of collagen fibers found in vivo. The gels are collapsed onto the porous surfaces by drawing the interfibril fluid out of the gel through the underside of the porous surface and then dried to form films. Dried collagen films made in this manner retain native fibrillar collagen structure and excellent diffusion characteristics. Native fibrillar collagen films produced according to the methods of the invention are useful as cell culture substrates. They have particularly advantageous properties for growth and differentiation of epithelial cells. This effect is synergistically enhanced by addition of butyric acid as a differentiation inducing agent.

Abstract: A human brain glycoprotein homologous to the mouse F3 and the chicken contactin/F11 adhesion molecules, nucleic acid sequences encoding the human brain glycoprotein and antibodies directed against the human brain glycoprotein.

Abstract: A novel Type I DNA polymerase derived from Bacillus pallidus is provided. The gene encoding the polymerase has been cloned and expressed. Characterization of the enzyme revealed, in addition to DNA polymerase activity, 5'-3' exonuclease activity, reverse transcriptase activity, strand displacing activity and the ability to initiate polymerization at a nick in double-stranded DNA. In certain constructs the domain encoding the 5'-3' exonuclease activity is deleted to provide an enzyme which is particularly useful for Strand Displacement Amplification.

Abstract: Primers and methods for adapter-mediated multiplex amplification of the IS6110 insertion element of Mycobacterium tuberculosis (M.tb) and the 16S ribosomal gene of Mycobacterium tuberculosis, useful for simultaneously detecting and/or identifying species of the M. tuberculosis complex and other clinically relevant Mycobacterium species. Multiplex Strand Displacement Amplification (SDA) is used in a single amplification reaction which is capable of simultaneously identifying M. tuberculosis and providing a screen for substantially all of the clinically relevant species of Mycobacteria. Also disclosed are methods for adapter-mediated multiplex amplification of multiple target sequences and a single internal control sequence for determination of sample efficacy or quantitation of the targets.

Abstract: Thermophilic Strand Displacement Amplification (tSDA) for amplification of nucleic acid target sequences in situ in cells in suspension, on slides or in tissues is described. Excellent specimen morphology is preserved, and either DNA targets, RNA targets, or both may be selectively amplified. In situ amplification by tSDA is compatible with immunochemical techniques, so that both amplification of target sequences and immunological staining can be performed on the same specimen.

Abstract: Cell culture substrates comprising dried films of native fibrillar collagen produced by a method in which collagen fibers are hydrolyzed in acid, solubilized, and reformed as gels on porous surfaces under non-physiologic salt conditions to produce large fibers with the striations characteristic of collagen fibers found in vivo. The gels are collapsed onto the porous surfaces by drawing the interfibril fluid out of the gel through the underside of the porous surface and then dried to form films. Dried collagen films made in this manner retain native fibrillar collagen structure and excellent diffusion characteristics. Native fibrillar collagen films produced according to the methods of the invention are useful as cell culture substrates. They have particularly advantageous properties for growth and differentiation of epithelial cells. This effect is synergistically enhanced by addition of butyric acid as a differentiation inducing agent.

Abstract: A human brain glycoprotein homologous to the mouse F3 and the chicken contactin/F11 adhesion molecules is provided. Also described are nucleic acid sequences encoding the human brain glycoprotein and antibodies directed against the human brain glycoprotein.