Abstract: The present invention is directed to a method of extracting nucleic acids from samples containing prokaryotic cells and/or eukaryotic cells by (a) lysing the cells under conditions that minimize RNA expression or enzymatic degradation of RNA; (b) dispersing and diluting the lysed cells; (c) mixing the dispersed and diluted lysed cells with a buffered solution containing cationic and anionic detergents; (d) separating non-nucleic acid contaminants from the nucleic acids by mixing with an organic extraction solvent to generate an emulsion that separates into an organic phase, an interface containing the contaminants and an aqueous phase containing the nucleic acids; (e) separating the phases, and retaining the aqueous phase; and (f) precipitating the nucleic acids in the aqueous phase.
Abstract: A novel limited primer extension reaction improves detection sensitivity and specificity in a variety of hybridization platforms. In the invention, a sequence of target DNA that lacks one of the four types of nucleic acid bases for a span of eight or more adjacent nucleotide positions is selected for use. This sequence is referred to as the extension complement sequence, or ECS. A primer with a sequence that is complementary to the target sequence that is immediately downstream (to the 3? side) of this ECS is used to initiate an extension reaction. Extension occurs using a DNA polymerase and standard deoxynucleoside triphosphates for three of the four types of nucleic acid bases. The fourth base, which is complementary to the base missing in the ECS, is either absent or present only in the form of a dideoxynucleoside triphosphate, which does not support further extension. In either case, the extension reaction does not proceed past the first occurrence in the template of the base that is missing in the ECS.
Abstract: A novel polymeric nucleic acid probe improves detection sensitivity and specificity in a variety of hybridization platforms. The probe is made up of multiple short nucleic acid sequences (referred to as monomers) attached together to form a long polymeric probe for use in hybridization applications. For applications requiring immobilization of the probes to a surface, the polymeric probes are similar to long DNA probes in that they can be immobilized to a variety of surfaces without need for a chemical modification to the end of the probe. Because target nucleic acids hybridize to the relatively short monomers in the polymeric probe, the polymeric probes are more specific than long DNA probes. In addition, polymeric probes also improve the signal-to-background ratio by increasing the number of accessible monomer oligonucleotide probes immobilized per unit area on a surface.