METHOD FOR POLYMERASE ACTIVITY DETERMINATION

• Main Authors: HAGSTROEM, ASA, MARGARETA, NEUMUELLER, THORD, MAGNUS, GRONOWITZ, JAN-SIMON, KAELLANDER, CLAS, FREDRIK, RUNESSON, KARLSTROEM, ROLF, ANDERS, LANGSTROEM-PERSSON, ULLA, M, WELTMAN, EVA, HELENA
• Format: Patent
• Language: English; French; German
• Published: 25.09.1991
• Subjects: ACCESSORIES, CLOSURES, OR FITTINGS THEREFOR; AFTER-TREATMENT OF THE SHAPED PRODUCTS, e.g. REPAIRING; BEER; BIOCHEMISTRY; CHEMISTRY; COMPOSITIONS OR TEST PAPERS THEREFOR; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL ORENZYMOLOGICAL PROCESSES; CONTAINERS FOR STORAGE OR TRANSPORT OF ARTICLES OR MATERIALS,e.g. BAGS, BARRELS, BOTTLES, BOXES, CANS, CARTONS, CRATES,DRUMS, JARS, TANKS, HOPPERS, FORWARDING CONTAINERS; CONVEYING; DERIVATIVES THEREOF; ENZYMOLOGY; HANDLING THIN OR FILAMENTARY MATERIAL; INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIRCHEMICAL OR PHYSICAL PROPERTIES; MAKING ARTICLES OF PAPER, CARDBOARD OR MATERIAL WORKED IN A MANNERANALOGOUS TO PAPER; MAKING CONTAINERS OF PAPER, CARDBOARD OR MATERIAL WORKED IN AMANNER ANALOGOUS TO PAPER; MEASURING; MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEICACIDS OR MICROORGANISMS; METALLURGY; MICROBIOLOGY; MUTATION OR GENETIC ENGINEERING; NUCLEIC ACIDS; NUCLEOSIDES; NUCLEOTIDES; ORGANIC CHEMISTRY; PACKAGES; PACKAGING ELEMENTS; PACKING; PERFORMING OPERATIONS; PHYSICS; PROCESSES OF PREPARING SUCH COMPOSITIONS; SHAPING OF MATERIAL IN A PLASTIC STATE, NOT OTHERWISE PROVIDEDFOR; SHAPING OR JOINING OF PLASTICS; SPIRITS; STORING; SUGARS; TESTING; TRANSPORTING; VINEGAR; WINE; WORKING OF PLASTICS; WORKING OF SUBSTANCES IN A PLASTIC STATE, IN GENERAL; WORKING PAPER, CARDBOARD OR MATERIAL WORKED IN A MANNER ANALOGOUSTO PAPER

L'invention a pour objet une méthode de détection quantitative de l'activité de polymérase d'acide nucléique dans un échantillon, comprenant l'incubation de l'échantillon avec un gabarit polynucléotide naturel ou complètement ou partiellement synthétique et une solution réactive contenant les substrats nécessaires y compris au moins un triphosphate nucléoside radiomarqué complémentaire au gabarit, la séparation du gabarit du substrat, et la mesure de la radioactivité introduite dans le gabarit, laquelle radioactivité est proportionnelle à l'activité de polymérase dans l'échantillon, et dans laquelle lorsqu'une amorce est nécessaire, celle-ci est soit hybridée au gabarit dès le début soit ajoutée pendant la détection. Selon l'invention, le gabarit est immobilisé à un porteur, ledit triphosphate nucléoside est marqué d'un isotope à rayonnement gamma, et la mesure de la radioactivité introduite est effectuée directement sur le gabarit immobilisé sans que celui-ci soit séparé du porteur. L'invention se rapporte également à un dispositif de détection de l'activité de polymérase d'acide nucléique, comprenant un gabarit lié au porteur et un substrat marqué d'un isotope à rayonnement gamma. The invention relates to a method of quantitatively determining nucleic acid polymerase activity in a sample, comprising incubation of the sample with a natural or completely or partially synthetic polynucleotide template and a reagent solution containing the necessary substrates including at least one radiolabeled nucleoside triphosphate complementary to the template, separation of the template from the substrate, and measurement of the radioactivity incorporated into the template, which radioactivity is substantially proportional to the polymerase activity in the sample, and wherein, when a primer is required, the latter is either hybridized to the template from the start or added during the determination. According to the invention the template is immobilized to a carrier, said nucleoside triphosphate is labeled with a gamma-radiating isotope, and the measurement of the incorporated radioactivity is performed directly on the immobilized template without release thereof from the carrier. The invention also relates to a kit for determining of nucleic acid polymerase activity, comprising a carrier-bound template and a substrate labeled with a gamma-radiating isotope.