Method for identifying the polymorphic sequences 4a/4b of the nitric oxide endothelial synthetase gene

• Main Authors: TODERAS ION, ISTRATI VALERIU, CAPROS NICOLAE, MOVILA ALEXANDRU, TODERAS LIDIA
• Format: Patent
• Language: English; Moldavian; Romanian
• Published: 31.12.2013
• Subjects: BEER; BIOCHEMISTRY; CHEMISTRY; COMPOSITIONS OR TEST PAPERS THEREFOR; COMPOSITIONS THEREOF; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL ORENZYMOLOGICAL PROCESSES; CULTURE MEDIA; DIAGNOSIS; ENZYMOLOGY; HUMAN NECESSITIES; HYGIENE; IDENTIFICATION; MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEICACIDS OR MICROORGANISMS; MEDICAL OR VETERINARY SCIENCE; METALLURGY; MICROBIOLOGY; MICROORGANISMS OR ENZYMES; MUTATION OR GENETIC ENGINEERING; PROCESSES OF PREPARING SUCH COMPOSITIONS; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; SPIRITS; SURGERY; VINEGAR; WINE

The invention relates to the medical molecular genetics, namely to a method for identifying the polymorphic sequences 4a/4b of the nitric oxide endothelial synthetase gene.Summary of the method consists in that is carried out the whole blood sampling, is extracted DNA with 0.7 M ammonium hydroxide, is maintained at room temperature for 5 minutes and incubated in a thermostat at the temperature of 100°C for 30 minutes with continuous stirring, then the extracted the DNA is amplified using sense NOS3: 5'-GCCCTATGGTAGTAGTGCCTTT-3' and antisense NOS3 primers: 5'-SCTCTTAGTGCTGTGGTCA-3', at the same time is carried out the initial denaturation at the temperature of 94°C for 3 min, then are performed 30 cycles with denaturation at the temperature of 94°C for 1 min, at the temperature of 54°C for 30 seconds, at the temperature of 72°C for 30 seconds and at the temperature of 72°C for 5 minutes, are separated the amplified DNA fragments in 1.8% agarose gel, are stained with 0.5 µg/mL solution of ethidium bromide and are identified the polymorphic sequences. Inventia se refera la genetica medicala moleculara, si anume la o metoda de identificare a secventelor polimorfe 4a/4b ale genei sintetazei endoteliale a oxidului nitric.Esenta metodei consta în aceea ca se colecteaza proba integrala de sânge, se extrage ADN cu hidroxid de amoniu 0,7 M, se mentine la temperatura camerei timp de 5 min si se incubeaza într-un termostat la temperatura de 100ºC timp de 30 min cu agitare continua, apoi ADN extras se amplifica cu utilizarea primerilor NOS3 sens: 5´-GCCCTATGGTAGTAGTGCCTTT-3´ si NOS3 antisens: 5´- CCTCTTAGTGCTGTGGTCA-3´, totodata se efectueaza denaturarea initiala la temperatura de 94°C timp de 3 min, apoi se efectueaza 30 de cicluri cu denaturarea la temperatura de 94°C timp de 1 min, la temperatura de 54°C timp de 30 s, la temperatura de 72°C timp de 30 s si la temperatura de 72°C timp de 5 min, se separa fragmentele amplificate de ADN în gel de agaroza de 1,8%, se coloreaza cu o solutie de bromura de etidiu de 0,5 µg/mL si se identifica secventele polimorfe.