| 摘要 |
FIELD: medicine; biotechnologies. ^ SUBSTANCE: method includes cloning of investigated sequence in gene gyrA DNA M. tuberculosis (MET) a method of polymerase chain reaction (PCR), with further denaturation of PTSR - a product at presence of the denaturating solution for obtaining of one-chained fragments of DNA. Further reveal in them mutations by division of the given fragments in a polyacrylamide gel. For carrying out of PCR the pair of primers has been picked up: 5 ' CTA TGC AAT GTT CGA TTC CGG CTT C 3 ' and 5 ' ACT GTC TCC TCG TCG ATT TCC CT 3 ', and amplification modes (1st stage - 95° - 4 minutes; 2nd stage - 95° - 30 sec, 65° - 40 sec, 72° - 40 sec (40 cycles); 3rd stage: 72° - 4 minutes; 10° - storage), allowing to clone DNA M. tuberculosis not only from evolved cultures, but also it is direct from biological secrets (spittle, BSA). Denaturation of the obtained amplicons is performed in a denaturating solution, destabilising a double spiral at heating. The electrophoretic separation of the obtained one-chained fragments of DNA perform in the 8 % polyacrylamide gel with about 5 % glycerine content at pressure of 400 volt within 5 hour at 8°C. Presence of mutations in an investigated gene is defined by comparison of degree of a divergence of the denaturated DNA threads of investigated and sensitive strains M. tuberculosis to fluoroquinolones. ^ EFFECT: method allows to spend definitions to shorter terms, it is accessible and safe. |
