Verifikace thermocyclerů a optimalizace podmínek PCR na Ústavu lékařské genetiky FNOL

Abstract

The thesis focuses on a comparative analysis of thermocyclers, devices used for amplifying selected DNA segments, according to specific assays. The amplified segments are subsequently analyzed using additional technologies, such as capillary electrophoresis, enabling the separation and quantification of the resulting products. The yield of polymerase chain reaction (PCR) is influenced by the quality and quantity of input DNA, the correct ratio of reagents, and the settings of the used system. Ensuring temperature stability in the block where the PCR reaction tubes are placed and rapidly adapting to temperature changes at recurring intervals are crucial. The aim of the thesis is to verify the thermocycler according to an internal procedure, based on the ČSN EN ISO 15189 - ed.3 standard, and to optimize PCR conditions using various concentrations of input DNA and different heating rates during PCR according to the set temperature profile in the thermocycler. All using certified kits from manufacturers, intended for the examination of cystic fibrosis (CF), dihydropyrimidine dehydrogenase (DPYD), and for determining the clinical diagnosis of Peutz-Jeghers syndrome (PJS) using SALSA MLPA Probemix P101 STK11. Temperature stability will be verified using the Multiplex ligation-dependent probe amplification (MLPA) method, where it is necessary to maintain a stable temperature for at least 16 hours. The resulting products will be analyzed using fragment analysis with capillary electrophoresis.