METHOD FOR DETERMINING NUCLEIC ACIDS BY REAL-TIME POLYMERASE CHAIN REACTION AND A DEVICE FOR THE IMPLEMENTATION THEREOF

摘要

The invention refers to molecular biology, medicine, biotechnology and is related to performance of Polymerase Chain Reaction and device for its implementation with real-time registration of reaction-product build-up. The task of the invention is solved as a result of using the method and device for its implementation for identification of nucleic acids by a real-time polymerase chain reaction including introduction of liquid samples containing nucleic acid into the reaction zones on the upper surface of the heat-conducting substrate of the microchip; isolation of the introduced samples from the atmosphere; contact of the nucleic acid of the sample with components of the polymerase chain reaction during thermocycling of the samples with heat removal through the outer surface of the microchip; fluorescent detection of the change of the quantity of the polymerase chain reaction products during thermocycling; identification of the quantity of the initial nucleic acid in the samples by the dynamic of growth of the fluorescent signal wherein the microchip used contains a heat-conducting substrate from a heat-conducting material with the thermal conductivity coefficient of more than 1 W/cm·K and the thermal diffusivity coefficient of more than 0.6 cm 2 /s while the reaction zones on the microchip surface are separated from the heat-conducting substrate by a layer of the passivating material covalently bound to the surface of the heat-conducting substrate, and the introduced samples are isolated by separating them from the atmosphere by a layer of liquid immiscible with water that is retained on the upper surface of the heat-conducting substrate by means of a peripheral barrier, with the ratio of the aggregate thermal mass of the microchip with the introduced samples and the layer of the liquid immiscible with water to thermal conductance of the microchip substrate not exceeding 0.04 s. The technical result is reduced duration of the analysis, higher reliability, accuracy, efficiency and cost-effectiveness of the analysis. 27 claims, 5 Figs.