The digital polymerase chain reaction (dPCR) is a novel method with numerous advantages over conventional PCR techniques, such as increased sensitivity, accuracy and specificity (Quan et. all, 2018). dPCR also enables multiplex approach, where multiple targets can be simultaneously detected and quantified within the same genome (Furuta-Hanawa et. all, 2019). The aim of this master's thesis was to develop, test and optimize a multiplex approach for evaluating the integrity of a chosen nucleic acid. As the nucleic acid research object, we used artificially synthesized construct double stranded DNA (gBlock). The objects of research were two artificial constructs in which we combined the sequence of 4 or 5 previously published amplicons and among them inserted target sites for the restriction enzymes. In the first part, with a gBlock with four amplicons, we showed that dPCR can determine the integrity of nucleic acids, what was confirmed by testing differently fragmented samples. In the second part, we used the gBlock containing five amplicons in which both ends consisted of the same target sequence (ITR region) mimicking the structure of adenovirus like viruses (AAV). We found that this gBlock is not suitable for dPCR analysis as we cannot determine if the ITR signal comes from beginning or end of gBlock. We also compared the results of two dPCR systems, QX ONE and QIAcuity, found that results coincide with minor differences due to variations in setting the baseline threshold. Another cause of deviations from expected results was random breaks at the fragile ends of the gBlock or imperfect cuts in the preparation of fragments. We also showed that increasing the concentration of targets gives better integrity due to more fragments can be arranged in the same partition.
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