Impact of thermal treatment on genomic DNA integrity and quantification bias in digital PCR

Preserving DNA integrity, defined as intact strands and correct sequence context, is crucial to the reliability of measurements and interpretations in molecular methods. The denaturation temperature of genomic DNA is influenced by several factors, including GC content, ionic strength, and the presence of stabilizing or destabilizing agents. In this work, we report the investigation of human genomic DNA denaturation behavior under low salt and high temperature conditions. We used digital PCR as the primary analytical tool for DNA stability which could quantify the extent of DNA strand separation. To ensure that observed changes reflected structural alterations rather than DNA degradation or evaporation, we employed complementary analytical approaches including quantitative PCR and isotope dilution liquid chromatography with tandem mass spectrometry. Despite the widespread use of heat inactivation protocols in restriction enzyme workflows, there remains a significant gap in our understanding of how these treatments affect genomic DNA structure. Our findings have important implications for the reliability and reproducibility of molecular biology procedures, particularly those that depend on precise DNA quantification or structural integrity.