Quantifying Uracil Content in DNA

In order to quantify the uracil content of DNA, a real-time quantitative PCR-based assay was used^{38 (link)}. Genomic DNA was isolated and digested with BamHI. DNA fragments of 5 kb were purified from gel. Real-time PCR was performed on a Mx3000P qPCR System (Agilent Technologies) using EvaGreen dye (Biotium) and PfuTurbo Hotstart DNA polymerase (Stratagene) and Mytaq Hotstart DNA polymerase (Bioline). A segment with 1017 base length defined by the primers (Table S41) was amplified during the PCR reaction. Two-fold dilution series were prepared from the DNA samples. Three parallel strains were used for each mutation in the experiments.