dUTP and dUTP Mixes

Carryover of previously amplified PCR products is the single most significant source of contamination in subsequent PCR. Several methods have been used to prevent carryover contamination, such as physical containment and chemical treatments.1 One of the most convenient and effective strategies is to include dUTP and uracil DNA glycosylase (UDG) in PCR reactions. In this system, dUTP is used to replace TTP in the PCR reaction so that all PCR products will contain dUTP. Before commencing each PCR, fully assembled reactions are treated with UDG, which cleaves the uracil base in dUTP -containing DNA (amplicon) but does not react with dUTP and has no effect on authentic DNA template containing thymidine bases or RNA. After initial treatment, UDG can be inactivated by heat denaturation prior to the PCR. New PCR product, amplified from authentic DNA template, though containing dUTP, is not destroyed. The degraded DNA is not suitable for use as a hybridization target or as a template for DNA polymerases so cannot be reamplified or serve as a contaminant in subsequent reactions.