In the context of PCR and DNA amplification, 'carryover contamination' refers to the accidental transfer of previously amplified DNA (amplicons) into new PCR reactions. This contamination can lead to false positives, where the detected signal is due to these contaminant DNA molecules rather than the target DNA from the sample being tested.

If preventing carryover contamination is a concern in your PCR process, pre-treatment with UDG is typically necessary and beneficial.

Hi Carla! Thanks for your reply, I was wondering about the method of detecting amplicons using dUTP labelled with a fluorophore. Why this type of nucleotide? Why not use labelled dTTP, for example?

The reason why dutp is used is to minimise pcr contamination. If pcr product (normal) gets into the working area or one of your reagents then it will amplify well and swamp all the other amplified samples and also lead to the no template pcr control being positive. If we use dUTP then we start every pcr with an incubation with UDG this reaction destroys all of the carried over contamination and the new pcr samples will be clear of contamination. It is convenient to destroy all U containing dna as this does not exist in nature but using any other base and an enzyme that degrades that base would destroy your starting dna unless the enzyme was fully inactivated before starting the pcr

While theoretically possible, labeling dATP, dCTP, or dGTP could modify the DNA structure or interfere with the efficiency of the DNA polymerase. Uracil (U) is naturally recognized by many enzymes as a substitute for thymine (T) in the context of DNA, which makes dUTP a convenient choice for such modifications without drastically affecting the overall structure and function of the DNA.

Thanks Carla Teixeira !