- This ready-to-use nucleotide mix is a premixed solution of the sodium salts of dATP, dGTP, and dCTP, each at a concentration of 10 mM, and dUTP at a concentration of 30 mM in water. This mix is optimized for use in all types of amplification reactions: PCR
- RT-PCR
- Prevention of carryover contamination
To allow decontamination of PCR or RT-PCR, dUTP in place of dTTP is incorporated into the PCR product. Subsequent reactions may then be treated with Uracil-DNA Glycosylase (UNG). Avoiding the need of re-opening the reaction vial, the vials are incubated at +20 °C, resulting in the degradation of potentially contaminating uracil-containing amplification products. During this step, template DNA and RNA remain unaffected, since normal DNA does not contain uracil, and RNA does not serve as a substrate for UNG. Before starting the actual thermocycling program, UNG is inactivated by incubation at +95 °C. Uracil -DNA Glycosylase, heat-labile is particularily useful, as it is fully inactivated already after incubation at +95 °C for 2 minutes. The natural enzyme from
E. coli requires incubating the reaction mixture for 10 minutes at +95 °C. The shorter heat treatment substantially reduces the risk for loosing the template nucleic acid, which typically is present at low concentrations only. This is of particular importance, when performing RT-PCR. We therefore recommend the use of Uracil-DNA Glycosylase.
It has been used in LightCycler PCR assay to identify B. pertussis and B.parapertussis in nasopharyngeal swabs. It has also been used in quantitative PCR (qPCR) assay.
