Cod Uracil-DNA Glycosylase

Cod UNG is the only UNG compatible with one-step RT-qPCR

In reverse transcriptase qPCR (RT-qPCR), RNA is the initial template. However, the problem of carry-over contaminants can be as much of a problem here as in regular PCRs. Following reverse transcription, cDNA is the template for the PCR. If your sample is contaminated with carry-over PCR products from previous PCRs, the primers cannot distinguish between cDNA and carry-over DNA, resulting in erroneous results.

In one-step RT-qPCR kits, RNA is added to a master mix containing both reverse transcriptase and polymerase, allowing cDNA synthesis and qPCR in the same tube. E. coli UNG/UDG has an optimal working temperature up to approx. 50˚C and generally retains its activity up to approx. 70˚C. This temperature range is not compatible with carry-over prevention in one-step RT-qPCR protocols, as E.coli UNG/UDG would remove uracil incorporated into the cDNA during reverse transcription, thereby causing degradation of template.

Recombinantly expressed Cod UNG from ArcticZymes was originally isolated from the cold-adapted organism Atlantic cod. Cod UNG is highly active at temperatures ranging from 20˚C to 40˚C, quickly lose activity at temperatures above 42˚C and is irreversibly inactivated already at 55˚C. Since the optimum temperature range of Cod UNG is considerably lower compared to that of E. coli UNG/UDG, Cod UNG is compatible with use in single tube RT-qPCRs. Carry-over prevention is simply carried out by adding Cod UNG to a final concentration of 0.01 U/µl and introduce a 5 minute incubation step at 25˚C prior to initiation of RT-qPCR. Alternatively, the concentration can be increased to 0.04 U/µl and the pre-incubation step omitted. Cod UNG will efficiently remove carry-over contamination during sample setup and cycler ramping.

Here we show that Cod UNG can be used for carry-over prevention in a commercial one-step RT-qPCR master mix containing dUTPs instead of dTTPs. Treatment with Cod UNG did not affect target cDNA, yielding the same Cq-values as untreated samples. For comparison, treatment with a generic UNG resulted in significant Cq delay, demonstrating incompatibility with one-step RT-qPCR.