PCR carry-over prevention
The high sensitivity of PCRs, and qPCRs in particular, makes the method prone to contamination, giving false or inaccurate results. Carry-over contaminants from previous PCRs are considered to be one of the major sources of false positive results. The contaminants may be carried over from previous amplification reactions due to aerosolizaton, contaminating pipettes, surfaces, gloves and reagents.
There are two common strategies to prevent carry-over contaminants when amplifying DNA and RNA. One is to have a separate lab for set-up and amplification, minimize the number of pipetting steps, and prevent opening of the tube after amplification. However, this is not always possible due to practical reasons. Moreover, it offers no guarantee for avoiding carry-over contamination.
The other most common strategy to prevent carry-over contamination is to partially or completely replace dTTP with dUTP during PCR amplification, thereby producing DNA containing uracil. Prior to initiating PCR, the PCR mixture is treated with Uracil-DNA Glycosylase (UNG). During the initial denaturation step temperature is elevated to 95°C, resulting in cleavage of apyrimidinic sites and fragmentation of carry-over DNA. As the template contains thymidine, it will not be affected by the UNG treatment. It is a prerequisite that all PCRs are carried out with dUTP substituting dTTP.
The benefit of using our Cod UNG is that it is irreversibly inactivated at 55ËšC. Cod UNG is the only UNG compatible with one-step RT-qPCR!
PCR carry-over prevention in 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. 50ËšC and generally retains its activity up to approx. 70ËšC. 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 20ËšC to 40ËšC, quickly lose activity at temperatures above 42ËšC and is irreversibly inactivated already at 55ËšC. 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 25ËšC 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.
Figure 1. Cod UNG compared to generic UNG in one-step RT-qPCR. One-step RT-qPCR was performed comparing Cod UNG and generic UNG to untreated control. Treatment with Cod UNG did not affect target cDNA, yielding the same Cq-values as untreated samples. Treatment with generic UNG resulted in significant Cq delay, demonstrating incompatibility with one-step RT-qPCR.
Figure 2. Cod UNG efficiently removes carry-over DNA. A spike with uracil-containing DNA was introduced into a RNA sample prior to performing one-step RT-qPCR. No pre-incubation step was introduced. Treatment with Cod UNG succeeded in removing the spiked amplicon to below detection limit.