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 (alkaline conditions) temperature is elevated to 95°C, resulting in cleavage of apyrimidinic sites and fragmentation of carry-over DNA. (Figure 1). 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.
Figure 1. Cleavage of uracil containing DNA before PCR.
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 loose 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.
Here we show that Cod UNG from ArcticZymes can be used for carry-over prevention in a commercial one-step RT-qPCR master mix containing dUTPs instead of dTTPs. The treatment did not reduce the sensitivity of the master mix.
Figure 2: Amplification plots, human HPRT1 (top) and bovine GAPDH (bottom). All plots show an average of three replicates. Human RNA spiked with uracil-containing bovine GAPDH amplicons was used as template for evaluating the performance of Cod UNG in a one-step RT-qPCR master mix. Bovine GAPDH amplicons were detected using specific primers and probe (HEX). Human RNA was detected using primers and probe (FAM) targeting the HPRT1 gene. The presence of amplicons (Bovine GAPDH) and human RNA (HPRT1) in samples treated with Cod UNG (0.01 U/µl) was compared to untreated controls using a multiplexing strategy.UNG treatment did not reduce the sensitivity of the master mix, while the carry-over contamination has been reduced to levels below the detection limit of a 45-cycle RT-qPCR.
- Add 0.2 U Cod UNG directly to your 20 µl RT-PCR reaction
- Preincubate at room temperature for 5 min
- Reverse transcribe your RNA at 50- 55°C
- Run your PCR
Cod UNG – The only true heat-labile Uracil-DNA Glycosylase
There are several commercially available Uracil-DNA glycosylases on the market today. Most of them are of bacterial origin and work well if you have no intention to further analyze the PCR products post-PCR. However, if you want to store your PCR products for downstream analysis such as cloning and sequencing, the reactivation of UNG and subsequent degradation of your PCR products are a problem with most of the commercially available UNGs. Cod UNG from ArcticZymes is the only commercially available UNG today which is completely and irreversibly inactivated by heat. This is illustrated in Figure 3, where various UNGs were tested for residual activity after heat inactivation. PCR was performed with dUTP and 1 Unit of 5 different commercially available UNGs. Post-PCR, the PCR products were incubated at room temperature for various time intervals, followed by heating and subsequent cooling. Gel electrophoreses of the PCR products revealed UNG reactivation, and thus severe degradation of PCR products of all UNGs tested, except for Cod UNG.
Figure 3. The only UNG that become completely and irreversibly heat-inactivated is Cod UNG.
Post-PCR sequence quality and integrity were further evaluated by sequencing the PCR products. PCR was performed with one of four different commercially available UNGs added to the mastermix. Post-PCR, the PCR products were incubated at room temperature or 4˚C at various time intervals. Samples were subsequently purified and sequenced. Sequence data were thoroughly analyzed with emphasis on reduced sequence quality as a result of UNG reactivation. As illustrated in both Figure 4 and Figure 5, samples treated with UNG showed severe degradation of PCR products due to UNG reactivation, except for samples treated with Cod UNG.
Figure 4. Chromatograms of sequenced PCR products pre-treated with 1 U Cod UNG (A) or 1 U E.coli UNG (B) and incubated at room temperature for 3 hours. Only Cod UNG leaves sequence quality intact.
Figure 5. UNG reactivation resulted in degraded sequences. Sequence data of PCR products pretreated with various UNGs and incubated at room temperature. All samples, except samples incubated with Cod UNG, demonstrated reactivation of UNG and severe degradation of PCR products.
Cod UNG works in all commercially available master mixes. Be sure that you have used dUTP containing dNTP mixes in your previous PCR experiments.
- Add 0.25 U Cod UNG directly to your 25 µl PCR reaction
- Pre-incubate for 5 min at room temperature
- Run your PCR
Store your PCR product at -20°C or 4°C degrees for as long you want, before analysis.
Purification and characterization of a cold-adapted Uracil-DNA Glycosylase from Atlantic cod (Gadus morhua). Lanes O, Guddal PH, Gjellesvik DR, Willassen NP. Comp Biochem Physiol B Biochem Mol Biol. 2000 Nov; 127(3):399-410