Enzymes that Unlock
Your Next Discovery
0

PCR Carry-Over Prevention

APPLICATION overview, CHALLENGES AND SOLUTION

See enzymes for

For Detailed Info Including:
  • Product overview
  • Performance data & figures
  • Specifications
  • Documents
  • FAQs
  • Ordering Info
  • Protocols
  • Publications

Application Overview

PCR carry-over contamination, is where amplified DNA from one reaction serves as an unintended template in subsequent reactions. This can lead to false-positive results affecting the accuracy and reliability of PCR, especially in sensitive qPCR applications.

ArcticZymes' Cod UNG addresses this challenge head-on. Designed to degrade uracil-containing DNA—a marker of carry-over when dUTP replaces dTTP—it ensures the integrity of PCR outcomes. Cod UNG transforms a molecular hurdle into a streamlined, reliable process

See enzymes for

For Detailed Info Including:
  • Product overview
  • Performance data & figures
  • Specifications
  • Documents
  • FAQs
  • Ordering Info
  • Protocols
  • Publications

Application Overview

PCR carry-over contamination, is where amplified DNA from one reaction serves as an unintended template in subsequent reactions. This can lead to false-positive results affecting the accuracy and reliability of PCR, especially in sensitive qPCR applications.

ArcticZymes' Cod UNG addresses this challenge head-on. Designed to degrade uracil-containing DNA—a marker of carry-over when dUTP replaces dTTP—it ensures the integrity of PCR outcomes. Cod UNG transforms a molecular hurdle into a streamlined, reliable process

THE PROBLEM These ENZYMEs SOLVE

for PCR carry-over contamination

PCR, especially qPCR, is highly sensitive, making it susceptible to contamination and consequently, false or inaccurate results. One major source of these false positives is carry-over contamination, where amplified DNA from a prior reaction inadvertently becomes a template in subsequent reactions. This can occur due to aerosolization, contaminated pipettes, surfaces, gloves, and even reagents. Cod UNG addresses this issue by targeting and degrading carry-over PCR-products, thereby preserving the accuracy and integrity of PCR results

THE PROBLEM These ENZYME SOLVES

for PCR carry-over contamination

PCR, especially qPCR, is highly sensitive, making it susceptible to contamination and consequently, false or inaccurate results. One major source of these false positives is carry-over contamination, where amplified DNA from a prior reaction inadvertently becomes a template in subsequent reactions. This can occur due to aerosolization, contaminated pipettes, surfaces, gloves, and even reagents. Cod UNG addresses this issue by targeting and degrading carry-over PCR-products, thereby preserving the accuracy and integrity of PCR results

PCR carry-over prevention figure
Fig . PCR carry-over prevention figure
PCR carry-over prevention figure

The Solution

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 PCR-products 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.

ArcticZymes Cod UNG is the the only UNG compatible with one-step RT-qPCR due to its irreversible inactivation at 55˚C.
By integrating Cod UNG into PCR protocols, labs can substantially mitigate false positives, aligning with ArcticZymes' commitment to enhancing the precision of molecular techniques.

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. 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 loses 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.

 PCR carry-over prevention figure  1
Fig 1. PCR carry-over prevention 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.

 PCR carry-over prevention figure 2
Fig 2. PCR carry-over prevention 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.

PCR carry-over prevention figure
Fig .  PCR carry-over prevention figure

Application Background