Application Overview
PCR carry-over contamination can lead to false-positive results and is commonly controlled using dUTP and UDG. However, PCR products are often needed for downstream applications such as sequencing, cloning, or further amplification. Conventional UDGs can remain active after PCR and gradually degrade these products.
Cod UNG eliminates carry-over contamination before amplification and is irreversibly inactivated during PCR, preserving amplicons for downstream use.
Application Overview
PCR carry-over contamination can lead to false-positive results and is commonly controlled using dUTP and UDG. However, PCR products are often needed for downstream applications such as sequencing, cloning, or further amplification. Conventional UDGs can remain active after PCR and gradually degrade these products.
Cod UNG eliminates carry-over contamination before amplification and is irreversibly inactivated during PCR, preserving amplicons for downstream use.
The Challenges These Enzymes Address
PCR carry-over contamination
PCR is highly sensitive, making it vulnerable to carry-over contamination from previous reactions. Even trace amounts of amplified DNA can generate false-positive results and compromise confidence in assay performance. Carry-over contamination is therefore routinely controlled using dUTP/UDG systems in research, diagnostic, and manufacturing workflows.
When PCR products need to be used afterwards
For workflows that require PCR products to be stored, sequenced, cloned, or used in downstream analysis, conventional UDGs can retain residual activity after PCR and continue degrading uracil-containing amplicons once the reaction is complete. To protect these products, additional measures such as inhibitor addition or immediate handling may be required. This adds complexity to the workflow and introduces extra steps that need to be managed and validated, particularly in regulated diagnostic and manufacturing environments and adds extra complexity when looking to automate.
Standard PCR: one enzyme, every stage
THE PROBLEM These ENZYME SOLVES
PCR carry-over contamination
PCR is highly sensitive, making it vulnerable to carry-over contamination from previous reactions. Even trace amounts of amplified DNA can generate false-positive results and compromise confidence in assay performance. Carry-over contamination is therefore routinely controlled using dUTP/UDG systems in research, diagnostic, and manufacturing workflows.
When PCR products need to be used afterwards
For workflows that require PCR products to be stored, sequenced, cloned, or used in downstream analysis, conventional UDGs can retain residual activity after PCR and continue degrading uracil-containing amplicons once the reaction is complete. To protect these products, additional measures such as inhibitor addition or immediate handling may be required. This adds complexity to the workflow and introduces extra steps that need to be managed and validated, particularly in regulated diagnostic and manufacturing environments and adds extra complexity when looking to automate.
Standard PCR: one enzyme, every stage
THE PROBLEM These ENZYMes SOLVE
PCR carry-over contamination
PCR is highly sensitive, making it vulnerable to carry-over contamination from previous reactions. Even trace amounts of amplified DNA can generate false-positive results and compromise confidence in assay performance. Carry-over contamination is therefore routinely controlled using dUTP/UDG systems in research, diagnostic, and manufacturing workflows.
When PCR products need to be used afterwards
For workflows that require PCR products to be stored, sequenced, cloned, or used in downstream analysis, conventional UDGs can retain residual activity after PCR and continue degrading uracil-containing amplicons once the reaction is complete. To protect these products, additional measures such as inhibitor addition or immediate handling may be required. This adds complexity to the workflow and introduces extra steps that need to be managed and validated, particularly in regulated diagnostic and manufacturing environments and adds extra complexity when looking to automate.
Standard PCR: one enzyme, every stage
The Solution
Cod UNG provides effective PCR carry-over contamination control while preserving PCR products for downstream use. Unlike conventional UDGs, Cod UNG is irreversibly inactivated during PCR cycling, eliminating the need for post-PCR inhibitors and allowing amplicons to remain intact during storage and downstream analysis. This simplifies workflows by removing additional post-PCR handling steps, making Cod UNG well suited to automated and high-throughput environments.
Amplicon storage & downstream use
The difference is demonstrated in Figure 1. While conventional UDGs can regain activity after heat treatment and continue degrading PCR products, Cod UNG remains permanently inactive, protecting amplicon integrity during storage and subsequent workflows without requiring additional intervention to protect the amplicon.
Various UDGs were tested for residual activity after heat inactivation. PCR was performed with dUTP and 1 Unit of 5 different commercially available UDGs. Post-PCR, the PCR products were incubated at room temperature for various time intervals, followed by heating and subsequent cooling. Gel electrophoresisof the PCR products revealed UDG reactivation, and thus severe degradation of PCR products of all UDGs tested, except for Cod UNG.
Once contamination control is handled, an intact amplicon is what makes the next step possible — whether that's:
- Next-generation sequencing
- Sanger sequencing
- Preamplification for low-input qPCR
- Cloning
Why amplicon preservation matters more than before
Traditional post-PCR applications such as cloning and Sanger sequencing are generally more tolerant of minor amplicon degradation. Today, as amplicon sequencing becomes routine in diagnostics, surveillance, and research, preserving PCR products is increasingly critical.
Residual activity from conventional UDGs can continue degrading uracil-containing amplicons after PCR, potentially affecting library preparation, sequencing quality, and downstream results while adding complexity to the workflow.
Sequence quality is lost when UDG reactivates after the reaction
The data in figure 2 show this across four commercially available UDGs at two timepoints: degradation is progressive, and Cod UNG is the only enzyme for which it does not occur. A conventional UDG that is only partially inactivated during PCR cycling regains activity at ambient temperature — during storage, during library preparation, or during a sequencing run. Cod UNG does not, because its inactivation above approximately 55°C is complete and irreversible.
UDG reactivation resulted in degraded sequences. Sequence data of PCR products pretreated with various UDGs and incubated at room temperature. All samples, except samples incubated with Cod UNG, demonstrated reactivation of UDG and severe degradation of PCR products.
Contamination control in PCR and qPCR protcol
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 0.2 U Cod UNG directly to your 20 µl PCR reaction.
- Pre-incubate for 5 min at room temperature.
- Run your PCR.
- Store your PCR product at -20°C or 4°C.
This protocol is a recommended starting point for evaluation and optimisation, not the only or necessarily optimal approach for every workflow. Reaction conditions may need adjustment depending on your specific assay, sample type, and equipment.
Application Background
The dUTP/UDG carry-over prevention system was introduced by Longo and colleagues in 1990 and remains the standard approach in PCR. Carry-over contamination was historically described as one of the most significant obstacles to routine nucleic acid amplification testing, and product integrity after treatment has become a practical concern of its own as PCR products are increasingly cloned, sequenced, or archived rather than discarded after a single use. Cod UNG's irreversible inactivation addresses that second, less commonly discussed requirement.
Manufacturing you can trust
Cod UNG has been supplied to diagnostic kit manufacturers for over two decades. Manufactured under ISO 13485, it offers the consistent quality and supply that regulated workflows depend on — contamination control that doesn't compromise sensitivity or performance, enabling robust assay development. Further details on manufacturing, specifications, and supply are available on the Cod UNG page.
