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Eliminate PCR Amplicon (but not real-time PCR?) Carry-Over With UNG

Use of UNG has been an elegant and smart option for anti-contamination control in PCR heavy labs for some time now. Surprisingly, despite its history of use, many people do not understand how UNG works or when to use it. In this article, I’ll reveal how UNG can be useful for PCR gel analysis. Also, I’ll share my concerns about the effectiveness of UNG for real-time PCR.

What Is UNG and How Does It Work?

UNG is the gene name for the E. coli enzyme Uracil-DNA-glycosylase (UDG) [1]. The acronyms UNG and UDG are used interchangeably in the literature. In this article, I’ll use UNG because that’s the most common term. Researchers commonly use UNG when performing PCR to eliminate amplicon carryover, because it excises uracil residues from DNA by cleaving the N-glycosylic bond. Also, UNG has the advantage of being able to act on dUTP-incorporated ss- or ds-DNA.

To use UNG in PCR contamination control, you need to:

  1. Use dUTP in place of dTTP in the dNTP mix.
  2. The UNG enzyme must be added to the PCR master mix.
  3. Prior to beginning thermocycling (this term refers to the heating, annealing, and cooling steps in PCR), a 2 minute 50°C hold prior to beginning that activates the UNG.
  4. Proceed with thermocycling as normal.

How Does UNG Help in Controlling Amplicon Contamination?

First off, you should know that UNG is helpful for preventing future contamination, but does not help with pre-existing contamination. You’ll see why below.

By using dUTP instead of dTTP in your PCR, all amplicons generated will have dUTP incorporated in them. In the future, if that amplicon becomes the source of contamination, using UNG prior to PCR specifically targets dUTP-containing DNA, resulting in excision of uracil, and prevents PCR contamination by a previous amplicon. The excision of uracil prevents the amplicon amplification by creating abasic sites in the amplicons. The abasic sites do not serve as good DNA templates for Taq polymerase. Therefore, the contaminated amplicons are prevented from being amplified further.

In short, the presence of dUTP in the DNA distinguishes the PCR product (i.e., an amplicon) from the native DNA template. Because the native template doesn’t have dUTP, it remains intact in the presence of the enzyme. But any amplicon DNA that contains dUTP is susceptible to enzyme action.

If you do a lot of PCR gel analysis, there is potential for amplicons to be aerosolized. So, UNG can really help to avoid the problems associated with amplicon carryover.  It’s use, along with proper PCR positive controls, can make troubleshooting easier.

But Is UNG Any Good for Real-Time PCR?

However, as more and more labs are moving into real-time PCR analysis for quantification purposes, I wonder if use of UNG is necessary.

Here is why I wonder about the usefulness of UNG

  1. In real-time PCR, most post-PCR analysis is done digitally. Generally, most analysis is performed using software that comes with the instrument or by using other software packages.
  2. Trying to remove samples from the PCR machine after the cycle can be difficult, if not impossible. This can make gel analysis of your amplicon very difficult. Therefore, more extreme precautions than what UNG can accomplish will have to be taken, like physical separation from other clean areas, de-gowning, multiple clean-risne procedures of everything that came in contact etc., (Ok, UNG might be of some help here after all this!)
  3. Most labs that do real-time PCR (should) have safe contained places to dispose of tubes, plates, or capillaries after the reaction is completed.

In light of the above, a false positive is often due to primary sample DNA contamination—not from amplicon contamination, so other protection, such as using filter pipette tips for specialty PCRs, might serve you better.  As mentioned earlier, UNG does not recognize native DNA and, therefore, would be ineffective as an anti-contamination weapon.

Besides, the temperature requirements for UNG incubation are different for enzymes from different vendors. Some at done at 55°C (AmpErase) and some are done at 20°C (Roche UNG). Primer-dimer formation during incubation at 55°C could be a big problem. Also, if you use the native enzyme, it has to be heated at 95°C for 10 minutes to be denatured. Improper use may result in active UNG in the PCR reaction, chewing up some of the early formed amplicons and skewing quantification numbers.

Because I don’t do any gel analysis after PCR, I am hesitant to use UDG at all. What do you think?

References:

  1. UniProtKB – P12295 (UNG_ECOLI). UniProt. http://www.uniprot.org/uniprot/P12295
  2. Schmidt M, Frey B, Kaluza K, Sobek H. (1996) Application of Heat-labile Uracil-DNA Glycosylase in Improved Carryover Prevention Technique. Biochemica.2: 13–5.

4 Comments

  1. Advocatus on March 3, 2011 at 3:49 pm

    I appreciate your article, Thank you. I was pondering the use of UNG as Applied Biosystems suggests the usage of Master Mixes with UNG with some of their assays. I’ll be performing RT before my qPCR and I’m unsure if dUTP is in my RT mix yet (or in my lab), so the inclusion of UNG would be potentially harmful.

    Thanks again

  2. John Mackay on October 12, 2010 at 10:32 pm

    I wouldn’t worry about the primer dimers Shoba – I seem to recall that UNG was published as a way to get *rid* of primer dimers (since they’ll be digested by the enzyme as well). Nice article – I think a lot of systems have UNG in them still perhaps due to SOPs etc. Some enzymes can’t handle dUTP as well as dTTP but perhaps that’s changing – I think a good idea to have dUTP in the mix in case you ever have to use UNG. But then again, it will only handle a limited amount of contamination (ie its easy to overwhelm it)

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