Use of UNG has been an elegant and smart option for PCR heavy labs in anti-contamination control for some time now. Today I’d like to tell you how this can be useful to you if you do PCR gel analysis, and also my concerns about whether it is effective 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], and the encoded enzyme, UDG, is commonly used when performing PCR to eliminate amplicon carry-over since it excises uracil residues from DNA by cleaving the N-glycosylic bond. It can 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 UDG enzyme must be added to the PCR master mix.

So, how does this help in controlling amplicon contamination?

Because, dUTP is used instead of dTTP, all amplicons generated during PCR will have dUTP incorporated in them. If that amplicon becomes the source of contamination, use of UDG prior to subsequent PCR will specifically target dUTP containing DNA resulting in excision of uracil. This creates abasic sites in the amplicons that do not serve as good templates for Taq polymerase activity. The contaminated amplicons are thus removed from being amplified further.

In short, the presence of dUTP in the DNA forms the basis of distinguishing the PCR product, i.e., an amplicon, from the native DNA template. Since the native template does not naturally have dUTP, the native template remains intact in the presence of the enzyme but any amplicon DNA that contains dUTP becomes susceptible to enzyme action.

If you do a lot of PCR gel analysis, there is potential for amplicons to get aerosolized so UNG can really help avoid the problems associated with amplicon carry-over.

But is it any good for rtPCR?

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

Here is why-

1. In real-time PCR, most post-PCR analysis is done digitally, ie using software that comes with the instrument or by using other software packages.
2. Even if gel-analysis is considered, it is difficult and almost impossible to remove samples from the reaction vessels currently used in most real-time instruments because of the size and shape of those vessels. Most users will be hesitant to do this. More extreme precautions than what UDG can accomplish will have to be taken, like physical separation from other clean areas, de-gowning, multiple clean-rise procedures of everything that came in contact etc., (Ok, UDG might be of some help here after all this!)
3. Most labs that do real-time PCR (should) have safe CONTAINED places to dispose tubes, plates or capillaries after reaction is completed.

In light of the above, false-positive result is often not from amplicon contamination, but due to primary sample DNA contamination. As mentioned earlier, UDG does not recognize native DNA and would therefore be ineffective as an anti-contamination weapon.

Besides, the temperature requirements for UDG incubation are different for enzymes from different vendors. Some at done at 55C (AmpErase(2)) and some are done at 20C (Roche UNG (3)). Primer-dimer formation during incubation at 55C would be a big problem. Also, if you use the native enzyme, it has to be heated at 95C for 10 minutes to be denatured (3). Improper use may result in active UDG in the PCR reaction chewing up some of the early formed amplicons skewing quantification numbers.

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

References:

1. http://www.uniprot.org/uniprot/P12295
2. http://www3.appliedbiosystems.com/cms/groups/mcb_support/documents/generaldocuments/cms_041542.pdf
3. Biochemica, No.2, 1996. Page 13.