SolisFAST® solutions with UNG enzyme for carryover contamination prevention

Nucleic acid amplification techniques such as PCR and LAMP are very sensitive methods. With optimized protocol and primers, the limit of detection can be as small as a few copies of the target sequence. PCR typically results in ~0.2 mg, and LAMP 10-20 mg of amplified product. Sensitivity and high yield are a good combo to create false positive results. Contamination can ruin your experiment leading to false discoveries, increasing background noise and even misdiagnosing. The contamination issue is vital in case of detection of infectious diseases. When misdiagnosed unnecessary treatment is applied and can cause a range of unwanted complications.

A combination of proactive methods and good laboratory practice helps us prevent contamination and perform accurate and sensitive analyses. As a part of the prevention strategy, carryover contamination from previous amplification of the same target sequence can be degraded by Uracil-N-Glycosylase (UNG). To make this method work, an amplicon has to contain Uracil residues. This is accomplished by including dUTPs in all amplification reactions. Since natural DNA sequences do not contain Uracil, then only the Uracil-containing DNA contaminants are degraded during the UNG treatment step and cannot be amplified in further analyses. It is advisable to use polymerases that can incorporate 100% of dUTP. For that we recommend SolisFAST® DNA polymerase, which very effectively incorporates dUTPs compared to regular Taq DNA polymerases. You can also be well assured that your RNA sample is not degraded during the UNG treatment! UNG is not able to degrade naturally Uracil-containing RNA sequences – other types of enzymes are needed for this. During UNG treatment step contaminants are degraded and cannot be amplified in further analyses. Note that it is proactive method and does not degrade other sources of contamination. [ref. 1, 2, 6]

For end-point PCR application SolisFAST® Master Mix with UNG is the perfect solution. It combines all the benefits:

• Proactive method to prevent carryover contamination. This Master Mix contains dUTPs and UNG enzyme in optimized concentration.
• Reduced pipetting steps. As it is a Master Mix you only have to add template, primers and water. You can analyze up to 18 targets in the same reaction, which makes it a cost-effective solution.
• Reaction setup at room temperature. SolisFAST® DNA Polymerase has a Stability TAG [ref 3, 4, 5] that significantly improves its thermostability. With that there is no need for uncomfortable ice boxes, which are a great source of contamination.

If you are performing qPCR, choose SolisFAST® Probe qPCR Mix with UNG. It has almost all the benefits listed above, with one difference – multiplexing up to 5 targets in one reaction. As an additional bonus, this optimized mix is inhibitor tolerant and compatible with TaqMan® and other hydrolysis probe types.

But there is more. We have great news for you, if your field is RT-(q)PCR, LAMP or some other molecular biology method that implements UNG. Solis BioDyne is launching new products: 1-step solutions for RNA detection with UNG in the mix and Uracil-N-Glycosylase standalone enzyme (Figure 1). Stay tuned for more information!

References

1. Aslanzadeh, J. (2004). Preventing PCR amplification carryover contamination in a clinical laboratory. Annals of Clinical & Laboratory Science, 34(4), 389-396.
2. Hu, Y. (2016). Regulatory concern of polymerase chain reaction (PCR) carryover contamination. Polymerase Chain Reaction for Biomedical Applications, 57-68.
3. Kahre, O., Artma, K., Kahre, T. (2015). Compositions for increasing polypeptide stability and activity, and related methods (European Patent No. 2501716 B1). European Patent Office.
4. Kahre, O., Artma, K., Kahre, T. (2016). Compositions for increasing polypeptide stability and activity, and related methods (US Patent No. 9,321,999 B2). U.S. Patent and Trademark Office.
5. Kahre, O., Artma, K., Kahre, T. (2017). Compositions for increasing polypeptide stability and activity, and related methods (South Korea Patent No. 10-1773636 B1). Korean Intellectual Property Office
6. Wang, Y., Wang, Y., Li, D., Xu, J., & Ye, C. (2018). Detection of nucleic acids and elimination of carryover contamination by using loop-mediated isothermal amplification and antarctic thermal sensitive uracil-DNA-glycosylase in a lateral flow biosensor: application to the detection of Streptococcus pneumoniae. Microchimica Acta, 185(4), 1-11.