Bioprospecting: finding new enzymes to improve molecular analysis

Bioprospecting in the Arctic

Tapping into the natural resources of the Atlantic Ocean: Uracil-DNA Glycosylase – a novel enzyme harnessing unique properties found in organisms adapted to extreme cold conditions.

Common problem: PCR carry over contamination

In diagnostic labs, PCR is a common tool that is used to amplify a segment of DNA. It is used every day to diagnose diseases, identify bacteria and viruses, match criminals to crime scenes, and much more. Unfortunately, the amplification that makes PCR so useful also makes it highly susceptible to contamination.
A major source of contamination stems from the amplification products and primers used in previous PCRs. While there are several ways by which diagnostic labs control for carry-over contamination, most of these methods require costly investments such as building separate labs for preparing DNA samples, performing PCR and analysing PCR results.

A simpler method is to swap dTTP with dUTP, and pre-treat with uracil-N-glycosylase (UNG), a selective degrader of uracil-containing DNA. This method ensures the eradication of contaminants, enabling anyone to perform PCR in the absence of strict physical isolation procedures.

The problem with typical UNG contamination control technology

There are several commercially available UNGs on the market, but most UNGs are either inefficient or very difficult to inactivate. Most UNGs are of bacterial origin, thus they can be heat-inactivated and will remain inactivated during PCR. However if the newly-copied DNA were to be kept at room temperature, or even at 4˚C, together with the UNG, the UNG will be reactivated and the PCR products will be destroyed. This represents a major issue to diagnostic labs because frequently, PCR is used to amplify DNA for further downstream processes such as sequencing or cloning.

Developing the ideal UNG enzyme

As fish are unable to regulate their body temperature, they require a metabolic system that allows them to thrive in the cold environments. Consequently, they have evolved enzymes that are active at extremely low temperatures, but inactive at higher temperatures. Such characteristics make these enzymes particularly useful for PCR applications.

ArcticZymes’ scientists analyzed the content of UNG in North Atlantic cod – the most common fish in Northern Norway. It was found that the cod liver was especially rich in UNG. Cod UNG is active at low temperatures and very sensitive to moderate heat. In contrast to other UNGs that reactivate after temperature normalization, Cod UNG is irreversibly inactivated at moderate temperatures. In addition, this enzyme has very high activity in conditions normally used in PCR reactions, improving the quality of PCR reactions.

To produce a molecular-grade enzyme with minimal batch-to-batch variation and very high purity, a recombinant production method had to be developed. Building upon years of experience in recombinant enzyme production, ArcticZymes isolated the gene encoding for cod liver UNG, characterized and cloned it into E. coli for expression. Later, an optimized purification protocol was developed that lead to quality yields of the enzymes, and a novel tool for contamination control was commercialized.

ArcticZymes’ unique combination of genetic engineering capabilities and sophisticated recombinant production skills made it possible to improve technology for control of PCR contamination in any environmental setting.