The selection of EA1 for use in ZyGEM’s DNA extraction kits was based on its ability to degrade tissues in buffer conditions compatible with PCR and on properties that enable the kits to use a simple temperature shift to activate and deactivate EA1. Of the decision to develop and commercialize EA1, Kinnon says, “We saw this as a powerful enzyme, so we thought we should commercialize it and get it to the market as quickly as possible, while also working to optimize our other enzymes. We plan to use the proceeds from sales of our reagent kits to research and further develop additional enzymes from our extremophile collection and to develop diagnostic products.”
EA1, which operates at 75°C, can degrade a range of proteins in animal tissues, including those known to be resistant to other enzymes, according to Kinnon, who adds that its versatility enables it to be used to extract DNA from a wide variety of sample types, while ZyGEM is also incorporating it in a number of specialized kits that are optimized for specific applications.
DNA from standard samples, for example, can be extracted using the prepGEM kits targeted to the basic research market, while DNA from cattle can be extracted using ZyGEM’s range of livestockGEM™ kits, and human DNA can be recovered using the forensicGEM kits.
As a result of the distinctive temperature characteristics of EA1, DNA extractions using ZyGEM’s kits can be conducted in a single closed tube, which speeds sample preparation time and increases ease of preparation, explains Kinnon.
“It also minimizes the chances of sample contamination and errors, reduces the amount of sample and reagent needed for successful extractions, and the ultimate cost of the process. The simplicity and flexibility of the approach make it easy to incorporate ZyGEM kits into standard laboratory DNA extraction procedures,” Kinnon says. The approach also works well with almost all off-the-shelf laboratory automation systems, he adds.
Carrying out DNA extractions at an elevated temperature also allows for more efficient digestion of proteins. Because EA1 is inactivated above 95°C, raising the temperature stops the reaction. The extracted DNA does not require further purification on columns or beads with solvents. “EA1 is also an ideal enzyme to incorporate into reagent kits for laboratory use because it can be stored in a refrigerator at 4°C for extended periods of time,” notes Kinnon.
When using ZyGEM’s kits for DNA extraction, samples are prepared on ice in a closed Eppendorf tube, then heated to 75°C to start the reaction. Heating the reaction mixture to 95°C stops the reaction, then the extracted DNA sample is cooled for use. All reagents contained in the kits are nontoxic, and the process can be automated by using any standard robotic liquid handling system.