WORKFLOWS

Many of the DNA extraction procedures used today are based on chemistries that are over 30 years old. These chemistries have evolved rather than been designed and as with most evolved systems, have gathered a complexity that makes them difficult to use, difficult to automate and liable to fail. In fact, a close look at traditional methods shows that nearly every step is intended to remedy a problem created in previous steps.

Most competing products on the market accept this historical dogma and attempt to use engineering to solve the problems. For automation, this approach requires dedicated robotics with magnets or vacuum manifolds and relatively costly columns or beads. Furthermore, all require numerous tube openings that potentially expose the sample to external contamination – a crucial weakness in forensic analysis or any analysis using generic primers.

ZyGEM researchers re-visited the underlying methods and asked, "Can we find an enzyme that is able to perform these steps in a single reaction?” The result of extensive screening of a large number of thermophilic proteases was the discovery of EA1 protease, the key ingredient in the prepGEM™ and forensicGEM™ product families. This enzyme has a pH profile, temperature profile, robustness and broad substrate specificity that make it ideal for DNA extraction in a closed tube.

The result is a system where engineering is replaced by using a simple temperature shift in a closed tube to modulate activity, providing significant advantages over existing approaches.

forensicGEM™ and prepGEM™ have another outstanding characteristic – minimal activity below 45°C. This means the enzyme is effectively 'deep frozen' at room temperature and so is very robust … But more importantly, it means that the enzyme can be used in combination with other enzymes – a feature not normally possible with proteases.

An example of this use is in the prepGEM™ Bacteria kit where the second enzyme is lysozyme.

…hands off, closed tube and all you need is a thermal cycler.