ZyGEM has taken a different approach to nucleic acid sample prep. Rather than using solid-phase extraction (SPE), ZyGEM offers an enzyme-based technology to strip the sample down to nucleic acid. While the technology is amenable to use on liquid handlers in the research lab, James Landers, Ph.D., was brought into ZyGEM as CSO to adapt the technology to microfluidics.
As professor of chemistry and mechanical engineering and assistant professor of pathology at the University of Virgina, Dr. Landers has abandoned SPE for ZyGEM’s enzymatic approach because of its simplicity and elegance, he said. With the adoption of microfluidics, the extraction process has been shrunk to sub-microliter volumes, which saves on enzyme and other reagent costs. In addition, the reaction kinetics are sped up; the entire process has been reduced to 35 minutes.
The reaction is controlled by temperature. The EA1 proteinase, a highly active metallo-endo-proteinase isolated from a thermophile in a volcanic vent, is inactive at room temperature. Peak activity occurs at 75°C where the enzyme works as a protease. Raising the reaction temperature to 94°C irreversibly kills enzyme activity.
Regarding the macro-to-micro interface, ZyGEM deals with real world samples; a buccal swab or blood droplet is collected in 100 µL volume. Of that starting material, only 1 µL is needed for downstream processing; the rest of the material can be stored for reanalysis, if necessary. Front-end concentration of the sample has not been determined to be necessary. From a buccal swab, ZyGEM routinely liberates purified DNA in the range of 6–12 ng/µL, which is in excess of the 4 ng/µL that is needed for PCR, Dr. Landers explained.
The ZyGEM approach has a narrow scope; the sample-prep method provides material that is “PCR-ready”, but is not limited to microfluidic devices. Kits are also available for use in microtiter plates on liquid handlers, where the enzyme approach is reportedly a dramatic improvement over SPE protocols as the number of sample-handling steps is reduced, avoiding loss of sample from transfer steps.
“It is important to remember that the ZyGEM process doesn’t help with finding a needle in a haystack. DNA that is liberated from the reaction comes from all sources in that sample. You must depend on the use of proper PCR primers to amplify up rare sequences in the mix or resort to SPE-based selection of target from the mix,” explained Dr. Landers.
“But with its simplicity and elegance, PoC applications are not far off. For example, where the challenge is to differentiate between viral and bacterial Encephalitis meningitis when the patient presents with similar symptoms, given the rapid kinetics of the sample prep of Cerebral Spinal Fluid, and subsequent PCR, you could determine the right course of treatment within the first two hours of hospitalization,” Dr. Landers claimed.