October 1, 2008 (Vol. 28, No. 17)

Tools for Research, Livestock Genotyping, and Forensics Are Based on EA1 Metalloproteinase

New Zealand-based ZyGEM opened its U.S. headquarters earlier this year, shortly after the company recruited Paul Kinnon as president and CEO. Kinnon’s mission is to expand ZyGEM’s enzyme-based life science product portfolio in North America and Europe.

ZyGEM develops and markets enzyme-based reagent kits for the high throughput extraction of DNA from samples. Some of the kits are intended for use in basic laboratory research, while others are optimized for specific applications such as extracting DNA samples from cigarette butts for forensic analysis or detecting a deadly fish virus in salmon.

ZyGEM’s current products use EA1 metalloproteinase, which was isolated from an extremophile bacterium that lives in a volcanic vent near the crater of Mount Erebus in Antarctica. Deserts, boiling mud pools, geothermal vents, desiccated dry valleys, and acid fumaroles in New Zealand and Antarctica serve as home to some of the more than 700 thermophilic bacteria and archaea and 1,000 fungi that comprise ZyGEM’s culture collection.

ZyGEM, established in 2005, has assembled its line of enzyme cultures based on collections originally started and perpetuated by predecessor companies New Zealand Enzyme and PacificGEM. At least 500 enzymes of interest have been isolated from thermophiles in ZyGEM’s library, and they operate at temperatures ranging from -40°C to 105°C and within a pH range of 2.8 to 10.9.

The enzymes hold promise for industrial, fine chemical, and molecular applications. “We are using the unique characteristics of our enzymes and technology to improve DNA extraction so that our customers can carry out their work more efficiently,” continues Kinnon.

EA1-Based Products

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.

Marketed Products

The prepGEM™ products extract DNA from tissue, hair, or blood in about 20 minutes, and the kits contain all reagents needed for optimized downstream PCR-based applications, Kinnon reports. The extracted DNA is suitable for PCR, qPCR, SNP analysis, STR analysis, genotyping, sequencing and mass spectrometry analysis.

The forensicGEM™ kits operate similarly and are designed to extract DNA from samples typically found at crime scenes, such as blood, saliva, tissue, hair, and cigarettes. “The kits contain cocktails with optimized buffers to allow flexible and specific applications for sample preparation,” says Kinnon.

In June, ZyGEM launched livestockGEM for genotyping cattle. “We don’t consider the basic research market as our only market,” explains Kinnon. “Our kits can be used in animal vectoring, food testing, and a number of other platforms.”

One such platform is livestock genotyping, which enhances cattle-breeding programs to produce better meat and milk quality and fitness traits that are of economic value to the livestock industry. ZyGEM has entered into a nonexclusive agreement to market these kits with Zee Tags.

“The livestockGEM kits reduce the time, cost, and complexity of obtaining DNA samples, which should resonate with the livestock industry’s growing interest in using genetic analyses for routine applications on an ongoing basis,” claims Kinnon.

Zee Tags sells ear tags with RFID technology to identify cattle, sheep, and other livestock. A Zee Tags Tissue Sampler allows farmers to take a small tissue sample from an animal when they insert an ear tag. The tissue sample is transferred to a tube for DNA extraction with livestockGEM.

The genotyping of cattle herds helps in identifying genes that contribute to high quality meat and milk. It also could help to spot genes that make animals susceptible to mad cow disease or Salmonella outbreaks.

Moving Toward Diagnostics

ZyGEM’s temperature-controlled and flexible technology platform lends itself to many different applications, maintains Kinnon. The technology “will allow us to move into the diagnostics market for sample preparation for clinical testing, and ultimately to genetic testing and personalized medicine.” Products in development at ZyGEM are moving in this direction. A key strategy of the company is to partner its technology widely across industries and sectors, integrating EA1 and other proprietary enzymes into other companies’ platform technologies.

For example, in June 2008, ZyGEM teamed up with Diagnotec to produce a molecular diagnostic test for the detection of infectious pancreatic necrosis virus (IPNV), a deadly pathogen that attacks salmon and other fish. IPNV is a highly contagious virus that is relatively uncommon in wild fish, but it can be devastating to large-scale fish farms.

ZyGEM also is developing an assay to fingerprint microorganisms using its enzymes, primers, and current instrumentation technologies, along with an approach to isothermal amplification and detection. The prototype fingerprinting method identifies 30 different bacteria in a single assay and takes less than a day to perform, according to Kinnon.

The method reportedly works with mixed populations and can differentiate microbes at the genus and species level.

“The approach is ideal for clinical, veterinary, biodefense, and environmental testing. Combining tests for microbes such as those that cause sexually transmitted diseases will give us a strong clinical platform for point-of-care detection products,” says Kinnon, who says he is open to talks with potential collaborators.

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