January 1, 2009 (Vol. 29, No. 1)

Generating Amplifiable DNA for Nucleic Acid Analysis

ZyGEM’s technologies are derived from a collection of extremophiles that thrive in extreme temperature and pH conditions. The company’s DNA-extraction technology is based on Erebus Antarctica 1 (EA1), an active metallo-endo proteinase from a Bacillius sp. discovered in a volcanic vent close to the crater of Mount Erebus in Antarctica. This EA1 proteinase has special characteristics that enable one-step, one-tube, temperature-controlled extraction (TCE) of DNA.

ZyGEM’s prepGEM™, forensicGEM™, and livestockGEM™ DNA Extraction Kits all utilize the EA1 enzyme. The EA1 TCE protocol of these kits is composed of simple temperature changes (Figure 1). By increasing the temperature of EA1 protease to its an optimal 75ºC activation temperature, this thermophilic proteinase degrades proteins by lysing cells, destroying nucleases, and stripping the DNA of proteins, to produce single-stranded DNA that is free of proteins.

The use of the elevated temperature allows more efficient digestion of some challenging proteins and sample types. Because of its narrow temperature profile, the enzyme is then irreversibly heat inactivated by increasing the temperature to 95ºC. The complete DNA-extraction system is carried out in a single reaction well or tube.

The EA1 TCE protocol streamlines laboratory workflow from DNA extraction to nucleic acid analysis to achieve time and cost savings. The single-tube DNA extraction means that, for most samples, the complete process can be automated using a peltier heating block on most automated liquid-handling systems. Alternatively, a PCR thermocycler can be programmed to perform TCE. The protocol generates DNA that can be used directly in PCR, qPCR, DNA sequencing, and genotyping by several methods (e.g., STR, SNP, microsatellite), and has been validated for STR and SNP genotyping.

Mesophilic proteinases such as Proteinase K are routinely used in DNA extraction but require the use of detergents such as SDS or chaotropic salts for isolating DNA. Without purification, these inhibitory components would be carried into the downstream reaction. Traditional proteinase protocols therefore utilize liquid extraction, solid-phase extraction (silica-based spin columns or membranes), or magnetic beads after protease digestion. These multistep methods make automation extremely challenging unless turn-key automated systems are used, create opportunities for introducing contaminants and errors, and consume large quantities of consumables and plastic ware. ZyGEM’s DNA Extraction Kits using EA1 TCE eliminates the need for additional reagents, consumables, and liquid-handling steps.

Figure 1. DNA extraction using ZyGEM’s EA1 proteinase technology

High-Throughput Applications

High-throughput EA1 TCE has many applications—from basic research, drug discovery and development, clinical research and diagnostics, to agriculture, animal and livestock, and forensics. Experiments employing forensicGEM illustrate the method’s high-throughput automation capabilities.

Forensic laboratories worldwide conduct DNA profiling of individuals for criminal DNA database development. These labs process thousands of samples, comparing database profiles against crime-scene DNA to generate cold hits. Often having a significant backlog of samples to process, many forensic laboratories seek streamlined automation in DNA extraction and STR analysis. The use of spin columns or magnetic beads is generally expensive and difficult to automate for high-throughput application.

In one set of experiments, an Eppendorf epMotion® 5075 robotic system, fitted with twin Peltier blocks and a thermal cycler (Figure 2) was used for forensicGEM DNA extraction of FTA card punches in 96-well format, followed by STR analysis using industry-standard STR kits. Data demonstrated (Figure 3) that this high-throughput automation of DNA extraction and STR analysis was ideally suited for forensic application and can streamline sample processing and analysis. The extraction process takes approximately 35 minutes with 4–5 minutes of technician time required initially for reaction preparation.

Figure 2. Set up of automated DNA extraction on the epMotion 5075 Liquid Handler for forensic FTA punches in 96-well format.


Traditional DNA sample preparation for genetic analysis assays can be a time-consuming, costly, and labor-intensive process. ZyGEM’s DNA-extraction technology using EA1 involves a temperature change without need for wash or elution steps. This simplicity lends itself to automated, high-throughput application using most existing liquid-handling systems and can simplify laboratory workflow from DNA extraction to nucleic acid analysis.

Figure 3. (A) Multiplex STR-PCR (STR Identifiler® PCR Kit, Applied Biosystems) analysis obtained from DNA extracted from liquid blood samples using forensicGEM.

Figure 3. (B) TaqMan® Real-time quantitative PCR trace

David Saul, Ph.D. ([email protected]), is senior scientist, and Nick Price, Ph.D., is technical director at ZyGEM. Web: www.zygem.com.

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