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Jan 1, 2011 (Vol. 31, No. 1)

Novel Methods Expedite Nucleic Acid Sample Prep

Technologies Focused at Both the Research and Clinical Markets Stir Excitement in the Field

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    Zygem is developing an integrated microfluidics system for forensic analysis. This system, which is based on the enzymes that drive the company’s business, will reportedly feature rapid turnaround time.

    Zygem (www.zygem.com) is developing an integrated microfludics system for forensic analysis, using the enzymes it has long been known for,” according to James Landers, Ph.D., professor of biochemistry at the University of Virginia and also CSO of the company. Dr. Landers is investigating short tandem repeats using Zygem’s microfluidics technology and hardware from Lockheed Martin.

    Dr. Landers says that the most important feature of the system is its rapid turnaround time. The company has worked with Lockheed Martin to speed the analytical process to as little as 60 minutes, and Dr. Landers believes that this time frame can be abbreviated further. The partners have concentrated on the three key processes—DNA separation, amplification, and detection—miniaturizing them, reducing the required steps and time frame, and increasing their sensitivity and robustness.

    The preparation chemistry takes advantage of an EA1 metalloproteinase from the company’s extremophile collection, which can degrade tissues in buffer conditions compatible with PCR and is inactivated by a simple temperature shift. As a result of the temperature characteristics of EA1, DNA extractions can be carried out at an elevated temperature in a single closed tube or as part of an integrated microfluidics system, improving its efficiency. Because EA1 is inactivated above 95°C, raising the temperature stops the reaction, leaving PCR-ready DNA available for downstream processing.

    Just the PCR amplification process alone is ordinarily a three and a half hour process when using conventional technology. The Zygem approach bundles up all three steps in a monolithic format, expediting the time required, according to Dr. Landers. Using special patented polymers for the separation component, Dr. Landers and his colleagues are able to achieve a 30-minute separation in 400 seconds, while still maintaining single-base resolution. In this fashion they are able to progressively drive down the turnaround time.

    The microfluidics device, which is the size of a microscope slide, is processed by an instrument about the size of a computer tower. Dr. Landers and his collaborators at Lockheed Martin look forward to a miniaturized system, the size of a laptop, and eventually to a handheld instrument for use in the field. However, at this point the platform is designed for a laboratory environment, so the size of the instrument is not a pressing concern.

    It is not clear at this time where the technology will fit into the overall picture of forensic investigation. There is no question that the microfluidics analysis of short-term DNA repeats as a means of identification can speed processing and decrease the backlog of cases in forensic labs around the world. The unknown is whether it would be of value at a crime scene.

    “We can envision a time in the future where our instrumentation could be used for onsite identification of a suspect,” Dr. Landers suggests. However, there is no consensus today on whether such technology would be welcome in a chaotic venue where DNA from many individuals would be dispersed. “Only when the technology is available will we know how these approaches will fit into the needs of crime-scene investigators.”

    “Portability, speed, and cost of analysis will be welcomed by the genetic analysis community, but there are issues concerning the use of this technology in the field,” Joan Bienvenue, Ph.D., a forensic scientist and program manager at Lockheed Martin, explains. “While you can find people on both sides of the fence, forensic typing for criminal purposes is presently carried out in incredibly well-controlled laboratory situations. The policy changes that would be required for use in the field are not trivial.”

    “Even in a very well-organized forensic laboratory, there are three to four instruments that are used, in an analysis that can take ten to twelve hours to process a DNA sample,” explains Paul Kinnon, CEO. “So the first step will be to put our system into use, and as the technology evolves it will migrate into other functions. This will enable investigators to move forward efficiently and get on with their jobs.”

    The multiplexibility of the technology opens up a wide range of possibilities for other applications. “With the system’s rapid turnaround, it would be possible to run a molecular diagnostics panel on a patient and discuss the results with him in less than an hour, rather than sending the patient home and doing the consult days later,” says Dr. Landers. “This could have a significant effect on management of patient anxiety while at the same time providing an opportunity for timely clinical intervention.”

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