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DOI: 10.1126/science.opms.p1000052

LIFE SCIENCE TECHNOLOGIES
Forensics Follows New Clues

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Forensic DNA analysis has already revolutionized criminal investigation. Now researchers and toolmakers are building faster, more sensitive DNA fingerprinting platforms while adding entirely new techniques to detectives' toolkits.

By Alan Dove



Inclusion of companies in this article does not indicate endorsement by either AAAS or Science, nor is it meant to imply that their products or services are superior to those of other companies.

On television, all forensic technology fits into a standard plotline. Someone commits a brilliant, dastardly crime, and a team of romantically entwined investigators—with limitless access to cutting-edge tools—finds and collars the perpetrator in under an hour, with time for commercial breaks.

Reality, of course, is considerably messier. The majority of criminals are not especially brilliant, but even their rudimentary efforts to cover their tracks can make an investigator's job infuriatingly difficult. New technology might help, but only if crime labs can adopt it without breaking their budgets or having to retrain an entire staff.

The problems are as diverse as the cases. DNA analysis forms the backbone of modern forensics, but labs processing DNA evidence operate under enormous technical and legal constraints. The slightest contamination of a sample, or just poor recordkeeping, can invalidate the evidence from multiple cases. Meanwhile, technicians are often working against the clock; if a prime suspect is in custody, there may be only a few hours to process evidence before he or she is released on bail.

DNA fingerprinting isn't the only challenge, though. Out in the field, investigators spend a lot of time searching for bodies, and then trying to identify them even if they're badly decomposed.

To address these problems, researchers working in corporate, university, and government labs are modifying established forensic technologies and are also developing entirely new ones. While some of the tools coming from this interdisciplinary field are targeted specifically at solving crimes, others may find a broad range of uses in basic and applied research.

CSI IN A CARTRIDGE

For researchers at Life Technologies in Carlsbad, California, developing new forensic technologies is a longstanding business interest. The company offers a range of DNA analysis products and reagents, including the popular PrepFiler DNA extraction system. "The PrepFiler kit chemistry is a magnetic bead based extraction system, but the magnetic particles are optimized, they have a multicomponent surface chemistry that really maximizes the binding of DNA to the beads in order to maximize recovery," says Lisa Calandro, senior staff scientist in the Human Identification and Forensics Group at Life Technologies.

The pre-made kit simplifies the DNA isolation somewhat, but the protocol still requires multiple steps, each of which introduces the opportunity for errors, spills, and contamination. To reduce those risks, Calandro and her colleagues have now developed an entirely automated version called AutoMate ExpressT.

"What the AutoMate ExpressT does is it takes that chemistry and puts it into a cartridge-based system, and then the extraction procedure is performed on a [benchtop] instrument," Calandro explains. Besides eliminating operator error from the protocol, the new system also makes it faster and simpler. A technician can simply cut the tip from a cheek swab, lyse the cells on it, centrifuge them through a separation column, and then apply the solution to the AutoMate ExpressT column. Completely purified DNA emerges from the other end.

While simple purification works well if the DNA came directly from a suspect, evidence from a crime scene requires special handling. Besides having much lower concentrations of DNA, crime scene samples may include mixtures of suspect and victim cells, as well as environmental chemicals that can inhibit polymerase chain reactions (PCR).

After purifying DNA from these samples, technicians might turn to a system such as the Life Technologies Quantifiler Duo, which quantifies the ratio of male to female DNA in a sample as well as detecting any PCR inhibitors. "This can be very helpful in sexual assault cases, where you have potentially very low amounts of male DNA," says Calandro.

Both the AutoMate ExpressT and Quantifiler systems are already in use by many forensics laboratories, but the company is also working on additional tools to speed the forensic DNA workflow. Though DNA evidence was previously considered a tool for solving homicide and assault cases, Calandro says police departments are increasingly using DNA fingerprints in property crimes such as burglary. That increases pressure on labs to boost their sample processing capacity, without compromising reliability.

The standards for DNA fingerprinting are also changing. Since 1994, when the U.K.'s Forensic Science Service (FSS) pioneered DNA fingerprinting (the FSS' closure was recently announced: http://scim.ag/g19eJX), European law enforcement officials have steadily added more genetic loci to the continent's standard testing protocols, with some variation between countries. As a result, European samples may be tested for matches at up to 16 distinct loci. Meanwhile, the U.S. Federal Bureau of Investigation (FBI) has settled on a standard protocol with 13 loci that partially overlap with the European set, but FBI officials are considering adding more.

Fortunately, forensics toolmakers are already addressing that challenge. Promega in Madison, Wisconsin for example, now offers the PowerPlex 16 system, a comprehensive DNA analysis platform that analyzes the 13 FBI loci plus three additional ones, overlapping with European testing standards and also providing gender identification. The company markets the system for a wide range of applications, from forensics and paternity testing to cell culture identification. Besides screening more loci than conventional kits, the PowerPlex 16 provides greater sensitivity and speed than traditional DNA fingerprinting protocols.

BOOK 'EM, DNA

Even with streamlined sample preparation, performing a complete DNA fingerprinting analysis can take a long time. "The overall process, in the way that is typically used in the industry, can take up to 14 days," explains Frederic Zenhausern, Ph.D., director of the Center for Applied NanoBioscience and Medicine at the University of Arizona in Phoenix.

Zenhausern and his colleagues in collaboration with the U.K. FSS have now developed a microfluidic device that can shorten the steps between purifying DNA and reading a fingerprint to two hours, while also making it more straightforward for technicians. "Everything is piloted by the computer, and it's a very simple user interface," says Zenhausern.

At the heart of the device is a microfluidic chip built from previously tested technologies. "What we do here is combine inside a lot of different components that have been developed in the past," says Zenhausern. Thermally activated polymers act as valves to move the sample from one step to the next, and onboard sensors allow the attached computer to control everything from reagent mixing to PCR and electrophoresis.

The chip is part of an enclosed, single-use cartridge. "All the pumping and valving systems are self-contained within the cartridge ... that was one of the key elements the forensic community really wanted, to really be sure there is no potential for cross-contamination," says Zenhausern. He adds that while the fluid volumes are small compared to traditional laboratory techniques, the team deliberately avoided pushing the boundaries of miniaturization; for forensics labs, reliability is more important than portability.

Indeed, confirming that the system is reliable enough to meet the rigorous standards for criminal evidence processing is the next major hurdle. "The Forensic Science Service in the United Kingdom has been really leading the development for us, they really have focused on the [reliability] measurements with the concept of where those measurements will go in the legal system," Zenhausern says.

Besides validating the new technique, the investigators are collaborating with a laboratory equipment vendor to mass-produce the microfluidic cartridges and their associated hardware. Ultimately, Zenhausern hopes to get the price per test down to twenty dollars or less, with a system that could be used in a police station's booking room rather than a crime lab. At that point, DNA testing could become as routine as a mug shot, allowing police to confirm or exclude a suspect before the booking process is even finished.

The system could also find uses outside of criminal justice. "If you change the design of the cartridge and the assay chemistry, the same instrumentation platform can be used for other applications in other market segments, like clinical diagnostics," says Zenhausern.

When the new microfluidic system reaches the market, it will already have competition. At PerkinElmer in Waltham, Massachusetts, product developers have been working hard to streamline forensic DNA analysis, resulting in the company's innovative JANUS workstations. The systems use PerkinElmer's proven liquid-handling automation platform to handle every step of DNA analysis, from purification through quantitative PCR setup. Because the system is modular, labs can acquire it piecemeal, and potentially add other processes in the future.

PAGING MR. HOFFA

While DNA evidence is useful for identifying perpetrators, in many cases police also have to grapple with the problem of identifying—or even finding—victims. Bodies buried in hidden graves are particularly challenging; once the soil has settled, the gravesite may be impossible to see. Ground-penetrating radar, search dogs, and soil sampling can help, but each technique comes with severe limitations. None of these methods can search large areas efficiently, and bodies buried underneath concrete slabs elude all of them.

Using a clever high-efficiency sampling technique, researchers at the National Institute of Standards and Technology (NIST) in Boulder, Colorado, may have found a better approach. The method builds on a standard air-sampling strategy used in a variety of environmental sensing experiments. "A lot of people use purge and trap, whereby they pull a sample into an empty tube and trap with liquid nitrogen anything that might be volatile. What we do is instead of using a one-sixteenth-inch tube, we use a very small capillary tube, the inside periphery of which is actually coated with very strong absorbent," explains Thomas Bruno, Ph.D., leader of Properties for Process Separations in the Thermophysics Division of NIST and principal investigator on the new work.

For grave soil detection, Bruno and his colleagues lined the capillary tube with aluminum oxide, which has a high affinity for the protein decomposition products that come from decaying flesh. A vortex tube connected to the system first chills the capillary to -40°C during sample collection then heats it to 160°C to elute the sample, which the device then quantifies in a sensitive colorimetric assay.

"If the compound is present at approximately 20 parts per billion in our substrate, we can detect enough or capture enough to get a solute concentration within 10 percent," says Bruno, adding that "if all you're interested in is a yes or no answer we can do it with two to three parts per billion, so it's extremely sensitive."

In the team's initial proof-of-concept studies, the device could detect the bodies of decaying rats even 20 weeks after they had been buried. The investigators could even detect bodies underneath concrete slabs, simply by drilling a hair-thin hole through the slab and inserting the capillary tube. Besides the potential for solving modern-day crimes, the system might also help unearth historical graves. Bruno says he's already been approached by a group hoping to recover the bodies of British explorer Robert F. Scott and two compatriots, who are believed to be entombed beneath an Antarctic glacier.

Because the key innovations lie in the sampling method, simple modifications could produce systems to sense a wide range of other chemicals. Indeed, the NIST researchers are already collaborating with the U.S. Department of Homeland Security on a portable, high-sensitivity explosive-detection system built on the same general design.

"We'll probably have 60 or 100 capillaries in sort of a bundle, and the idea for Department of Homeland Security is: 'can you use this to sample a larger volume of air that might be inside of a cargo container on board a ship?'" says Bruno.

TIMING TEETH FROM TRINITY

Applied research such as Bruno's has produced many of the recent innovations in forensics, but basic science and serendipity have contributed as well. At the Lawrence Livermore National Laboratory in Livermore, California, for example, senior scientist Bruce Buchholz and his colleagues stumbled onto a new forensic tool while trying to answer a fundamental question in neuroscience.

"Maybe seven or eight years ago, I started working with Kirsty Spalding and Jonas Frisen at the Karolinska Institute, and we were looking at using the 14C bomb pulse to date turnover of neurons," explains Buchholz. The "bomb pulse" was the global spike in atmospheric 14C from aboveground nuclear testing at the height of the Cold War. People worldwide have taken the isotope into their tissues at predictable rates from the food supply, providing a potential clock for dating various cells' life-spans.

Looking for a control tissue to compare with the neurons, Buchholz and his colleagues realized that the mineral component of tooth enamel does not turn over. As a result, the level of 14C in the enamel reveals when it was formed; someone born in 1955 grew teeth with a much higher 14C level than someone born in 1985. Because scientists have tracked the bomb pulse rigorously over the years, the team can analyze a single tooth and determine an individual's birth date to the nearest year. Combining the method with an established technique called aspartic acid racemization could reveal both the approximate birth date and the approximate date of death of a cadaver.

Aboveground nuclear testing ended in 1963, so the technique will become less useful over time. Eventually, the isotope will dissipate and decay until 14C levels return to their pre-pulse state.

Nonetheless, testing 14C levels will likely remain useful for a while. "I suspect in 20 years it's going to be so flat that it's not going to be usable for something that's contemporary," says Buchholz, but he adds, "There's still a lot of applications for people who've been alive over the course of the pulse."

Whether future scientists' foray into forensics are accidental, like Buchholz's, or a deliberate career choice, like Callandro's, there is little doubt that researchers in the field will continue to push the technology in new directions. As they do, real world crime scene investigators can look forward to more happy endings and fewer cliffhangers.


Featured Participants



Federal Bureau of Investigation
www.fbi.gov

Forensic Science Service
www.forensic.gov.uk

Karolinska Institute
www.ki.se

Lawrence Livermore National Laboratory
www.llnl.gov

Life Technologies
www.lifetechnologies.com





National Institute of Standards and Technology
www.nist.gov

PerkinElmer
www.perkinelmer.com

Promega
www.promega.com

U.S. Department of Homeland Security
www.dhs.gov

University of Arizona
www.arizona.edu




Note: Readers can find out more about the companies and organizations listed by accessing their sites on the World Wide Web (WWW). If the listed organization does not have a site on the WWW or if it is under construction, we have substituted its main telephone number. Every effort has been made to ensure the accuracy of this information. Inclusion of companies in this article does not indicate endorsement by either AAAS or Science, nor is it meant to imply that their products or services are superior to those of other companies.

Alan Dove is a science writer and editor based in Massachusetts.

DOI: 10.1126/science.opms.p1000052



This article was published as a special advertising feature in the 04 March 2011 issue of Science magazine.


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