Genetic Engineering & Biotechnology News

AUG 2018

Genetic Engineering & Biotechnology News (GEN) is the world's most widely read biotech publication. It provides the R&D community with critical information on the tools, technologies, and trends that drive the biotech industry.

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Page 26 of 33 | Genetic Engineering & Biotechnology News | AUGUST 2018 | 25 develop a microfluidic chip that grabs the sperm cells while allowing everything else to be washed away, all in one go. Previously, investigators tried using separa- tion procedures incorporating sperm-binding antibodies, but these antibodies often come away empty-handed because they grasp at sur- face antigens that can begin to degrade within hours. Seeking more durable handholds, Dr. Demirci and his colleagues turned to lectins, highly stable proteins that bind sperm cells to oligosaccharides that coat egg cells. The sperm cell's surface is studded with many lectin molecules, which boost the sperm-capturing efficiency of the research- ers' oligosaccharide-covered chip surface. The researchers spent several years optimiz- ing their chip's surface chemistry and design- ing a flowthrough to retain the highest pos- sible percentage of sperm cells. They also teamed up with George Dun- can, Ph.D., a DNA supervisor at the Broward County Sheriff's Office Forensic Laboratory, to test the chip under real-world conditions. Even when the chip was used on decade-old swabs, it successfully snagged sperm, dem- onstrating up to 92% capture efficiency. The chip, Dr. Demirci asserts, is easy to use and less dependent on human labor than pre- vious protocols. "This will be the advantage in the long run," he adds, "when the technol- ogy is translated and commercialized." Smaller, Cheaper, faSTR Once the relevant DNA is isolated, it is cut into fragments, which are analyzed to create a DNA profile. Over the last 10 years, innova- tors have created "rapid DNA" systems that have cut the time between cheek swab and DNA profile down to 90 minutes. Though technically portable, these machines still weigh a hefty 150 lb. "We refer to that as 'two-man luggable,'" says James P. Landers, Ph.D., a professor at the University of Virginia (UVA). Besides maintaining UVA appointments in chemistry, mechanical engineering, and pa- thology, he holds executive posts at ZyGEM US and MicroGEM. Challenged to design a system that could fit in a backpack, Dr. Landers and his team, aided by UVA's Applied Research Institute, embarked on what Dr. Landers calls "a three-year trek to deliver something para- digm shifting." The effort was sponsored by the Department of Defense. By radically reimagining the mechanics that could be used in a rapid DNA system, the team created a device weighing just 10 pounds. "It's roughly the size of two reams of paper," Dr. Landers points out. In this device, which is called "faSTR," a spinning microflu- idic disc puts fluids into motion, making it un- necessary to resort to vacuum pumps or other bulky hardware. "The device is the size of an old-fashioned CD," Dr. Landers notes. "But it has fairly complex fluidic architecture." The much smaller dimensions of the new system required some fine-tuning to optimize the biochemistry. According to Dr. Landers, the faSTR system can complete a short tan- dem repeat (STR) analysis from a swab in 35 minutes. Naturally, a system that is smaller and faster than its predecessor is going to be more expensive, right? Not necessarily. "We've pioneered a method for making microfluidic discs out of overhead transparen- cies," Dr. Landers states. A pattern of microflu- idic channels is created by printing toner onto the disc everywhere except where the channels will be. Production of the discs can involve a dozen layered transparencies, with different channel patterns printed onto the different lay- ers, allowing three-dimensional designs. Handheld, while-you-wait DNA profil- ing, such as that provided by faSTR, could transform the forensic collection of genetic information. How useful that information will be in solving crimes depends, in some cases, on the availability of searchable data- bases of DNA profiles for comparison. Fighting Crime with Genetic Genealogy Once DNA has been collected from a crime scene and analyzed, it can be com- pared to DNA taken directly from a suspect. If no suspect is available, authorities can search databases full of DNA "fingerprints" in hopes of finding a fingerprint that matches the DNA from the crime scene. Law-enforcement databases typically con- tain DNA fingerprints taken from prior of- fenders, so if these databases fail to produce a match, investigators may need to widen their search. They may even consider delving into ancestry databases, which represent new troves of DNA information. A new genetic genealogy service has been unveiled by Parabon NanoLabs. Investiga- tors can work with the company to check their sample against DNA profiles that have been uploaded to a public database such as GEDmatch. Some DNA profiles may closely match the DNA from a crime scene. The more closely two profiles match, the more likely they come from closely related indi- viduals, and perfectly matching profiles may even point to the same individual. Ellen Greytak, Ph.D., director of bioin- formatics at Parabon, emphasizes that not all DNA genealogy databases are fair game for law enforcement searches. "There are a number of companies that let you use their DNA-testing services," she says. "These companies maintain their own private databases. Law enforcement has no access to those databases at all." Company-specific database restrictions also affect customers. For example, if two family members get tested by two different companies, they can't compare their results. Enter GEDmatch. The company doesn't do its own DNA testing. It just accepts files, which customers upload so they can compare their information with other customers' infor- mation. Customers must expressly allow their GEDmatch files to be publicly searchable; only then can these files be searched by com- panies such as Parabon. Starting with a DNA sample left at a crime scene by an unknown suspect—or by an un- identified victim—Parabon searches the da- tabase for relatives as far removed as a third cousin or a shared great-great-grandparent. The company has partnered with genetic gene- alogist CeCe Moore, best known for her work finding the biological relatives of adoptees. To- gether, the partners use genetic ties to build a family tree that will, if all goes well, lead back to the individual whose DNA has been obtained. Recently, the team used this method to solve a 31-year-old murder case in Washing- ton state. Two persons were in the database who were both second cousins to the suspect but genetically unrelated to each other. Us- ing genealogical records, Moore constructed both family trees and found where they in- tersected by marriage, leading to a positive identification of the suspect. Proteins Do What DNA Can't Finally, when DNA is hard to come by, researchers can turn to protein. "Protein is in- trinsically more stable," says Glendon Parker, Ph.D., founder and CEO of Protein-Based Identification Technologies. Over time, DNA degrades until the remaining segments are too short to be amplified by PCR. Some forensic samples, such as fingerprint residues and hair strands, may yield too little DNA for analysis. Still, DNA has become the go-to molecule for forensic identification because protein assays require a certain amount of finesse. Proteins denature and lose their three-dimen- sional structure, making enzyme assay results inconsistent. Protocols for analyzing DNA, on the other hand, became more streamlined and reproducible. "The whole field moved over to DNA and for very good reasons," Dr. Parker says. "In the meantime, the field of proteomics has gone through a revolution." Protein-based identification employs the same principle at work in DNA profiling. Es- sentially, the technique detects single-amino- acid changes, brought about by single-nucle- otide changes in the DNA. A protein contain- ing single-amino-acid polymorphisms (SAPs) is digested with an enzyme, creating a set of peptides roughly 8–30 amino acids long. Then, mass spectrometry is used to determine the weights of the various peptides. Finally, the pattern of fragment sizes reveals changes in the amino acid sequence. "The pattern is complex, but it's a direct function of the se- quence of amino acids," Dr. Parker explains. Investigators can create a unique personal protein signature if they test enough of these genetically variable peptides. "It's another way of reading what's in their DNA," Dr. Parker declares. He and his team found that testing around 60 different peptides gives a power of discrimination of one in a billion. So far, he says, over 300 different genetically variable peptides have been catalogued. Considering that humans shed hair con- stantly, should people worry that their hair will reveal their personal genetic footprint? Not yet, says Dr. Parker. "At this point," he notes, "we don't have the databases we would need." Protein-based identification can't be used to search for a completely un- known suspect, only to compare the crime scene sample to a given individual. Tradi- tional detective work won't be supplanted anytime soon, it seems. Translational Medicine At Stanford University, a microfluidic chip has been developed that efficiently extracts sperm from rape kit samples. The chip's channels, which are coated with Sialyl-Lewis X , a carbohydrate ligand for sperm-egg binding, will capture a perpetrator's sperm, but not a victim's epithelial cells, which are easily washed away. After sperm are captured on the chip, they are lysed, and sperm DNA analysis commences. You Can Run, but Your DNA Can't Hide Continued from page 1 ZyGEM US, the microfluidics and forensics arm of MicroGEM, is developing the faSTR Profiling System, a field DNA analysis device that can complete a short tandem repeat (STR) analysis from a swab in 35 minutes. Currently focused on criminal justice and military applications, faSTR will eventually extend to clinical diagnostics, potentially as a home-based testing system.

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