Lab freezers are like mom's attic: cluttered with the property of people long gone. At home, that might include baseball cards and old shoes; in the lab, it's samples that graduate students and postdocs left behind. Mom can clean out her attic easily enough, but what about in the lab?
"I probably have stuff that goes back 20 years," in the freezers, says Jeffrey Bada, director of NASA Specialized Center in Research and Training in Exobiology at Scripps Institution of Oceanography. "I'll keep a sample as long as somebody knows what it is."
"It's a huge issue," agrees Kris Klueg, assistant scientist and cDNA/vector lab manager,
NOT NEW TO ALL
That DNA samples needn't be wet to be usable is nothing new; forensic scientists have known it for years. "You can get perfectly reasonable information from DNA extracted, for example, from blood stains on a shoe," Hogan says. The Royal Canadian Mounted Police (RCMP) notes in its most recent annual report: "Biological samples can be developed into DNA profiles from evidence that is decades old."
Today's dry storage systems stem from research conducted nearly 20 years ago by Leigh Burgoyne and Craig Fowler at Flinders University, Australia. The pair developed a mixture of chemicals that, when infused into a cotton paper, could protect DNA from nucleases and other degradative processes. They called their process FTA (fast technology for analysis of nucleic acids).
FTA paper contains chemicals to lyse cells, denature proteins, inactivate nucleases (the major cause of degradation), and hold the sample to the paper through physical entanglement, says Rob McPheeters, technical marketing manager for biosciences at Whatman, the Brentford, UK, company that licensed the technology from Flinders University.
The resulting DNA is extremely stable at room temperature, freeing up valuable freezer space for more current projects. McPheeters says DNA dried 14 years ago onto FTA paper has been successfully amplified. The samples also can be shipped through the regular mail without a biohazard label or any special handling.
Another advantage, McPheeters says, is that the paper inactivates bacteriophage. Thus, samples that develop a phage infection can be recovered: simply extract the DNA from the paper and reclone it. Finally, dry samples don't suffer the damage inherent in repeated freeze-thaw cycles.
Migration itself is trivial: Thaw the sample, apply it to FTA paper, dry, and file the card in a storage cabinet. "It's very easy to do," Klueg says. "If it's in bacteria, you grow the culture, concentrate it, take it and put in on the card and let it dry." According to Hogan, "Ninety percent of the work of migration is trying to find the samples and getting them organized."
Some groups have embraced the technology, especially forensic scientists. Biological specimens such as buccal swabs and blood samples can be applied directly to the cards for convenient storage and stabilization in the field. The RCMP has more than 80,000 FTA cards on file. "We have yet to find anything that is simpler to user for the police, easier to store, and more cost effective (than) the FTA card format," says Ron Fourney, director, RCMP DNA Databank. The US Department of Defense has 4.5 million cards in its catalog.
But access to those samples is inefficient, as the sample retrieval step wasn't automated, says Hogan. Consequently, adoption of the technology has generally been slow.
DYNAMIC AND PERSONAL
That could be changing, however, thanks to new automated storage systems and consumables from Whatman and GenVault. In April Whatman launched its new EasyClone 384-plate system (produced by GenVault), which adapts FTA technology to a 384-well microtiter plate format. Though Whatman already offered 96-well CloneSaver versions of its FTA cards, the new plates can be processed in automated workflows to simplify sample storage and retrieval.
Proteins: The Next Generation
Dry archival technologies are now being vetted for protein storage. "There's no equivalent to PCR for protein," says Mike Hogan, chief scientific officer at GenVault. "Most of the tests don't require protein to remain properly folded, just that it remain intact."
"Experts told us they needed two things from a protein dry-storage system," says Hogan: the ability to fully recover the protein, and to recover it at the same concentration at which it was stored. "We found no way to use a paper-type substrate to do that."
"There is, however, an extremely sophisticated sponge industry out there... offering extremely well-defined pores and elastic properties," he notes. In the system currently being tested, sponges are put into each well of a 96-well plate. Sample, such as serum, is then applied and allowed to dry, and finally reconstituted by adding buffer.
In testing Hogan routinely recovers about 90% of the total fluid volume at its original concentration. He estimates the protein will remain stable in a room temperature dry-storage system "for many months." Work to determine a more specific shelf life is ongoing. Nevertheless, Hogan expects to release a dry protein-storage system later this year. "We're investigating sponges for DNA also," says GenVault CEO Mitch Eggers.
GenVault offers its own 384-well FTA solution, called GenPlates. Each GenPlate holds either 384 aliquots of an individual DNA sample, or 40 aliquots each of six samples per plate. A 12-sample plate is in development. "The ultimate subdivision would have each well independent, with a different clone in each well," says Dave Wellis, GenVault's senior vice president for marketing and sales. This system would allow researchers to keep the samples from clinical trials on a single plate.
GenPlates can hold DNA from blood, buffy coat, cell suspensions, buccal mouthwash, and purified genomic DNA, and it is developing storage for DNA from buccal swabs. These plates can be stored, and samples retrieved, in one of GenVault's automated storage systems.
The Dynamic Archive GV10 series system has a starting capacity of 21,000 Gen-Plates; the series GV100 has 56,000 plates. Both configurations can be expanded for additional storage and include a barcode reader, integrated sample-management software, and a computer and server to maintain the database. The system can store 300,000 DNA samples in 390 square feet, versus the 2,400 square feet needed for freezers.
According to Mitch Eggers, CEO and cofounder of GenVault, such a system can reduce labor costs by a factor of 10, and overall costs of storage and retrieval by a factor of four. Indeed, using the Dynamic Archive, a single worker can store and retrieve as many as 200 plates per hour.
GenVault can store your samples at its own GLP-compliant, IRB-approved facility for $1 per plate per month. But if you prefer storing your samples at your own facility, the Personal Archive system ($99,950) stores 990 384-well plates; that's 380,160 DNA samples stored in less than 114 cubic feet of space. Storing the same number of samples in freezers would require about 60 freezers and 1,500 cubic feet of storage, at a cost of about $546,000 for the freezers alone (based on the cost of a Revco, 86°C, 25 cubic foot upright freezer, quoted at $9,100 at bestlabdeals.com)
CHAIN OF CUSTODY
Yet, as important as saving space is, verifying a sample's chain of custody and identification is perhaps even more so. In Ottawa, the RCMP integrated its sample control database with the forensic process several years ago. The resulting system, called STaCS, links each sample to a printed barcode on each FTA card for full traceability. Sample control is thus independent of human bias, which has improved logistics, so that DNA analysis is being used in high-volume crimes such as breaking and entering, and DNA from unsolved cases is being reexamined. From May 2003 to May 2004, the RCMP reported 1,872 cases in which a sample from a crime scene matched a DNA profile on its convicted offender list.
GenVault uses oligonucleotide-based tags called GenCodes, preloaded onto each paper before the DNA sample is added, to provide an internal tag of each sample's provenance. "The tag is removed in solution with the DNA, so you can always identify the sample," he explains. "This makes the chain of custody more robust."