Applied Bio and Orchid Target Forensics Labs

Determining the identity of a person through a DNA sample is rarely straightforward, especially if the sample comes from a decomposed, burned, or otherwise degraded source. A body that has been submerged under water for months, for example, may contain DNA fragments that are too small to be analyzed by conventional means. Two companies, Foster City, Calif.-based Applied Biosystems http://www.appliedbiosystems.com, and Princeton, NJ-based Orchid Biosciences http://www.orchid.com, have worked out methods to deal with such samples. Applied Biosystems' new Quantifiler™ Human DNA and Y Human Male DNA Quantification kits enable investigators to rapidly determine if a sample contains DNA suitable for conventional analysis. Designed for real-time PCR (RT-PCR) quantification of DNA in a sample, the kits use automation to reduce hands-on time, says associate product manager Karen Cormier. And, because the technique is PCR-based, the kits provide a more comparable indicator of how much of the DNA in the sample can be amplified in downstream, PCR-based steps such as short tandem repeat (STR) analysis, she says. Current quantification methods employ membrane hybridization and involve a large number of manual steps. Yet frequently, investigators cannot determine quantitatively if the DNA they extract is suitable for later PCR-based testing. "At the moment the quantification test just tells you how much DNA is there, but that doesn't mean to say that it's useful DNA," says Peter Gill, research consultant with the Home Office Forensic Science Service in London. Since genomic analysis of DNA is time consuming and expensive, any technique that helps investigators eliminate unidentifiable samples is a plus, says Gill. RT-PCR based methods, says Barry Duceman, director of biology, New York State Police Office of Forensics, feature "cool technology that can take a sample that's been through the wringer, and tell you if you want to go ahead" with downstream analysis. Degraded DNA is not the only problem plaguing attempts at human identification. Cormier explains that many forensic samples are inhibited in some way. "You can imagine, going to a crime scene and having to scrape the goo off the sidewalk, there's other things that can get introduced into the DNA and sometimes come through in the extraction process," she says. To address this problem, the Quantifiler kits include an internal PCR control that indicates if a sample contains such inhibitors. Cormier adds that the Quantifiler Y Human Male DNA kit is the first and only commercially available kit able to quantify the human male portion of a DNA sample. SNP-BASED ALTERNATIVE While the Quantifiler kits can help labs determine how much of a sample is usable, investigators could be at a loss if the sample is largely degraded. The most commonly used DNA identification technique is based on STR analysis, in which regions containing tandemly repeated DNA elements are amplified by PCR. Degraded DNA lacks regions of sufficient length to be analyzed by this method. Orchid BioSciences instead uses SNP scoring (with SNPs from public databases) to analyze such samples. The Federal Bureau of Investigation (FBI) recently awarded Orchid two grants to develop its proprietary technology further. Bob Giles, executive director of research and development, Orchid Cell-mark, Dallas, explains that in STR analysis, "the fragments that are being copied need to be in [the] 200-to-300 base pair range, at least, to produce a profile from a DNA sample." But the fragments Orchid is analyzing from degraded DNA samples are far shorter, he says, only 50 to 90 base pairs in length. The technology to be developed for the FBI will permit identification of male DNA via Y-chromosome SNPs, as well as increase the throughput of SNP typing for forensic applications. Giles notes that Orchid's SNP technology has been used to analyze "thousands" of degraded samples from the World Trade Center disaster site and that it has been accredited for use as a standalone system for paternity testing. But don't expect SNP technology to replace STR technology any time soon. Barry Duceman notes that despite the advantages, it may be difficult for SNP-based methods to gain widespread use, as laboratories have accumulated upwards of 1 million DNA samples for the national DNA index using STRs. As STRs and SNPs examine different portions of DNA molecules, a SNP-based profile cannot be searched against an STR-based profile. Were SNP-based technology to be widely adopted, all these samples would need to be reanalyzed – a costly prospect, says Duceman. Still, Duceman sees immediate potential for SNP identification as an adjunct to traditional techniques, a sentiment Giles echoes. "As an additional technology for forensic scientists to use in those situations where their current technology is not successful, I think it's a very big step," says Giles. "It's just another thing they have available to them to help solve crimes." - Aileen Constans (additional reporting by Elizabeth Haserick) Identifying World Trade Center Victims Using SNP Technology DNA COLLECTION & PREPARATION Orchid Cellmark receives two sets of samples for DNA analysis – (A) DNA extracted from remains recovered from the World Trade Center site, and (B) reference samples obtained from either the victim’s personal items or close family members. The extracted genetic material is then amplified in preparation for SNP analysis. Figure 1 SNP DETERMINATION Using its SNP analysis technology, Orchid Cellmark determines which SNPs are present in each DNA sample, generating a specific pattern of SNPs for that sample. Figure 2 SAMPLE COMPARISON Since every individual has a unique pattern of SNPs, scientists can compare the patterns of SNP markers obtained from the World Trade Center and reference samples to identify matches. When a potential match is obtained, scientists confirm the identification. Figure 3

Applied Bio and Orchid Target Forensics Labs

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Aileen Constans

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February 2004

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