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According to Westen, Gerretsen & Maat (2008), "DNA identification is a tool used in a growing number of mass disasters and forensic investigations. When the identity of a person cannot be established with traditional identification methods, for example facial recognition, dactyloscopy, or odontolgoy, DNA analysis may offer a solution." Hard tissues (bone and teeth) are the preferable samples when body putrefaction or other environmental insults preclude DNA preservation in soft tissue (Alonso et al. 2005). Identification of remains in mass disasters is difficult, but DNA has made if possible if handled properly.
Mass disasters create a tremendous problem when it comes to the identification of remains. Comingling of individuals, fire damage, mutilation caused by the damage, and decomposition are only a few of the barriers that workers can face. Mass disasters can be dated back years, if not centuries. Some of the larger quantity and most memorable include The World Trade Center (2001) and the South East Asia tsunami (2004). In both of these mass disasters DNA was the primary form of identification. Butler states, "DNA testing has a major advantage in that it can be used to identify each and every portion of the remains recovered from the disaster site, provided 1) that there is sufficient intact DNA present to obtain a DNA type and 2) a reference sample is available for comparison purposes from a surviving family member or some verifiable personal item containing biological material."
Unfortunately there is not a lot of information available about the collection of DNA from bone samples. According to Westen, Gerretsen & Maat (2008), "Many publications and protocols have been presented on Methodology for isolating DNA from tissue samples for genotyping purposes in the laboratory. In contrast, limited information is available about the collection of bone and tooth samples, and advice on the prevention of contamination for these samples is often contradictory."
The primary source of information I was able to locate on the collection of DNA from bones came from an article titled: Femur, rib, and tooth sample collection for DNA analysis in disaster victim identification (DVI). The article was written by Westen, Gerretsen & Maat (2008) and primarily focused on the tsunami victims in 2004. The protocol implemented was from the Thai tsunami victim identification (TTVI) committee. After all initial work was done, such as, photographs and removal of the jaw bone and teeth samples were collected by all individuals. To take the bone sample from the femur, the skin was cut all the way to the femur during several incisions. Once at the femur, it was cleaned of all muscle tissue. After exposing the femur a hacksaw was used to remove a sample of the femur. For extraction of DNA from the ribs the same protocol was implemented with the exception that scissors were used to cut the rib rather than a hacksaw. With tooth extraction, a healthy tooth had to be used and must be free of caries, fillings, or any artificial modifications. Preferred extraction would consist of a canine, an upper incisor, or a molar with intact roots (Westen, Gerretsen & Maat, 2008).
With all extractions it is important to implement cross-contamination protocol. For the collection of the previous bones the same process was used for all. The protocol used was again from the TTVI. It consisted of five steps being completed prior to exposure of the bone that are pretty basic standards involving cleaning of tools and surfaces. However, in the incision of the femur, it required several different levels of cuts to the skin to cut back the level of contamination. The bodies had all been placed on top of each other, leading to a high possibility of contamination. Cutting and then cleaning all tools reduces the risk of cross contamination which is crucial in DNA identification with remains.
"Two types of reference samples are usually collected for DNA comparison with mass disaster remains: appropriate family references and direct references, such as personal effects, or antemortem biological specimens, such as biopsies and bloodstain cards", according to Alonso et al, 2005. Budowle, Bieber, & Eisenberg state, "As long as sufficient genomic DNA can be recovered, items that were previously handled or contacted may potentially serve as a reliable known reference sample for a direct DNA-based identification. Direct comparisons are straightforward and often require typing of fewer genetic loci to effect the reliable and valid identification than are needed in situations requiring indirect identification using kinship analyses."
After samples have been extracted from bone and reference samples have been obtain the process of testing the DNA begins. In an article written by Alonso et al (2005) it states, "Multiplex PCR amplifications of a variable number of autosomal short tandem repeats (STRs) loci is at present the preferred technology for DNA identification of mass disaster victims mainly due to its simplicity, adequate sensitivity, and high discrimination power." He goes on to say, "Severely degraded DNA samples could contain only very short DNA template molecules (under 150 bp) making conventional STR typing unsuccessful." This was one of the challenges in the World Trade Center natural disaster that began the development of new PCR typing that use very short DNA sequences. Newer methods of DNA typing data can increase success of obtaining genetic typing DNA from highly degraded samples (Budowle et al, 20005). Two types of these alternative tests are: mini-STRs, which reduce the size of STR amplicons and nuclear single nucleotide polymorphism (nuSNPs). These methods implement the use of smaller DNA target regions than are possible with the standard STR kits used within the forensic community.
The most informative genetic marker for identity testing is STR loci. Budowle et al, 2005 explains, "To improve success in STR typing of degraded DNA, the PCR primers for the STR loci can be repositioned so they reside closer to the repeat region." The amplified product will be reduced in length, and if smaller than some of the fragmented DNA, genetic characterization of the sample may then be possible. Techniques are improving and the amounts of DNA necessary keep getting smaller and smaller. This is very important because the amount of DNA contained in bones is not always high.
Once the DNA has been extracted from the bone and tested the job is far from complete. The sample then has to be compared to the reference sample that was obtained, if a reference could be located. One of the problems associated with reference samples is when a complete DNA profile is completed, and identification may not be possible if there are insufficient reference samples for comparison (Budowle et al, 2005). Familial samples may not be close enough in kin to determine an identity. For example, if no immediate family members are available for references and half-siblings are used the genetic information might be reduced so much a comparison cannot be completed. Another problem is the collection of biological material from a disaster site is sometimes anything but trivial (Butler, 2005). His example is if a plane crashes over an ocean, especially in deep water, then recovery of the remains can be quite difficult. Additionally Butler states, "By the very nature of the disaster, there is typically damage done to the biological samples and hence the DNA molecules contained therein."
An additional factor with the results is the amount of DNA that was extracted from the bone. The quantity can vary depending on the bone it is removed from. For example, Alonso et al (2001) states, "In our experience and according to others (14-16), the quality of DNA extracted from teeth is usually higher than that of DNA from bones. In addition, the quality of DNA obtained from long bones is higher than that extracted from skull or ribs."
Even though it is difficult, the main purpose of DNA typing in mass disasters is to provide identification of the remains. Due to many factors this obviously cannot happen in each case. As previously stated some of the factors can be the amount of DNA extracted, the reference samples, and the quality after it is tested. DNA has been critical in the identification of individuals in many of the mass disasters that our generation can recall. In the world trade center disaster, the Human Identification Project obtained usable profiles in about 75% of cases (Budimlija et al, 2003). Budimlija et al. goes on to say, "The success rate of nuclear DNA analysis on bone samples were similar to other identification efforts dealing with compromised samples."
As previously stated, identification of remains in mass disasters is difficult, but DNA has made it possible if handled properly. I briefly explained mass disasters and some of the difficulties that arise with them in the collection of DNA for identification, as well as demonstrated some basic methods for avoiding the cross contamination of the samples. Reference samples were explained and why they are important to the processing and the amounts of DNA that are extracted from the bones. I also explained the difficulties that come with identification and the sample comparisons. Finally, I presented a percentage based number of identifications that were obtained during the world trade center mass disaster. The amount of information I provided is just concerning the aspect of mass disasters and DNA extracted from bones for identification. This does not even scratch the surface of importance in the use of DNA from bones. DNA has been used for many other purposes, but I felt it was important to focus on one of the aspects that have greatly impacted the United States.