Anthropologists use 300,000-year-old ancient DNA (aDNA) to help inform their studies regarding human evolution and population. By virtue of time, this aDNA arrives at the lab highly degraded.
In the modern forensic world, this degradation, which results in highly fragmented and damaged DNA, is also seen in victims of war, disaster victim cases, mass fatality cases and even some cold cases.
This led Elena Zavala, a graduate student at Max Planck Institute for Evolutionary Anthropology, to ask the question: can methods from ancient DNA be used to improve the success rate for forensic identification?
“After receiving a Master’s in forensic science, I chose to do my Ph.D. in ancient DNA specifically to learn about methods used in this field and see if they are applicable to modern forensics,” said Zavala during a presentation at ISHI 32.
In her session, Zavala presented the results of her recent study comparing three common forensic extraction methods to one of the most widely used ancient DNA extraction methods.
In 2015, the Armed Forces DNA Identification Laboratory (AFDIL) validated a next-generation sequencing protocol for degraded mtDNA in the hopes of identifying soldiers from the Korean War. After testing on 153 samples, AFDIL was disappointed to find an overall success rate of only 44%.
That’s when they decided to bring in reinforcements—namely Zavala and the ancient DNA extraction methods she has spent her Ph.D. pioneering.
“Not only have we been using NGS, but we’ve also taken every single step along the laboratory workflow and optimized all these methods for the recovery and retention of degraded DNA specifically,” said Zavala.
AFDIL gave Zavala 15 previously tested long bones from unidentified soldiers from WWII and the Korean War. The bones were buried, subjected to an insecticide/deodorant for transport, soaked in a 40 to 50% formaldehyde mixture for 3 to 5 days, underwent a fungicide treatment and were finally reburied.
“This is good for the human corpse,” said Zavala, “but not so much for DNA.” Suffice to say, the DNA Zavala was trying to analyze was indeed highly degraded.
For the study, Zavala used the bones to test different extraction protocols: 3 common forensic extraction protocols, 1 aDNA extraction protocol, the same aDNA extraction protocol at a different incubation, and finally a combined forensic and aDNA extraction protocol.
In all three forensic DNA extraction protocols, the bone pellet completely dissolved and went into the solution during the initial lysis step. However, that was not the case with the aDNA method. So, Zavala used the method to incubate at 37 C, and then reextracted the same bone pellet and completed the extraction protocol a second time at 56 C—just for comparison’s sake.
According to the study results, the aDNA method at 56 C performed better than all three forensic protocols, and the aDNA method at 37 C outperformed two of three forensic extraction methods.
“Theoretically, if we skipped the pre-extraction step at 37 degrees and started right way with the 56 degrees, all this DNA would have potentially been released all at once. If we combine the two, the aDNA method is significantly better than all other extraction methods,” said Zavala.
Overall, Zavala and her team found that the ancient DNA extraction method tested in the study recovered between 2 and 37x as many DNA fragments that mapped to the human genome than any of the other extraction protocols.
These results led AFDIL to begin the process of validating the ancient DNA extraction protocol (56 C incubation) for their casework going forward.
Zavala said she and her team will continue working on the project to test the impacts of hybridization capture on the amount of mtDNA that can be recovered.