The effect of chemical compromise on the recovery of DNA from skeletonized human remains: A study of three World War II era incidents recovered from tropical locations.

The use of DNA extracted from skeletonized human remains is a common challenge for those working in human identification. Thermal age and chemical compromise should be considered prior to performing DNA testing on skeletonized remains. Both heat and chemical contamination may cause damage to the DNA present in the osseous materials and a subsequent increase in both the difficulty and expense of DNA testing. For this study, three World War II era mass fatality events involving the US military, the USS Oklahoma, the Battle of Tarawa, and the Cabanatuan Prison Camps, were examined for the overall success of DNA testing using five DNA modalities: Sanger sequencing of mitochondrial DNA, AmpFlSTR® MiniFiler™; PowerPlex® Fusion; a modified AmpFlSTR® Yfiler™; and a Next Generation Sequencing (NGS) protocol. Decedents from the three chosen incidents were buried in tropical environments and have the same approximate post mortem interval of 75 years, however, the chemical conditions that decedents were exposed to at each of the incidents vary. Remains from the USS Oklahoma were soaked in fuel oil and salt water immediately post-mortem; Cabanatuan Prison Camp remains were treated with a 'hardening' compound; and those from the Battle of Tarawa were not treated. Skeletal elements from each incident were compared across the 5 tested DNA modalities for success. Chemical insult to skeletal materials appears to have the greatest impact on every modality of DNA testing examined.

Getting Ahead: Extraction of DNA from Skeletonized Cranial Material and Teeth.

Between 1990 and 2018, the Defense POW/MIA Accounting Agency submitted 2177 cranial elements and 1565 teeth to the Armed Forces Medical Examiner System-Armed Forces DNA Identification Laboratory for DNA testing. In an effort to identify missing United States service members, materials were recovered from wartime losses inclusive of World War II, the Korean War, and Southeast Asia. Using four different DNA extraction protocols, DNA testing was performed using mitochondrial DNA Sanger sequencing, modified AmpFlSTR® Yfiler™, AmpFlSTR® MiniFiler™, PowerPlex® Fusion, or Next Generation Sequencing. This paper aims to provide optimal strategies for the DNA testing of skeletonized cranial materials. Cranial elements produced the most consistent results in Sanger sequencing using an organic purification; however, teeth were most successful for the same platform with an inorganic purification. The inverse is true for STR testing of cranial bones. Of the cranial elements, the temporal provided the most consistent results.

Extraction of DNA from Skeletonized Postcranial Remains: A Discussion of Protocols and Testing Modalities.

This paper provides a retrospective of the DNA analysis performed by the Armed Forces Medical Examiner-Armed Forces DNA Identification Laboratory between 1990 and 2018. Over 13,000 postcranial osseous materials, comprised of wartime losses from World War II, the Korean War, and South-East Asia, were examined by the following: mitochondrial DNA sequencing, a modified AmpFlSTR® Yfiler™, AmpFlSTR® MiniFiler™, PowerPlex® Fusion, or NGS. Four different DNA extraction protocols were used: incomplete demineralization coupled with an organic purification; complete demineralization with an organic purification; complete demineralization with an inorganic purification using QIAquick PCR Purification Kit; and a protocol designed specifically for use with next-generation sequencing. In general, complete demineralization coupled with an organic purification was the optimal extraction protocol for sequencing of mitochondrial DNA, regardless of the osseous element tested. For STR testing, demineralization paired with an inorganic purification provided optimum results, regardless of kit used or osseous element tested.

Determination of materials present in skeletonized human remains and the associated DNA: Development of a GC/MS protocol.

DNA testing of skeletonized human remains is considered to be challenging, especially when the remains have been exposed to inhibitory materials during decomposition. Inhibitors affect the processing of DNA, either by preventing efficient extraction or interfering with down-stream PCR-based processes. Limited studies have been performed on real-world samples that have been exposed to such inhibitors. This paper presents the development of a gas chromatography/mass spectrometry (GC/MS) protocol for the evaluation of materials present in skeletonized human remains recovered from the field, as well as the DNA extracted from the same materials. Twenty-one bone samples and seventeen DNA extracts were evaluated across three solvents and multiple GC/MS parameters to determine the optimal conditions for the recovery of trace materials present. The aim of this work is to provide a technique that can determine the presence of inhibitors prior to DNA extraction, allowing analysts to optimize removal of inhibitory materials.

Extraction of DNA from Skeletal Remains.

Acquisition of DNA from skeletal remains can be a delicate process. With the advent of improved extraction buffers that provide complete demineralization of the osseous materials, extraction of total genomic DNA from nearly any skeletal element is possible. This chapter describes both traditional organic and more newly developed inorganic extraction methods for fresh and dried skeletal remains.

Field contamination of skeletonized human remains with exogenous DNA.

The Armed Forces DNA Identification Laboratory reports the mitochondrial DNA (mtDNA) sequences of over 800 skeletal samples a year for the Joint POW/MIA Accounting Command-Central Identification Laboratory. These sequences are generated from degraded skeletal remains that are presumed to belong to U.S. service members missing from past military conflicts. In the laboratory, it is possible to control for contamination of remains; however, in the field, it can be difficult to prevent modern DNA from being transferred to skeletal elements and being carried forward through the analysis process. Four such cases are described here along with the controls in place in the laboratory to eliminate the possibility of the exogenous DNA being reported as authentic. In each case, the controls implemented by the laboratories prevented the false reporting of contaminant exogenous DNA from remains that were either faunal or human, but lacked endogenous DNA.

Mystery solved: the identification of the two missing Romanov children using DNA analysis.

One of the greatest mysteries for most of the twentieth century was the fate of the Romanov family, the last Russian monarchy. Following the abdication of Tsar Nicholas II, he and his wife, Alexandra, and their five children were eventually exiled to the city of Yekaterinburg. The family, along with four loyal members of their staff, was held captive by members of the Ural Soviet. According to historical reports, in the early morning hours of July 17, 1918 the entire family along with four loyal members of their staff was executed by a firing squad. After a failed attempt to dispose of the remains in an abandoned mine shaft, the bodies were transported to an open field only a few kilometers from the mine shaft. Nine members of the group were buried in one mass grave while two of the children were buried in a separate grave. With the official discovery of the larger mass grave in 1991, and subsequent DNA testing to confirm the identities of the Tsar, the Tsarina, and three of their daughters--doubt persisted that these remains were in fact those of the Romanov family. In the summer of 2007, a group of amateur archeologists discovered a collection of remains from the second grave approximately 70 meters from the larger grave. We report forensic DNA testing on the remains discovered in 2007 using mitochondrial DNA (mtDNA), autosomal STR, and Y-STR testing. Combined with additional DNA testing of material from the 1991 grave, we have virtually irrefutable evidence that the two individuals recovered from the 2007 grave are the two missing children of the Romanov family: the Tsarevich Alexei and one of his sisters.

DNA identification of "Earthquake McGoon" 50 years postmortem.

This report describes the genetic identification of James "Earthquake McGoon" McGovern, a WWII fighter ace who perished in Laos while providing supplies to French troops during the French Indochina war. Because reference samples were unavailable for all of the potential casualties, testing of the entire mitochondrial genome, autosomal STRs and Y-chromosomal STRs was performed to increase the genetic information available for analysis. Kinship analyses performed on the evidentiary data and numerous indirect family references for McGovern excluded other possible casualties and definitively established McGovern's identity. This particular case demonstrates the practical utility of novel research technologies and aggressive genetic typing protocols in the identification of aged, degraded remains.