Diagnosing post-mortem treatments which inhibit DNA amplification from US MIAs buried at the Punchbowl

The US military is committed to recovering and identifying the remains of unknown military service members. Casualties of the Korean War were exhumed from the National Memorial Cemetery of the Pacific, or Punchbowl, and submitted to the Armed Forces DNA Identification Laboratory (AFDIL) for mtDNA sequencing. Contrary to AFDIL's experience on other samples from this era, most failed to yield amplifiable DNA. Suspicion fell on mortuary practices that may have been applied to the remains, evidenced by a white powder found with the bones, and general records suggesting the use of formaldehyde-based stablizing agents. To improve the chances of successful identification of the unknown individuals, we looked for the causes underlying this failure. We did this by examining the state of the collagen, the most abundant biomolecule in bone, by using differential scanning calorimetry (DSC) and transmission electron microscopy (TEM). The DSC analyses showed collagens with a range of different thermal stabilities. When these results were compared with the DNA amplification results, a clear correlation between elevated thermal stability and amplification failure was evident. TEM analysis revealed that fibril integrity was maintained after thermal and acid treatments in the samples which failed amplification. Together these two approaches implicate a stabilization agent as the cause of problems with DNA analysis, presumably due to excessive cross-linking. Following the initial study, the ability of DSC to rapidly identify problem samples was tested in a blind study of 14 samples, the method successfully identifying all the problematic samples from Punchbowl. Within this unusual context, DSC analysis is a useful method to assess the likelihood of successful DNA extraction and amplification.