Author: Suni Edson
Edson, Suni, 2019 Process improvements for the extraction of DNA from skeletonized human remains, Flinders University, College of Science and Engineering
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The extraction of DNA from skeletonized human remains is a challenging issue faced by laboratories world-wide. When confronted with large sets of commingled remains, or even single sets of skeletonized remains, forensic laboratories must make a determination which skeletal element will provide the best opportunity for the recovery of high quality DNA. This thesis outlines the difficulties surrounding the extraction of DNA from skeletonized human remains and provides guidance for both sampling of osseous materials as well as a novel technique by which practitioners may identify PCR-inhibiting materials co-extracting with DNA.
In the first half of the thesis, a large set of skeletal elements was surveyed for success. The remains were recovered from world-wide locations by the Defense POW/MIA Accounting Agency (DPAA) and submitted to the Armed Forces Medical Examine System - Armed Forces DNA Identification Laboratory (AFMES-AFIL) for DNA testing. Samples recovered range from those lost during the Vietnam Conflict (1955-1975), Korean War (1950-1953); and the United States involvement in World War II (1941-1945). Elements surveyed had DNA extracted using four different extraction protocols, two of which involve an organic purification of the DNA removed from the remains. The DNA was tested using five different strategies: Sanger sequencing of mitochondrial DNA (mtDNA); a modified AmpFlSTR® Yfiler™ procotocol; AmpFlSTR® MiniFiler™; PowerPlex® Fusion; and Next Generation Sequencing (NGS).
The goal in the first few chapters was to provide a framework for practitioners to make decisions about both the osseous sampling strategy and the associated DNA testing strategy. By examining a large set of remains gathered from a myriad of conditions, recommendations can be made that are applicable in a variety of circumstances. In general, a DNA extraction protocol involving the complete demineralization of the skeletal material coupled with an organic purification is optimal for skeletal remains that are chemically compromised or contain a high level of fats. This is also the preferred extraction technique when testing with Sanger sequencing of mtDNA.
For the second half of the thesis, a novel technique is presented for the analysis of skeletal material and the associated DNA. A gas chromatography / mass spectrometry (GC/MS) protocol was developed to ascertain the compounds present in recovered skeletal materials and the associated DNA. The protocol involves the use of a solvent extraction (acetonitrile and dichloromethane) of osseous detritus removed from the surface of a bone during the standard cleaning prior to pulverization for DNA extraction. The material removed is typically discarded as medical waste, but in this new protocol, it provides an insight both into the person from which the skeletal materials originated and the environment in which decomposition occurred.
By comparing the GC/MS profiles generated from the skeletal materials to that of the extracted DNA, it was determined that there is little to no carry-over of compounds from the osseous element to the DNA. The results indicate that the extraction protocols currently in use are highly effective at removing any chemical compounds endogenous to the remains, as carry-over was seen in less than 0.1% of the samples tested.
The outcome of this work is a framework practitioners may use to evaluate groups of skeletonized remains for the osseous element that will provide the best results for the testing strategy being employed. The GC/MS testing performed would seem to indicate that the extraction protocols currently in use are effective at the removal of potential PCR inhibitors and other endogenous materials. This result should encourage practitioners to revisit DNA extraction protocols to perhaps reduce the stringency of available extraction techniques thereby reducing DNA loss while still maintaining sample purity.
Keywords: DNA, skeletonized human remains, Gas Chromatogrphy/Mass Spectrometry, human identification, DNA extraction
Subject: Biology thesis
Thesis type: Doctor of Philosophy
Completed: 2019
School: College of Science and Engineering
Supervisor: Adrian Linacre