Highly efficient and automated extraction of DNA from human remains using a modified EZ1 protocol

Abstract

Bones and teeth often represent the only sources of DNA available for identifying human remains. DNA in bones and teeth is generally better preserved than that in soft tissues because of the presence of hard connective tissue with a high level of calcium. Because of the extensive mineralisation, the choice of an efficient DNA extraction procedure is important to minimise the sampling of a high level of minerals and to remove polymerase chain reaction (PCR) inhibitors. Some protocols are available for DNA extraction from bones and teeth as part of the Qiagen EZ1 DNA Investigator Kit using the EZ1 Advanced XL automated purification platform. To improve the efficiency of DNA extraction from skeletal remains, the present study focuses on a modification to these already available protocols. In this study, different bones and teeth collected between 1 and 50 years after death were subjected to DNA extraction using the standard EZ1 protocol, a supplementary protocol, and a modified protocol. The modified approach included a decalcification step, whereas the Qiagen protocols worked directly on non-decalcified powder. In all three procedures, 150 mg samples were used for DNA extraction. We evaluated the quantity of DNA recovered from samples, the presence of any PCR inhibitors co-extracted, the level of DNA degradation, the quality of short tandem repeat (STR) profiles, and the reproducibility of the modified procedure. When compared with the other protocols, the modified protocol resulted in the best recovery of DNA that was free of PCR inhibitors. Additionally, the STR profiles were reliable and of high quality. In our opinion, the decalcification step increases DNA recovery by softening tissues, which allows lysis solutions to act more effectively. Furthermore, the use of two lysis solutions and the variation added to the EZ1 purification step allow for DNA recovery with quality and quantity superior to those of the previously available Qiagen-based protocols. These findings may be helpful solutions to the problems commonly encountered when dealing with difficult samples, such as bones and teeth. KEY POINTSBones and teeth often represent the only sources of DNA for identifying human remains. The choice of an efficient DNA extraction procedure is important for maximizing DNA recovery and removing PCR inhibitors. This study focuses on modifications to the previously available Qiagen-based protocols. The modified protocol enabled the best recovery of DNA, and both quality and quantity were superior to those of the previously available Qiagen-based protocols. The STR profiles obtained from samples extracted using the modified protocol were reliable and of high quality.

Assessment of the efficiency of DNA isolation and profiling applying a temperature‐driven method in human remains

“…As a result, several researchers tried to improve the techniques toward this goal. Barbaro et al [2] developed a modified EZ1 protocol to improve the AE of bones and teeth with different postmortem intervals. This modification was successful with respect to the original protocol, increasing the DNA yield and decreasing the inhibition and degradation indices.…”

“…If DNA is the chosen method for identification, this relies on the possibility of obtaining enough sample from bones and/or teeth of good quality to get a full DNA profile to match a potential missing person. However, depending on the scenario, human remains are exposed to environmental factors, such as ultraviolet (UV) light, humidity, and temperature, as well as different types of soils, inducing the degradation of DNA and introducing inhibitors that could also hamper the polymerase chain reaction (PCR) [2].…”

“…Due to their position within the maxillary and mandibular bones, teeth are protected from the environment [3]. Enamel, the most external layer of the tooth, is the hardest component and the most highly mineralized material in the human body, protecting the other layers (dentin and pulp) from these environmental insults and increasing the chances of getting high-quality and good quality DNA yields [2].…”

“…Others applied a silica-based approach [3,7,8] or magnetic beads [9,10], as well as the classical phenol-chloroform methodology [11][12][13]. Other researchers applied automated extraction to avoid introducing external contamination to the samples, as less human manipulation is required [2,8,14].…”

An analysis of data curated from 5 years of identifying human remains

“…Extraction workflows often begin with demineralization of the bone powder during an incubation with EDTA [13][14][15] followed by cellular disruption with proteinase K/SDS digestion or high concentrations of chaotropic salt [4,[8][9][10][11][12][13] and gDNA was generally recovered by binding to silica [12,13].…”