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Genetic Identification of Human Remains in Mexico
Increasing DNA Extraction Efficiency from Degraded Tissues Using the Promega Maxwell® RSC 48 Instrument
Valentina Leonie Birne1, Franziska Holz1, Marcel A. Verhoff1, Christoph G. Birngruber1, Richard Zehner1 1 Institute of Legal Medicine, University Hospital Frankfurt, Goethe University, Frankfurt am Main, Germany
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Introduction
1. Identification of unknown deceased in Mexico
In Mexico, violence and crime have increased since the beginning of the “War on Drugs” in 2006 [1], accompanied by rising numbers of homicides that are often linked to drug-trafficking groups [2]. Furthermore, the National Registry of Missing and Unlocated Persons (Mexico) has been counting an increasing number of missing people from 2006 until today [3]. A total of 113,574 people are currently reported missing in Mexico and cannot be located. Almost every day, unknown dead bodies are found and delivered to Mexican Forensic Medical Services for identification. They may come from hidden mass graves that are frequently found by authorities or searching relatives. In more than a few cases, the bodies not only show signs of advanced decomposition, but also have been dismembered, burned, or treated with acid to complicate the identification process. Combined with financial and personal shortage, this vast number of bodies to be identified is a major burden for laboratories, authorities, and the society.
2. German-Mexican International Collaboration
Project “CoCiMex” (DAAD)
The CoCiMex project [4] was founded in 2021 as German-Mexican University Collaboration to support the Mexican Forensic Medical Services in the identification process of unknown deceased in Mexico. The Institute of Legal Medicine Frankfurt, Germany, was among others part of this project. The research project was supported by the German Academic Exchange Service (DAAD) with funds from the German Cooperation for Sustainable Development GmbH (GIZ) on behalf of the German Federal Foreign Office (DAAD Project ID: 57594060).
Project “ID-Mex” (UNFPA)
As follow-up project after the end of the CoCiMex cooperation, the United Nations Population Fund (UNFPA) and Goethe University Frankfurt am Main signed a cooperation agreement at the end of June 2023 to support the Mexican government in identifying unknown bodies in Mexico. The current project is called ID-Mex “Identification in Mexico”.
Which Tissue to Take?
During a research stay in Mexico as part of the CoCiMex project (DAAD), DNA samples from body parts that have been exhumed from a hidden mass grave were taken, including bone, teeth, hair, nail, tendon, muscle, and no longer differentiable soft tissue. DNA had to be extracted from body parts with heavy postmortem changes and, for different reasons, indeterminate storage conditions. Our laboratory was faced with a new challenge since we had rarely worked with such degraded tissues. The question was: Which tissue is most appropriate for DNA analysis while also considering time and cost efficiencies. Solid material such as teeth or bones are the gold standard as DNA material for genetic analysis of altered human remains, but the analysis is time- and cost consuming [5]. Different tissues such as kidney, liver, skeletal muscle, cartilage, intervertebral discs, and Achilles tendons were tested as alternative DNA material, proposed, and approved for DNA analysis of samples with an extended post-mortem interval. However, there are always cases in which the above-mentioned tissues cannot be used due to the decomposition of the corpses or simply because the body part to be examined has no nails, liver, or kidney. We wanted to explore if tendons could serve as a potential alternative DNA source, since tendons are present in every part of the body and can be easily collected and extracted without time-consuming sample preparation as in the case of bones. Our efforts focused on improving DNA extraction from bone and tendon samples using the Maxwell® FSC DNA IQ™ Casework Kit.
MATERIALS AND METHODS
To increase the efficiency of DNA extraction from these post-mortem altered tissues, several bone, tendon, and nail samples were analysed with modified protocols using the Promega Maxwell® RSC 48 instrument with the Maxwell® FSC DNA IQ™ Casework Kit combined with the Bone DNA Extraction Kit for bone samples and the Casework Extraction Kit for other tissues. DNA quantification was performed with the Promega PowerQuant® System qPCR assay and the Promega PowerPlex® Fusion System was used for STR allele calling. Not all extracted samples were analysed by STR analysis, as the focus of this study was on increasing the efficiency of DNA extraction. 10 representative bone and tendon samples were selected for STR analysis to illustrate the effects of the different extraction protocols on allele calling.
1. DNA Extraction from Bone Samples
Following the publication from Duijs and Sijen [6], the Bone DNA Extraction Kit protocol was adapted and tested with different bone samples. The main differences of the adapted protocol compared to the user guidelines were: a) the double extraction, i. e., the use of two 100 mg sample powders in separate tubes, b) the incubation step over night instead of 2.5 hours, c) the elution in 33µL elution buffer instead of 50µL, and the final pooling of the two extracts after the elution step.
2. DNA Extraction from Tendons and Other Tissues
In addition, the Casework Extraction Kit protocol for DNA extraction from tendons was adapted. The main differences of the adapted protocol compared to the user guidelines were: a) the double extraction, i. e., the use of two tendon samples in separate tubes, b) the extended incubation step with incubation in the shaker, and c) the pooling of the two lysates in one cartridge before the automated extraction. Both protocols used an elution volume of 35µL.
Figure 1: Overview of DNA bone extraction with the Bone DNA Extraction Kit. User guidelines (grey) and adapted protocol (magenta).
Figure 2: Overview of DNA soft tissue extraction with the Casework Extraction Kit. User guidelines (grey) and adapted protocol (blue).
RESULTS
1. Increasing DNA Extraction Efficiency from Bones
A higher DNA concentration was achieved in 13 of 17 analysed samples when bones were extracted according to the adapted protocol (pooling of two extracts). However, the completeness of the STR-profiles was not improved for every sample analysed, as the profile was already complete, or no DNA could be extracted. Overall, the adapted protocol achieved a 50% increase in extraction yields compared to the user guidelines.
Figure 3: Results of DNA analysis of bones extracted with the Bone DNA Extraction Kit (Promega) according to the user guidelines (grey) and the adapted protocol (magenta). A Bone extraction efficiency. B Completeness of STR profiles: 10 of 17 extracted samples were selected to illustrate the effects of the different extraction protocols on allele calling.
As a further option and for cost reasons, it was also tested, with five bone samples, whether the extraction performance could be increased simply by reducing the elution from 50µL to 33µL, while the remaining extraction steps were performed according to the user guidelines. The DNA output could be increased, however the adapted bone DNA extraction protocol using two pooled extracts resulted in higher DNA concentration values.
Figure 4: Results of DNA analysis of bones extracted with the Bone DNA Extraction Kit (Promega) according to the user guidelines (black), reduced elution volume from 50µL to 33µL (blue) and the adapted protocol (magenta).
2. Increasing DNA Extraction Efficiency from Tendons
Tendon extraction according to the adapted protocol (pooling of two lysates in one cartridge) resulted in higher DNA concentration values for all 21 tissue samples analysed compared to the tendons extracted according to the user guidelines. This also led to a higher rate of called STR loci in 6 of 10 analysed samples.
In some cases, there was a very high increase in extraction efficiency, e.g. from 0,2pg/µL to 27pg/µL. Overall, the adapted extraction protocol led to an average increase in extraction efficiency of 868%.
Figure 5: Results of DNA analysis of tendons extracted with the Casework Extraction Kit (Promega) according to the user guidelines (grey) and the adapted protocol (blue). A Tendon extraction efficiency. B Completeness of STR profiles: 10 of 21 extracted samples were selected to illustrate the effects of the different extraction protocols on allele calling.
3. Nail Extraction
We tested the adapted Casework Extraction Kit protocol not only on tendons, but on all the material we collected from the bodies from Mexico: muscle, hair, nail, and soft tissues that could no longer be differentiated. The adapted protocol led to higher DNA concentration values for almost every tissue. However, this was not the case for fingernails and toenails, where the adapted protocol led to a 34% decrease in extraction performance.
Figure 6: DNA extraction efficiency of nails extracted with the Casework Extraction Kit (Promega) according to the user guidelines (grey) and the adapted protocol (green).
CONCLUSION
The adapted protocol for DNA extraction from bone was able to increase the efficiency of DNA extraction, as could the adapted protocol for soft tissue in tendon. However, the extraction outcome of nail samples was lower when the adapted soft tissue protocol was used. The total amount of DNA extracted from nail samples was higher than the DNA quantities extracted from bone or tendon samples. It is possible that the Casework Extraction Kit, which was developed for challenging casework samples and low DNA quantities, is not the appropriate extraction kit to increase the extraction efficiency of samples with high DNA concentrations in the first place.
Genetic analysis of degraded tissue for identification purposes can be challenging due to the small amount of DNA and progressive DNA degradation. Therefore, maximising the DNA extraction outcome is a good starting point for high quality STR profiling. One way to increase the amount of extracted DNA is to increase the initial amount of the sample to be analysed during the extraction process. In automated/ half-automated extraction the input amount of the samples is often limited by the space available in the machine and preset reagent quantities. However, it is always possible to adapt commercially available kits for samples with special requirements, e. g. for highly decomposed samples with low DNA quantities.
To summarise, two alternative protocols for the DNA extraction of bone and tendon samples were implemented, which allow an easy pooling of samples ensuring cost and time efficient processing of forensically challenging samples when using the Maxwell® FSC DNA IQ™ Kit.
Valentina Leonie Birne PhD Research Assistent
In 2021, I started my Master Thesis on Forensic DNA Phenotyping and the molecular prediction of hair, eye and skin colour at the Institute of Legal Medicine in Frankfurt am Main, Germany. Following my Master’s Degree in Molecular Biosciences, I was lucky enough to work as a research assistant for the German-Mexican cooperation “CoCiMex”, a research project supported by the German Academic Exchange Service (DAAD) on behalf of the German Federal Foreign Office (Project ID: 57594060). Since 2023, I have been working as PhD candidate in the department of Forensic Biology at the Institute of Legal Medicine in Frankfurt am Main, Germany. In my research, I focus on the genetic identification of human remains and work as research assistant for the project “ID-Mex”, a project financed by the Unites Nations Population Fund (UNFPA) with the aim of identifying unknown deceased persons in Mexico.
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