Author: Thien Ngoc Le
Le, Thien Ngoc, 2022 Human bone cells: detection and STR profiling, Flinders University, College of Science and Engineering
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DNA encased in bone is a potential substrate for human identification in forensic casework if there is a need to associate human remains with reference samples. The source of DNA within bones is embedded in a calcium-based matrix that needs to be removed to allow subsequent DNA analyses. An initial step in DNA isolation may be to drill into the bone and create a fine powder. Decalcification is required to remove calcium from the powder and aid in the release of the cells containing DNA. The removal of calcium is performed in a solution containing a high molarity of EDTA. It is not possible to monitor how many cells, and therefore the amount of DNA, are present in the powder and then in the EDTA-rich solution. An outcome may be that some bone samples have no DNA, or insufficient DNA yields, for subsequent DNA profiling. The ability to record cells at the early stage of DNA isolation from bone, and therefore enhance DNA recovery, is the central part of this thesis. Cells were recorded as being present using a DNA binding dye (Diamond nucleic acid dye, or DD) and presented in the published paper ‘A novel approach for rapid cell assessment to estimate DNA recovery from human bone tissue’ in the journal Forensic Science Medicine & Pathology.
Archived osteochondral tissues may be a potential template for short tandem repeat (STR) profiling as these samples have an abundance of nucleated cells, such as chondrocytes and osteocytes. DNA within these cells were rapidly visualised by use of a mini portable microscope after the application of the same DNA staining dye (DD). Stained cellular material appeared as fluorescent green images, and at the expected size and morphology of a bone cell. The number and location of the stained cells was recorded, along with areas within the bone matrix where there was an absence of cells. A standard DNA extraction method was performed to collect DNA from eleven bone sections (5 µm thickness). DNA yields were quantified by real-time polymerase chain reaction (qPCR). Ten of the eleven samples (91%) returned a low-level degradation status and subsequently generated complete STR profiles (based on the targeting of 15 STR loci). The remaining other sample reported a higher degradation metric than the others and subsequently recorded an incomplete STR profile. There was therefore a significant correlation between the number of DD-stained cells and the number of STR alleles amplified (p < 0.05) (publication I, chapter 2). The inference is that staining sections of bone with a nucleic acid dye can determine the prevalence of bone cell nuclei and then significantly increase the chance of generating a DNA profile.
This thesis also investigated the use of a simple means to release bone cells from the bone matrix such that the cells could then be easily collected and used as a template for DNA profiling (‘DNA profiling from human bone cells in the absence of decalcification and DNA extraction’ was published in the Journal of Forensic Sciences). Nine stored tibial tissues (aged between 3 – 5 years between sample collection and analyses) were part of this study. Using these samples, approximately ~ 20 mg was removed and fragmented using a clean pestle, this was without the use of liquid nitrogen or any specific device. The fragmented remains were stained with DD to visualise the matrix-free cells. These cells were collected and subjected directly to qPCR. A standard DNA extraction process was performed in parallel to compare the two sets of results. Processing the nine samples by both methods resulted in informative STR profiles with minimal indication of inhibitors. By adding the stained cells directly to the qPCR and also as a template for a direct PCR approach, circumvented the decalcification step and DNA extraction process, resulted in STR profiles from tibia samples being generate within 8 hours (publication II, chapter 3).
Keywords: human bone; bone cells; osteocytes; diamond nucleic acid dye; STR typing, matrix-free bone cells; decalcification, DNA extraction
Subject: Biotechnology thesis
Thesis type: Masters
Completed: 2022
School: College of Science and Engineering
Supervisor: Adrian Linacre