Optimization of a method for the extraction of DNA from human skeletal remains for forensic casework
- WCU Author/Contributor (non-WCU co-authors, if there are any, appear on document)
- Sherri Marie Deaton (Creator)
- Institution
- Western Carolina University (WCU )
- Web Site: http://library.wcu.edu/
- Advisor
- Mark Wilson
Abstract: Obtaining full DNA profiles from bone can be challenging due to the inherently low quantity and quality of DNA. An efficient extraction protocol is required to maximize DNA recovery from bone samples while minimizing the coextraction of the PCR inhibitors naturally present in bone.DNA extraction from bone involves three discrete steps: demineralization, lysis, and purification. Demineralization involves incubating bone that has been pulverized into a powder in a chelating agent in order to sequester the divalent metal cations that comprise the mineral matrix of bone tissue. This is followed by a lysis step, in which the demineralized bone material is incubated in a buffer that disrupts the cellular membrane, releasing the DNA and intracellular components. Purification is performed after lysis to separate the DNA from proteins and cellular debris, as well as to remove PCR inhibitors.Several variables were examined in this study, including the use of three different lysis buffers, two different bone types, two different materials for the end caps and impactor bar used in the pulverization process, and the interactions between these variables. The effectiveness of each modification in the procedure was assessed using qPCR, which was used to measure mtDNA recovery in copies of mtDNA per gram of bone material, as well as by the assessment of inhibition using an internal positive control. The actual effectiveness of each lysis buffer varied considerably with bone types examined. In addition, a novel modification to the pulverization step was found to significantly enhance mtDNA recovery by reducing inhibition detected in samples.The findings of this work suggest that the current methodology is not fully optimized, and additional emphasis needs to be placed upon removing inhibitors but also investigating the effects of the bone lysate on the pH and ionic strength on the recovery of DNA from bone material. Preliminary work is presented from this study in developing a method for DNA extraction from human bone and elucidating the sources of inhibition that are introduced via the extraction method itself as well as those naturally present in bone tissue.
Optimization of a method for the extraction of DNA from human skeletal remains for forensic casework
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Created on 6/1/2015
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Additional Information
- Publication
- Thesis
- Language: English
- Date: 2015
- Keywords
- Bone, Extraction, Forensic, mtDNA
- Subjects
- Mitochondrial DNA -- Analysis -- Technique -- Evaluation
- Bone -- Analysis -- Technique -- Evaluation
- Forensic genetics -- Technique -- Evaluation