A comparison of DNA extraction methods from hair shafts: mitochondrial DNA analysis using next generation sequencing and nuclear DNA analysis using InnoQuant DNA quantification kit and InnoTyper 21 DNA typing kit
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Forensic crime laboratories receive hair shafts as evidentiary samples and process them for DNA evidence as a means of identification of individuals. The Armed Forces DNA Identification Laboratory (AFDIL) provides DNA analysis to aid in the medico-legal death investigation of fallen service members and contributes to research and education in the field of forensic DNA analysis. The AFDIL receives hair samples from family members of service individuals as a reference standard as well as remains from both past and present day conflicts for identification. Often times nuclear DNA is too highly degraded in hair shafts to be obtained successfully. There are many more copies of mitochondrial DNA in a cell than nuclear DNA. Therefore, it is common practice to target mitochondrial DNA instead of nuclear DNA when processing a hair shaft for DNA evidence. Mitochondrial DNA also plays an important role at the AFDIL in familial identification when a self-reference standard is not available. The current validated protocol for extracting mitochondrial DNA (mtDNA) at the AFDIL is a manual digestion with a micro-tissue grinder followed by a 24-hour incubation step and an organic phenol-chloroform extraction. The first part of this study compares the currently validated organic extraction method with the new QIAGEN QIAamp® Fast DNA Tissue Kit extraction method. The QIAamp® Fast DNA Tissue Kit extraction is a mechanical, chemical, and enzymatic protocol involving a 2 mL disruption tube with a stainless steel bead which aides in disruption of the hair followed by a silica-column based purification. The QIAamp® Fast DNA Tissue Kit is a single day protocol which reduced lab processing time by more than half (7 hours to 3 hours). When results from six different hair samples extracted with the current organic method were compared with the same samples extracted with the QIAamp® Fast DNA Tissue Kit method, it was found that the two methods are comparable for mtDNA recovery, amplification, and sequencing. Recently, researchers at InnoGenomics© (New Orleans, LA) have developed nuclear DNA quantification and amplification kits that target small DNA fragments, creating the potential to obtain sufficient nuclear DNA data from samples containing highly degraded DNA. Instead of targeting loci that contain short tandem repeats (STRs), the InnoQuant™ DNA quantification kit and InnoTyper 21™ DNA typing kit (InnoGenomics©, New Orleans, LA) targets retrotransposable elements (REs). The second part of this study compares nuclear DNA extraction methods from hair shafts such as the QIAamp® Fast DNA Tissue Kit, a direct lysis procedure using ZyGEM® prepGEM™ enzyme, and direct lysis procedures using pronase. Nuclear DNA extracts were quantified InnoQuant™ and InnoTyper 21™ for DNA typing. Full profiles were obtained using the InnoTyper 21™ amplification kit from samples with as low as 0.0331 ng of DNA which were extracted with 0.05 mg/mL. However, no single extraction method demonstrated consistently higher DNA concentrations or more complete DNA profiles.