2026-09-23T06:22:50Zhttps://shareok.org/server/oai/request

oai:shareok.org:11244/3425812026-06-01T14:59:25Zcom_11244_323718com_11244_47149col_11244_324596

Creecy, James Deen, Beth A. Haynie, Michelle L., 1975- Porterfield, Caitlin 2026-05-19T14:55:14Z 2026-05-19T14:55:14Z 2026 (Alma MMSId)9983181494502196 https://shareok.org//handle/11244/342581 (OCoLC)1592157790 Wildlife forensic investigations increasingly rely on molecular species identification, yet current polymerase chain reaction (PCR)-based methodologies are constrained by the need for species-specific primer sets, high reagent costs, and inefficiencies introduced by co-extracted nuclear and bacterial DNA. This study presents a universal, PCR-independent protocol for the isolation and sequencing of mitochondrial DNA (mtDNA) from non-human mammalian whole blood samples for forensic wildlife applications. Using Solid Phase Reversible Immobilization technology in conjunction with Exonuclease V enzymatic digestion (Mseek protocol), linear nuclear DNA was selectively digested and removed from tissue extracts, yielding purified circular mitochondrial genomes suitable for downstream next-generation sequencing (NGS) without PCR amplification. Co-extracted bacterial DNA was also effectively reduced through this protocol. Isolated mtDNA extracts from four felid species -- Acinonyx jubatus (cheetah), Panthera uncia (snow leopard), Panthera leo (lion), and Panthera onca (jaguar) -- were sequenced on the Illumina NextSeq 2000 platform and successfully mapped to their respective NCBI reference genomes. Post-Mseek samples demonstrated substantially higher mitochondrial mapping percentages (2.3–25.4%) compared to pre-Mseek samples (0.05–0.1%), with analysis times reduced from hours to seconds. These results demonstrate that targeted mtDNA isolation prior to NGS significantly increases sequencing efficiency, reduces per-sample cost, and eliminates dependency on known primer sequences, advancing the feasibility of a truly universal molecular method for species identification for forensic wildlife investigations. All rights reserved by the author, who has granted UCO Chambers Library the non-exclusive right to share this material in its online repositories. Contact UCO Chambers Library's Digital Initiatives Working Group at diwg@uco.edu for the permission policy on the use, reproduction or distribution of this material. Wildlife forensics--Technique Animal genetics--Technique Mitochondrial DNA Nucleotide sequence Deoxyribonucleic acid Forensic science Forensics Mitochondrial DNA Wildlife A universal method for the extraction, enrichment, and sequencing of mitochondrial genomes for the purposes of forensic wildlife investigations Academic theses M.S., Forensic Science Jackson College of Graduate Studies open.access