Resolving mtDNA mixtures by denaturing high-performance liquid chromatography and linkage phase determination |
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| Affiliation: | 1. University of Denver, Department of Biological Sciences, 2190 E. Iliff Avenue Rm 102, Denver, CO 80208, USA;2. Sun Yat-sen University, Department of Forensic Medicine, 74 Zhongshan Rd., Guangzhou, Guangdong 510089, PR China;3. Mitotyping Technologies, LLC, 2565 Park Center Blvd. Suite 200, State College, PA 16801, USA;1. University of Lausanne, School of Criminal Justice, Institute of Forensic Science, 1015 Lausanne-Dorigny, Switzerland;2. University of Lausanne, University Center of Legal Medicine Lausanne and Geneva, Rue du Bugnon 21, 1011 Lausanne, Switzerland;1. Section of Forensic Genetics, Department of Forensic Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, 2100 Copenhagen, Denmark;2. Forensic Genetics Unit, Institute of Forensic Sciences, University of Santiago de Compostela, Spain;3. Department of Mathematical Sciences, Aalborg University, DK-9220 Aalborg East, Denmark;1. ESR Ltd., Private Bag 92021, Auckland 1142, New Zealand;2. California Department of Justice, Jan Bashinski DNA Laboratory, Richmond, CA 94804, United States;1. Institute of Forensic Sciences Luis Concheiro, Genomics Medicine Group, University of Santiago de Compostela, Spain;2. Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Genomics Medicine Group, CIMUS, University of Santiago de Compostela, Spain;3. Forensic Genetics Laboratory, Institute of Legal Medicine, Università Cattolica del Sacro Cuore, Rome 001684, Italy;4. Instituto Nacional de Toxicología y Ciencias Forenses, Departamento de Sevilla, Spain;5. Center of Excellence in Genomic Medicine Research (CEGMR), King Abdulaziz University, Jeddah, Saudi Arabia;1. Institute of Legal Medicine, Innsbruck Medical University, Innsbruck, Austria;2. Central Institute for Blood Transfusion & Immunological Department, Innsbruck, Austria;3. Blood Transfusion Service Zürich, SRC, Schlieren, Switzerland;4. Penn State Eberly College of Science, University Park, PA, USA |
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| Abstract: | Mitochondrial DNA (mtDNA) sequencing can provide crucial information to forensic investigators when the quantity and quality of DNA would otherwise be limiting. The difficulty of analyzing mtDNA mixtures, however, has been a significant obstacle to its broader use in forensics. Denaturing high-performance liquid chromatography (DHPLC) in combination with direct sequencing makes it possible to determine the linkage phase of individual amplicons in a mixture. The reliability of the approach is based, in part, on the strong correlation between a change in the relative quantities of different DNA amplicons in a given mixture versus a change in the relative electrophoretic peak heights at mixed base positions. Using standard operating procedures previously validated for use in forensic laboratories, this approach enables sequence-specific fractionation of natural (heteroplasmic) or situational (multi-contributor) DNA mixtures prior to direct sequencing. As a novel approach for the rapid and accurate analysis of DNA mixtures, DHPLC may aid criminal investigators by making it possible to obtain definitive mitochondrial DNA results from otherwise challenging samples. |
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