Optimising direct PCR from anagen hair samples |
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| Institution: | 1. School of Biological Sciences, Flinders University, Australia;2. Forensic Science South Australia, Adelaide, Australia;1. Armed Forces DNA Identification Laboratory, Armed Forces Medical Examiner System, United States;2. American Registry of Pathology, United States;3. Flinders University, Australia;4. National Institute of Standards and Technology, United States;1. Laboratorio de Genética Molecular de Cruz Vital – Cruz Roja Ecuatoriana Quito, Ecuador;2. Laboratorio GENES Ltda, Medellin, Colombia;1. Institute of Legal Medicine, Faculty of Medicine, University of Cologne, Cologne, Germany;2. IPATIMUP – Institute of Molecular Pathology and Immunology of the University of Porto, Porto, Portugal;3. Forensic Genetics Unit, Institute of Legal Medicine, University of Santiago de Compostela, Santiago de Compostela, Spain;1. IPATIMUP, Institute of Molecular Pathology and Immunology of the University of Porto, Portugal;2. Faculty of Sciences of the University of Porto (FCUP), Portugal;3. Center of Biological and Natural Sciences, Federal University of Acre (UFAC), Rio Branco, AC, Brazil;4. DNA Diagnostic Laboratory (LDD), State University of Rio de Janeiro (UERJ), RJ, Brazil |
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| Abstract: | Anagen hairs are in the active growth phase, and when forcefully removed, may contain an intact root or sheathing. The hair root or sheathing is a source of nucleic DNA and can be amplified using direct PCR. Human identification STR kits are optimised to a small range of input DNA for PCR. Anagen hairs are unable to be quantified prior to amplification and can exhibit characteristics of an over-loaded DNA sample when analysed. The aim of this study was to optimise direct PCR for anagen hair sampling. Two separate modifications to the downstream processes were carried out in order to determine the most effective method at minimising PCR artefacts. Decreasing the cycle number from the standard 29 cycles to 27 cycles when using the NGM? kit displayed the best results for this method. However, decreasing the cycle number may increase allelic drop-out and would be costly for laboratories to perform an in-house validation. Diluting the PCR product during electrophoresis analysis minimises the effects of PCR artefacts in the same way decreasing the cycle number does. Diluting the PCR product is the most cost-effective method and does not increase the chance of allelic drop-out. |
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| Keywords: | Direct PCR Anagen hair Human identification Forensic casework NGM? STR profiling |
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