A new technology in mtDNA sequencing: Success rates vs time |
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| Authors: | F Balsa L Andrade M Carvalho A Serra V Lopes AM Bento H Afonso Costa C Oliveira L Batista MJ Anjos F Corte-Real |
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| Institution: | aForensic Genetics Service, Centre Branch, National Institute of Legal Medicine, I.P., Coimbra, Portugal;bNational Institute of Legal Medicine, I.P., Coimbra, Portugal;cFaculty of Medicine, University of Coimbra, Portugal |
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| Abstract: | Study of mitochondrial DNA (mtDNA) control region is a current practice in forensic genetics. In our service, mtDNA analysis is performed in many evidentiary specimens. Evaluation of this methodology is important to improve quality, increase efficiency and decrease artefacts, in order to reduce costs and time consuming.A case with 12 reference samples (bucal swabs) and 190 telogenic hair specimens extracted with DNA IQ™ System Tissue and Hair Extraction Kit (Promega) is reported. HVS-1 and HVS-2 control regions were sequenced with BigDye® Terminator v1.1 Kit (Applied Biosystems), using BetterBuffer (Microzone Limited), followed by a simple bead purification method (XTerminator) to remove unincorporated terminators. Application of this procedure had success in 180 hair samples within a very short time comparing to dRhodamine/ethanolic precipitation sequencing strategy and also demonstrated that better results are achieved with clean sequence data closer to the primer.The quality of data produced by the BigDye/BetterBuffer/XTerminator (BDX) procedure has been demonstrated to be very high. Besides that the BDX procedure can significantly reduce overall processing time and cost per reaction. This new methodology has additional advantages like fewer reagent transfers and smaller amounts of DNA. |
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| Keywords: | mtDNA BetterBuffer XTerminator Sequencing Methodology |
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