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Mitochondrial sequence analysis for forensic identification using Pyrosequencing technology
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
Uppsala University, Disciplinary Domain of Science and Technology, Physics, Department of Physics and Astronomy, Applied Nuclear Physics.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Genomics.
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2002 (English)In: BioTechniques, ISSN 0736-6205, Vol. 32, no 1, 124-6, 128, 130-3 p.Article in journal (Refereed) Published
Abstract [en]

Over recent years, requests for mtDNA analysis in the field of forensic medicine have notably increased, and the results of such analyses have proved to be very useful in forensic cases where nuclear DNA analysis cannot be performed. Traditionally, mtDNA has been analyzed by DNA sequencing of the two hypervariable regions, HVI and HVII, in the D-loop. DNA sequence analysis using the conventional Sanger sequencing is very robust but time consuming and labor intensive. By contrast, mtDNA analysis based on the pyrosequencing technology provides fast and accurate results from the human mtDNA present in many types of evidence materials in forensic casework. The assay has been developed to determine polymorphic sites in the mitochondrial D-loop as well as the coding region to further increase the discrimination power of mtDNA analysis. The pyrosequencing technology for analysis of mtDNA polymorphisms has been tested with regard to sensitivity, reproducibility, and success rate when applied to control samples and actual casework materials. The results show that the method is very accurate and sensitive; the results are easily interpreted and provide a high success rate on casework samples. The panel of pyrosequencing reactions for the mtDNA polymorphisms were chosen to result in an optimal discrimination power in relation to the number of bases determined.

Place, publisher, year, edition, pages
2002. Vol. 32, no 1, 124-6, 128, 130-3 p.
National Category
Medical and Health Sciences
URN: urn:nbn:se:uu:diva-92987PubMedID: 11808686OAI: oai:DiVA.org:uu-92987DiVA: diva2:166327
Available from: 2005-04-29 Created: 2005-04-29 Last updated: 2011-10-26Bibliographically approved
In thesis
1. Sensitive Forensic DNA Analysis: Application of Pyrosequencing and Real-time PCR Quantification
Open this publication in new window or tab >>Sensitive Forensic DNA Analysis: Application of Pyrosequencing and Real-time PCR Quantification
2005 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The field of forensic genetics is growing fast and the development and optimisation of more sensitive, faster and more discriminating forensic DNA analysis methods is highly important. In this thesis, an evaluation of the use of novel DNA technologies and the development of specific applications for use in forensic casework investigations are presented.

In order to maximise the use of valuable limited DNA samples, a fast and user-friendly Real-time PCR quantification assay, of nuclear and mitochondrial DNA copies, was developed. The system is based on the 5’ exonuclease detection assay and was evaluated and successfully used for quantification of a number of different evidence material types commonly found on crime scenes. Furthermore, a system is described that allows both nuclear DNA quantification and sex determination in limited samples, based on intercalation of the SYBR Green dye to double stranded DNA.

To enable highly sensitive DNA analysis, Pyrosequencing of short stretches of mitochondrial DNA was developed. The system covers both control region and coding region variation, thus providing increased discrimination power for mitochondrial DNA analysis. Finally, due to the lack of optimal assays for quantification of mitochondrial DNA mixture, an alternative use of the Pyrosequencing system was developed. This assay allows precise ratio quantification of mitochondrial DNA in samples showing contribution from more than one individual.

In conclusion, the development of optimised forensic DNA analysis methods in this thesis provides several novel quantification assays and increased knowledge of typical DNA amounts in various forensic samples. The new, fast and sensitive mitochondrial DNA Pyrosequencing assay was developed and has the potential for increased discrimination power.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2005. 44 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 33
Genetics, forensic, sensitive DNA analysis, DNA quantification, mtDNA, Real-time PCR, Pyrosequencing, Genetik
National Category
Medical Genetics
urn:nbn:se:uu:diva-5775 (URN)91-554-6234-0 (ISBN)
Public defence
2005-05-21, Rudbecksalen, Rudbecklaboratoriet, Uppsala, 09:00
Available from: 2005-04-29 Created: 2005-04-29Bibliographically approved

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