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Mitochondrial sequence analysis for forensic identification using Pyrosequencing technology
Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi.
Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Fysiska sektionen, Institutionen för fysik och astronomi, Tillämpad kärnfysik.
Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet.
Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi, Genomik.
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2002 (Engelska)Ingår i: BioTechniques, ISSN 0736-6205, E-ISSN 1940-9818, Vol. 32, nr 1, s. 124-6, 128, 130-3Artikel i tidskrift (Refereegranskat) 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.

Ort, förlag, år, upplaga, sidor
2002. Vol. 32, nr 1, s. 124-6, 128, 130-3
Nationell ämneskategori
Medicin och hälsovetenskap
Identifikatorer
URN: urn:nbn:se:uu:diva-92987PubMedID: 11808686OAI: oai:DiVA.org:uu-92987DiVA, id: diva2:166327
Tillgänglig från: 2005-04-29 Skapad: 2005-04-29 Senast uppdaterad: 2017-12-14Bibliografiskt granskad
Ingår i avhandling
1. Sensitive Forensic DNA Analysis: Application of Pyrosequencing and Real-time PCR Quantification
Öppna denna publikation i ny flik eller fönster >>Sensitive Forensic DNA Analysis: Application of Pyrosequencing and Real-time PCR Quantification
2005 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
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.

Ort, förlag, år, upplaga, sidor
Uppsala: Acta Universitatis Upsaliensis, 2005. s. 44
Serie
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 33
Nyckelord
Genetics, forensic, sensitive DNA analysis, DNA quantification, mtDNA, Real-time PCR, Pyrosequencing, Genetik
Nationell ämneskategori
Medicinsk genetik
Identifikatorer
urn:nbn:se:uu:diva-5775 (URN)91-554-6234-0 (ISBN)
Disputation
2005-05-21, Rudbecksalen, Rudbecklaboratoriet, Uppsala, 09:00
Opponent
Handledare
Tillgänglig från: 2005-04-29 Skapad: 2005-04-29 Senast uppdaterad: 2018-01-13Bibliografiskt granskad

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