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Sensitive Forensic DNA Analysis: Application of Pyrosequencing and Real-time PCR Quantification
Uppsala universitet, Medicinska vetenskapsområdet, Medicinska fakulteten, Institutionen för genetik och patologi.
2005 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
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.

sted, utgiver, år, opplag, sider
Uppsala: Acta Universitatis Upsaliensis , 2005. , s. 44
Serie
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 33
Emneord [en]
Genetics, forensic, sensitive DNA analysis, DNA quantification, mtDNA, Real-time PCR, Pyrosequencing
Emneord [sv]
Genetik
HSV kategori
Identifikatorer
URN: urn:nbn:se:uu:diva-5775ISBN: 91-554-6234-0 (tryckt)OAI: oai:DiVA.org:uu-5775DiVA, id: diva2:166330
Disputas
2005-05-21, Rudbecksalen, Rudbecklaboratoriet, Uppsala, 09:00
Opponent
Veileder
Tilgjengelig fra: 2005-04-29 Laget: 2005-04-29 Sist oppdatert: 2018-01-13bibliografisk kontrollert
Delarbeid
1.
Posten ble ikke funnet. Det kan skyldes at posten ikke lenger er tilgjengelig eller det er feil id i adressefeltet.
2. Nuclear and mitochondrial DNA quantification of various forensic materials
Åpne denne publikasjonen i ny fane eller vindu >>Nuclear and mitochondrial DNA quantification of various forensic materials
2006 (engelsk)Inngår i: Forensic Science International, ISSN 0379-0738, E-ISSN 1872-6283, Vol. 164, nr 1, s. 56-64Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

Due to the different types and quality of forensic evidence materials, their DNA content can vary substantially, and particularly low quantities can impact the results in an identification analysis. In this study, the quantity of mitochondrial and nuclear DNA was determined in a variety of materials using a previously described real-time PCR method. DNA quantification in the roots and distal sections of plucked and shed head hairs revealed large variations in DNA content particularly between the root and the shaft of plucked hairs. Also large intra- and inter-individual variations were found among hairs. In additions DNA content was estimated in samples collected from fingerprints and accessories. The quantification of DNA on various items also displayed large variations, with some materials containing large amounts of nuclear DNA while no detectable nuclear DNA and only limited amounts of mitochondrial DNA were seen in others. Using this sensitive real-time PCR quantification assay, a better understanding was obtained regarding DNA content and variation in commonly analysed forensic evidence materials and this may guide the forensic scientist as to the best molecular biology approach for analysing various forensic evidence materials.

Emneord
quantification, real-time PCR, forensic materials, hair, nuclear DNA, mitochondrial DNA
HSV kategori
Identifikatorer
urn:nbn:se:uu:diva-92985 (URN)10.1016/j.forsciint.2005.11.024 (DOI)000242666700006 ()16427750 (PubMedID)
Tilgjengelig fra: 2005-04-29 Laget: 2005-04-29 Sist oppdatert: 2017-12-14bibliografisk kontrollert
3. Rapid quantification and sex determination of forensic evidence materials
Åpne denne publikasjonen i ny fane eller vindu >>Rapid quantification and sex determination of forensic evidence materials
2003 (engelsk)Inngår i: Journal of Forensic Sciences, ISSN 0022-1198, E-ISSN 1556-4029, Vol. 48, nr 6, s. 1280-1287Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

DNA quantification of forensic evidence is very valuable for an optimal use of the available biological material. Moreover, sex determination is of great importance as additional information in criminal investigations as well as in identification of missing persons, no suspect cases, and ancient DNA studies. While routine forensic DNA analysis based on short tandem repeat markers includes a marker for sex determination, analysis of samples containing scarce amounts of DNA is often based on mitochondrial DNA, and sex determination is not performed. In order to allow quantification and simultaneous sex determination on minute amounts of DNA, an assay based on real-time PCR analysis of a marker within the human amelogenin gene has been developed. The sex determination is based on melting curve analysis, while an externally standardized kinetic analysis allows quantification of the nuclear DNA copy number in the sample. This real-time DNA quantification assay has proven to be highly sensitive, enabling quantification of single DNA copies. Although certain limitations were apparent, the system is a rapid, cost-effective, and flexible assay for analysis of forensic casework samples.

HSV kategori
Identifikatorer
urn:nbn:se:uu:diva-92986 (URN)14640271 (PubMedID)
Tilgjengelig fra: 2005-04-29 Laget: 2005-04-29 Sist oppdatert: 2017-12-14bibliografisk kontrollert
4. Mitochondrial sequence analysis for forensic identification using Pyrosequencing technology
Åpne denne publikasjonen i ny fane eller vindu >>Mitochondrial sequence analysis for forensic identification using Pyrosequencing technology
Vise andre…
2002 (engelsk)Inngår i: BioTechniques, ISSN 0736-6205, E-ISSN 1940-9818, Vol. 32, nr 1, s. 124-6, 128, 130-3Artikkel i tidsskrift (Fagfellevurdert) 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.

HSV kategori
Identifikatorer
urn:nbn:se:uu:diva-92987 (URN)11808686 (PubMedID)
Tilgjengelig fra: 2005-04-29 Laget: 2005-04-29 Sist oppdatert: 2017-12-14bibliografisk kontrollert
5. Coding mtDNA analysis for increased forensic discrimination power using Pyrosequencing technology
Åpne denne publikasjonen i ny fane eller vindu >>Coding mtDNA analysis for increased forensic discrimination power using Pyrosequencing technology
Manuskript (Annet vitenskapelig)
Identifikatorer
urn:nbn:se:uu:diva-92988 (URN)
Tilgjengelig fra: 2005-04-29 Laget: 2005-04-29 Sist oppdatert: 2010-01-13bibliografisk kontrollert
6. Quantification of mtDNA mixtures in forensic evidence material using pyrosequencing
Åpne denne publikasjonen i ny fane eller vindu >>Quantification of mtDNA mixtures in forensic evidence material using pyrosequencing
Vise andre…
2006 (engelsk)Inngår i: International journal of legal medicine (Print), ISSN 0937-9827, E-ISSN 1437-1596, Vol. 120, nr 6, s. 383-390Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

Analysis of mtDNA variation using Sanger sequencing does not allow accurate quantification of the components of mtDNA mixtures. An alternative method to determine the specific mixture ratios in samples displaying heteroplasmy, consisting of DNA contributions from several individuals, or containing contamination would therefore be valuable. A novel quantification system for mtDNA mixture analysis has been developed based on pyrosequencing technology, in which the linear relationship between incorporated nucleotides and released light allows quantification of the components of a sample. Within five polymerase chain reaction fragments, seven variable positions in the mtDNA control and coding region were evaluated using this quantification analysis. For all single nucleotide polymorphisms quantified in this study, a linear relationship was observed between the measured and expected mixture ratios. This mtDNA quantification assay is an easy to use, fast and accurate quantification system, with the ability to resolve and interpret major and minor mtDNA components in forensic mixture samples.

Emneord
mixtures, mitochondrial DNA, pyrosequencing, quantification, forensic material
HSV kategori
Identifikatorer
urn:nbn:se:uu:diva-92989 (URN)10.1007/s00414-005-0072-8 (DOI)000241522300013 ()16453148 (PubMedID)
Tilgjengelig fra: 2005-04-29 Laget: 2005-04-29 Sist oppdatert: 2017-12-14bibliografisk kontrollert

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