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  • 1.
    Allen, Marie
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Genomics.
    Andréasson, Hanna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Mitochondrial D-loop and coding sequence analysis using pyrosequencing2005In: Methods in Molecular Biology, ISSN 1064-3745, E-ISSN 1940-6029, Vol. 297, p. 179-196Article in journal (Refereed)
    Abstract [en]

    In forensic casework analysis, mitochondrial deoxyribonucleic acid (DNA) often is used when the evidence material contains scarce amounts of DNA. Here, a mitochondrial DNA typing system for D-loop and coding region analysis based on pyrosequencing is described. Pyrosequencing is a real-time, single-tube sequencing-by-synthesis method, in which a cascade of enzymatic reactions yields detectable light. This pyrosequencing system has a higher resolution than the D-loop analysis performed routinely today as it also covers informative positions in the mitochondrial coding region. The system is composed of 16 polymerase chain reaction (PCR) fragments and 24 pyrosequencing reactions with a turn around time for a 96-well plate of less than 3 h after PCR.

  • 2.
    Allen, Marie
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Divne, Anna-Maria
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Universal tag arrays in forensic SNP analysis.2005In: Methods in Molecular Biology, ISSN 1064-3745, E-ISSN 1940-6029, Vol. 297, p. 141-154Article in journal (Refereed)
    Abstract [en]

    Microarray-based single nucleotide polymorphism (SNP) genotyping enables simultaneous and rapid detection of a large number of markers and is thus an attractive method for forensic individual acid identification. This assay relies on a one-color detection system and minisequencing in solution before hybridization to universal tag arrays. The minisequencing reaction is based on incorporation of a fluorescent dideoxynucleotide to a primer containing a tag-sequence flanking the position to be interrogated. This one-color system detects C and T polymorphisms in separate reactions on multiple polymerase chain reaction targets with the fluorophore TAMRA coupled to the respective dideoxynucleotide. After incorporation, tagged primer sequences are hybridized through their complementary sequence on the array, and positive signals are detected by a confocal laser-scanner.

  • 3.
    Allen, Marie
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Divne, Anna-Maria
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Calloway, Cassandra
    Erlich, Henry
    Author´s response:  2006In: Journal of Forensic Sciences, ISSN 0022-1198, E-ISSN 1556-4029, Vol. 51, no 4, p. 937-938Article in journal (Other academic)
    Abstract [en]

    The mitochondrial hypervariable regions I and II have proven to be a useful target for analysis of forensic materials, in which the amount of DNA is limited or highly degraded. Conventional mitochondrial DNA (mtDNA) sequencing can be time-consuming and expensive, limitations that can be minimized using a faster and less expensive typing assay.We have evaluated the exclusion capacity of the linear array mtDNA HVI/HVII region-sequence typing assay (Roche Applied Science) in 16 forensic cases comprising 90 samples. Using the HVI/HVII mtDNA linear array, 56% of the samples were excluded and thus less than half of the samples require further sequencing due to a match or inconclusive results. Of all the samples that were excluded by sequence analysis, 79% could be excluded using the HVI/HVII linear array alone. Using the HVI/HVII mtDNA linear array assay, we demonstrate the potential to decrease sequencing efforts substantially and thereby reduce the cost and the turn-around time in casework analysis.

  • 4.
    Allen, Marie
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Genomics.
    Engström, A-S.
    Meyers, S.
    Handt, O.
    Saldeen, Tom
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Forensic Medicine.
    von Haeseler, A.
    Pääbo, S.
    Gyllensten, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Genomics.
    Mitochondrial DNA sequencing of shed hairs and saliva on robbery caps: sensitivity and matching probabilities1998In: Journal of Forensic Sciences, ISSN 0022-1198, E-ISSN 1556-4029, Vol. 43, no 3, p. 453-464Article in journal (Refereed)
    Abstract [en]

    Sequencing of mitochondrial DNA (mtDNA) has been used for human identification based on teeth and skeletal remains. Here, we describe an amplification system for the mtDNA control region (D-loop) suited for the analysis of shed hair, which constitutes the most common biological evidence material in forensic investigations. The success rate was over 90% when applied to evidence materials such as shed hair, saliva stains and saliva on stamps. The analysis of evidence materials collected from three similar robberies revealed the presence of mtDNA sequences identical to those of the suspects in the three crimes. The use of mtDNA control region sequences for individual identification was evaluated. The probability of identity by chance for the mtDNA types of the suspects in the robberies was found to vary between Pr = 0.017 - < 0.0017, depending on the reference population used, emphasizing the need for large population databases to obtain the appropriate estimate.

  • 5.
    Allen, Marie
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Genomics.
    Eriksson, Inger
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Liu, Limin
    Gyllensten, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    High resolution genetic typing of the class II HLA-DRB1 locus using group-specific amplification and SSO-hybridisation in microplates1998In: Hereditas, ISSN 0018-0661, E-ISSN 1601-5223, Vol. 129, no 2, p. 161-167Article in journal (Refereed)
    Abstract [en]

    The HLA-DRB1 locus is one of the most polymorphic HLA class II loci and rapid and accurate typing of this polymorphism is important both in bone-marrow transplantation, analysis of disease association and in forensic medicine. The allelic variation at DRB1 is characterized by combinations of a limited number of amino-acid motifs, reducing the resolution of a typing strategy based on a single PCR and subsequent analysis of polymorphic motifs. In the present paper we describe a strategy for typing of DRB1 based on eight allele-specific PCRs followed by sandwich hybridization to immobilized probes in a microplate format. The combined approach results in a rapid typing system with very high resolution. Using a rapid DNA extraction protocol, a complete HLA-DRB1 typing can be performed in less than a day.

  • 6.
    Allen, Marie
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Genomics.
    Kalantari, M.
    Ylitalo, Natalie
    Pettersson, B.
    Hagmar, B.
    Scheibenflug, L.
    Johansson, B.
    Pettersson, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Gyllensten, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    HLA DQ-DR haplotype and susceptibility to cervical carcinoma: indications of increased risk for development of cervical carcinoma in individuals infected with HPV 181996In: Tissue Antigens, ISSN 0001-2815, E-ISSN 1399-0039, Vol. 48, no 1, p. 32-37Article in journal (Refereed)
    Abstract [en]

    The association of HLA class II DQB1 and DRB1 alleles with the development of cervical carcinoma was studied in 150 Swedish patients using PCR-based HPV and HLA typing. The association of cervical carcinoma with alleles encoding the DQ3 antigen, previously found among German and Norwegian patients, was not observed in the Swedish patients. Five DQ-DR haplotypes were indicated to be positively associated with development of cervical carcinoma in the Swedish patients. Two of these HLA associations were specific for HPV 18 infected patients, suggesting that the ability of the oncogenic HPV 18 to cause more rapid-transit tumors than other high risk HPV types may be due to a deficiency in antigen presentation by the HLA molecules encoded by carried on these haplotypes.

  • 7.
    Allen, Marie
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Liu, Limin
    Gyllensten, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    A comprehensive polymerase chain reaction-oligonucleotide typing system for the HLA class I A locus1994In: Human Immunology, ISSN 0198-8859, E-ISSN 1879-1166, Vol. 40, no 1, p. 25-32Article in journal (Refereed)
    Abstract [en]

    A comprehensive system for genetic typing of the HLA class I A locus is described, based on PCR amplification and typing with nonradioactively labeled SSO probes. Exons 1-3 of the A locus are amplified and typing is performed with a set of 30 nonradioactively labeled oligonucleotide probes. This system resolves 34 of 39 known alleles and 561 (94%) of 595 possible genotypes. Among a sample of 354 individuals from Sweden and China, 97.5% of the genotypes were resolved. Probes were directed preferentially at replacement substitutions in foreign antigen-binding sites, in order to detect not only the known alleles but also new combinations of polymorphic motifs, indicative of previously unrecognized alleles. Three individuals were found with a new combination of polymorphic motifs, suggesting the presence of at least one previously undescribed allele in the populations sampled. This typing system is useful for disease association studies, tissue typing, and in forensic medicine.

  • 8.
    Allen, Marie
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Saldeen, T.
    Pettersson, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Gyllensten, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Genetic typing of HLA class II genes in Swedish populations: application to forensic analysis1993In: Journal of Forensic Sciences, ISSN 0022-1198, E-ISSN 1556-4029, Vol. 38, no 3, p. 554-70Article in journal (Refereed)
    Abstract [en]

    In an attempt to determine the value of DNA based typing of HLA class II loci to forensic analysis, allele and genotype frequencies at DQA1, DQB1, DPB1, and DRB1 were determined in samples from two Swedish populations using hybridization with sequence specific oligonucleotides to PCR amplified DNA. Significant allele frequency differences were observed at the DQB1 and DRB1 loci between the two populations, as well as between one of the Swedish and a Norwegian population. The average heterozygosity varies between 0.74 to 0.91 and the power of discrimination between 0.90 to 0.98, with the highest values obtained for the DRB1 locus. The probability of genotype identity by chance differs on average 2% between the populations. When applied to a paternity case with one parent deceased and a criminal case, typing of class II loci proved in both cases informative. Analyses of DR and DQ genes does not increase the power of discrimination, due to strong linkage, but offers through the reconstruction of putative haplotypes an internal control for the consistency of the typing results at several loci. Typing of the DRB1 and DPB1 loci was found to result in an approximate combined average probability of genotype identity by chance of one in a thousand.

  • 9.
    Allen, Marie
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Genomics.
    Saldeen, Tom
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Forensic Medicine.
    Gyllensten, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Genomics.
    Allele-specific HLA-DRB1 amplification of forensic evidence samples with mixed genotypes1995In: BioTechniques, ISSN 0736-6205, E-ISSN 1940-9818, Vol. 19, no 3, p. 454-463Article in journal (Refereed)
    Abstract [en]

    A major problem in analyzing forensic casework samples is the presence of genetic material from more than one individual in the material evidence. For instance, in sexual assault cases the evidence (vaginal swabs) usually contains a majority of vaginal epithelial cells and varying amounts of sperm cells from the perpetrator. Samples with mixed genotypes are also common among other biological evidence materials such as nail scrapes and mixed bloodstains. We have developed an allele-specific amplification system for the highly polymorphic HLA class II DRB1 locus that permits the detection of individual alleles in a sample with mixed genotypes, independent of the initial frequency of the alleles. Using a set of eight allele-specific amplification primers and typing the amplified fragments with sequence-specific probes, most of the 60 DRB1 alleles can be resolved. The method is highly specific and sensitive, with the potential for amplifying 15 copies of a particular allele in a background of 3 x 10(5) copies of other alleles. The method was successfully applied to three forensic cases, where the material evidence consisted of sperm stains on panties, nail scrapes and bloodstains on skin. Thus the DRB1 allele-specific amplification system can be employed for the unambiguous determination of the presence of individual alleles in materials suspected to contain mixed genotypes, even when the alleles of interest constitute only a small fraction of the total DNA

  • 10.
    Allen, Marie
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Genomics.
    Saldeen, Tom
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Forensic Medicine.
    Gyllensten, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Genomics.
    PCR-based DNA typing of saliva on stamps and envelopes1994In: BioTechniques, ISSN 0736-6205, E-ISSN 1940-9818, Vol. 17, no 3, p. 546-552Article in journal (Refereed)
    Abstract [en]

    In forensic cases involving mail bombs, extortion, kidnapping or threatening letters, biological evidence such as the saliva used to attach the stamp and seal the envelope could be used for genetic analysis. We have developed a highly sensitive semi-nested PCR method for the HLA-DRB1 locus; suitable for the analyses of very limited amounts of DNA. When applied to a set of stamps and envelopes with saliva from control individuals, typing results were consistent with those obtained using hairs drawn from the same individuals. No interference was found due to DNA from the fingerprints of people handling the letters. The system was applied to three forensic cases with threatening letters. The first case resulted in an exclusion of the suspect. In the second case, the suspect could not be excluded (probability of identical genotype by chance > 0.01). These results demonstrate that biological evidence in cases with threatening letters is amenable to genetic typing.

  • 11.
    Allen, Marie
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Sandberg-Wollheim, Magnhild
    Sjögren, Karin
    Erlich, Henry A.
    Petterson, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Gyllensten, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Association of susceptibility to multiple sclerosis in Sweden with HLA class II DRB1 and DQB1 alleles1994In: Human Immunology, ISSN 0198-8859, E-ISSN 1879-1166, Vol. 39, no 1, p. 41-8Article in journal (Refereed)
    Abstract [en]

    The association of MS with HLA class II alleles was studied by PCR-based typing of the DQA1, DQB1, DRB1, and DPB1 loci in 94 Swedish patients with relapses and remissions of the disease. The haplotype DRB1*1501-DQA1*0102-DQB1*0602 was found to be positively associated and three haplotypes were found to be negatively associated with MS. Linkage disequilibrium makes it difficult to assess whether DRB1 or DQB1 plays the primary role in the disease association, while the association with DPB1 and DQA1 appears to be secondary to that of DQB1 and DRB1. Two of the three haplotypes negatively associated with MS carry the DQB1*0301 allele. Also, the negatively associated DRB1*0401-DQA1*0301-DQB1*0301 haplotype differs from those with nonassociated DRB1*0401-DQA1*0301-DQB1*0302 haplotype only at DQB1. These results suggest that DQB1 alleles, as well as some DRB1 alleles, are involved in susceptibility and protection to MS. In searching for sequence motifs in the DR beta chain associated with MS susceptibility, all DRB1 alleles on haplotypes positively associated with MS, including the DRB1*1501, were found to encode a Val at position 86 of the DR beta chain. Also, DRB1 alleles that are negatively associated with MS all encode a Gly at position 86, suggesting that the residue at position 86 may be critical in conferring susceptibility and protection to MS. Finally, when the effect of the DRB1*1501 haplotype was removed there was no support for the hypothesis that MS is associated with a putative DQ-alpha beta heterodimer, encoded for by certain DQA1 and DQB1 alleles.

  • 12.
    Andreasson, H
    et al.
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Genetics and Pathology.
    Asp, A
    Alderborn, A
    Gyllensten, U
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Genetics and Pathology.
    Allen, M
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Genetics and Pathology.
    Mitochondrial sequence analysis for forensic identification using pyrosequencing technology.2002In: Biotechniques, Vol. 32, p. 124-Article in journal (Refereed)
  • 13.
    Andréasson, H.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Genomics.
    Gyllensten, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Genomics.
    Allen, Marie
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Genomics.
    Real-Time DNA Quantification of Nuclear and Mitochondrial DNA in Forensic Analysis2002In: BioTechniques, ISSN 0736-6205, E-ISSN 1940-9818, Vol. 33, no 2, p. 402-411Article in journal (Refereed)
    Abstract [en]

    The rapid development of molecular genetic analysis tools has made it possible to analyze most biological materialfound at the scene of a crime. Evidence materials containing DNA quantities too low to be analyzed using nuclear markers can be analyzed using the highly abundant mtDNA. However, there is a shortage of sensitive nDNA and mtDNA quantification assays. In this study, an assay for the quantification of very small amounts of DNA, based on the real-time Taq-Man assay, has been developed. This analysis will provide an estimate of the total number of nDNA copies and the total number of mtDNA molecules in a particular evidence material. The quantification is easy to perform, fast, and requires a minimum of the valuable DNA extracted from the evidence materiaL The results will aid in the evaluation of whether the specific sample is suitable for nDNA or mtDNA analysis. Furthermore, the optimal amount of DNA to be used in further analysis can be estimated ensuring that the analysis is successful and that the DNA is retained for future independent analysis. This assay has significant advantages over existing techniques because of its high sensitivity, accuracy, and the combined analysis of nDNA and mtDNA. Moreover, it has the potential to provide additional information about the presence of inhibitors in forensic samples. Subsequent mitochondrial and nuclear analysis of quantified samples illustrated the potential to predict the number of DNA copies required for a successful analysis in a certain typing assay.

  • 14.
    Andréasson, Hanna
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Allen, Marie
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Genomics.
    Rapid quantification and sex determination of forensic evidence materials2003In: Journal of Forensic Sciences, ISSN 0022-1198, E-ISSN 1556-4029, Vol. 48, no 6, p. 1280-1287Article in journal (Refereed)
    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.

  • 15.
    Andréasson, Hanna
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Asp, Allan
    Uppsala University, Disciplinary Domain of Science and Technology, Physics, Department of Physics and Astronomy, Applied Nuclear Physics.
    Alderborn, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy.
    Gyllensten, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Genomics.
    Allen, Marie
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Genomics.
    Mitochondrial sequence analysis for forensic identification using Pyrosequencing technology2002In: BioTechniques, ISSN 0736-6205, E-ISSN 1940-9818, Vol. 32, no 1, p. 124-6, 128, 130-3Article in journal (Refereed)
    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.

  • 16.
    Andréasson, Hanna
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Nilsson, Martina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Budowle, B.
    Frisk, Stine
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Allen, Marie
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Quantification of mtDNA mixtures in forensic evidence material using pyrosequencing2006In: International journal of legal medicine (Print), ISSN 0937-9827, E-ISSN 1437-1596, Vol. 120, no 6, p. 383-390Article in journal (Refereed)
    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.

  • 17.
    Andréasson, Hanna
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Nilsson, Martina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Lundberg, Hans
    Allen, Marie
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Genomics.
    Nuclear and mitochondrial DNA quantification of various forensic materials2006In: Forensic Science International, ISSN 0379-0738, E-ISSN 1872-6283, Vol. 164, no 1, p. 56-64Article in journal (Refereed)
    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.

  • 18.
    Andréasson, Hanna
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medicinsk genetik och genomik.
    Nilsson, Martina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medicinsk genetik och genomik.
    Styrman, Hanna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medicinsk genetik och genomik.
    Pettersson, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Allen, Marie
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medicinsk genetik och genomik.
    Forensic mitochondrial coding region analysis for increased discrimination using pyrosequencing technology2007In: Forensic Science International: Genetics, ISSN 1872-4973, E-ISSN 1878-0326, Vol. 1, no 1, p. 35-43Article in journal (Refereed)
    Abstract [en]

    Analysis of mitochondrial DNA (mtDNA) is very useful when nuclear DNA analysis fails due to degradation or insufficient amounts of DNA in forensic analysis. However, mtDNA analysis has a lower discrimination power compared to what can be obtained by nuclear DNA (nDNA) analysis, potentially resulting in multiple individuals showing identical mtDNA types in the HVI/HVII region. In this study, the increase in discrimination by analysis of mitochondrial coding regions has been evaluated for identical or similar HVI/HVII sequences. A pyrosequencing-based system for coding region analysis, comprising 17 pyrosequencing reactions performed on 15 PCR fragments, was utilised. This assay was evaluated in 135 samples, resulting in an average read length of 81 nucleotides in the pyrosequencing analysis. In the sample set, a total of 52 coding region SNPs were identified, of which 18 were singletons. In a group of 60 samples with 0 or 1 control region difference from the revised Cambridge reference sequence (rCRS), only 12 samples could not be resolved by at least two differences using the pyrosequencing assay. Thus, the use of this pyrosequencing-based coding region assay has the potential to substantially increase the discriminatory power of mtDNA analysis.

  • 19. Bogdanowicz, Wiesław
    et al.
    Allen, Marie
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medicinsk genetik och genomik.
    Branicki, Wojciech
    Lembring, Maria
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Gajewska, Marta
    Kupiec, Tomasz
    Genetic identification of putative remains of the famous astronomer Nicolaus Copernicus2009In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 106, no 30, p. 12279-12282Article in journal (Refereed)
    Abstract [en]

    We report the results of mitochondrial and nuclear DNA analyses of skeletal remains exhumed in 2005 at Frombork Cathedral in Poland, that are thought to be those of Nicolaus Copernicus (1473-1543). The analyzed bone remains were found close to the altar Nicolaus Copernicus was responsible for during his tenure as priest. The mitochondrial DNA (mtDNA) profiles from 3 upper molars and the femurs were identical, suggesting that the remains originate from the same individual. Identical mtDNA profiles were also determined in 2 hairs discovered in a calendar now exhibited at Museum Gustavianum in Uppsala, Sweden. This calendar was the property of Nicolaus Copernicus for much of his life. These findings, together with anthropological data, support the identification of the human remains found in Frombork Cathedral as those of Nicolaus Copernicus. Up-to-now the particular mtDNA haplotype has been observed only 3 times in Germany and once in Denmark. Moreover, Y-chromosomal and autosomal short tandem repeat markers were analyzed in one of the tooth samples, that was much better preserved than other parts of the skeleton. Molecular sex determination revealed that the skeleton is from a male individual, and this result is consistent with morphological investigations. The minimal Y-chromosomal haplotype determined in the putative remains of Nicolaus Copernicus has been observed previously in many countries, including Austria, Germany, Poland, and the Czech Republic. Finally, an analysis of the SNP located in the HERC2 gene revealed the C/C genotype that is predominant in blue-eyed humans, suggesting that Copernicus may have had a light iris color.

  • 20. Budowle, B.
    et al.
    Gyllensten, Ulf
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Chakraborty, R.
    Allen, Marie
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Forensic analysis of the mitochondrial coding region and association to disease.2005In: International journal of legal medicine (Print), ISSN 0937-9827, E-ISSN 1437-1596, Vol. 119, no 5, p. 314-315Article in journal (Refereed)
  • 21.
    Bus, Magdalena
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Genomics. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medicinsk genetik och genomik.
    Allen, Marie
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Genomics. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medicinsk genetik och genomik.
    Collecting and Preserving Biological Samples from Challenging Environments for DNA Analysis2014In: Biopreservation and Biobanking, ISSN 1947-5535, E-ISSN 1947-5543, Vol. 12, no 1, p. 17-22Article in journal (Refereed)
  • 22.
    Bus, Magdalena M.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medicinsk genetik och genomik. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Edlund, Hanna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medicinsk genetik och genomik. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Allen, Marie
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medicinsk genetik och genomik. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Forensic Analysis of Mitochondrial and Autosomal Markers Using Pyrosequencing®.2015In: Methods in Molecular Biology, ISSN 1064-3745, E-ISSN 1940-6029, Vol. 1315, p. 379-396Article in journal (Refereed)
    Abstract [en]

    Forensic casework analyses often face challenges, such as limited genetic material with or without fragmentation and damage. To compensate for low amounts and degradation, shorter amplicons are often applied in the analysis. Also, a change of markers might be necessary using mitochondrial instead of autosomal markers. In addition, forensic research often involves analysis of large number of samples for marker evaluation and population-database compilation. Therefore, a flexible, robust but also rapid method for the detection of variation is highly useful. Pyrosequencing(®) is a rapid, reliable, easy-to-use method for sequence analysis. The method is well suited for rapid forensic analysis of a few targets or analysis of a single target in many samples. It allows sequencing of very short amplicons, which facilitates analysis of degraded DNA. Here we present the use of Pyrosequencing, a robust method for sensitive forensic analysis of mitochondrial DNA, autosomal STRs, and Y-chromosome STRs and SNPs.

  • 23.
    Bus, Magdalena M.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medicinsk genetik och genomik.
    Karas, Ognjen
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medicinsk genetik och genomik.
    Allen, Marie
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medicinsk genetik och genomik.
    Multiplex pyrosequencing of InDel markers for forensic DNA analysis2016In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 37, no 23-24, p. 3039-3045Article in journal (Refereed)
    Abstract [en]

    The capillary electrophoresis (CE) technology is commonly used for fragment length separation of markers in forensic DNA analysis. In this study, pyrosequencing technology was used as an alternative and rapid tool for the analysis of biallelic InDel (insertion/deletion) markers for individual identification. The DNA typing is based on a subset of the InDel markers that are included in the Investigator (R) DIPplex Kit, which are sequenced in a multiplex pyrosequencing analysis. To facilitate the analysis of degraded DNA, the polymerase chain reaction (PCR) fragments were kept short in the primer design. Samples from individuals of Swedish origin were genotyped using the pyrosequencing strategy and analysis of the Investigator (R) DIPplex markers with CE. A comparison between the pyrosequencing and CE data revealed concordant results demonstrating a robust and correct genotyping by pyrosequencing. Using optimal marker combination and a directed dispensation strategy, five markers could be multiplexed and analyzed simultaneously. In this proof-of-principle study, we demonstrate that multiplex InDel pyrosequencing analysis is possible. However, further studies on degraded samples, lower DNA quantities, and mixtures will be required to fully optimize InDel analysis by pyrosequencing for forensic applications. Overall, although CE analysis is implemented in most forensic laboratories, multiplex InDel pyrosequencing offers a cost-effective alternative for some applications.

  • 24.
    Bus, Magdalena M.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medicinsk genetik och genomik. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Lembring, Maria
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medicinsk genetik och genomik. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Kjellstrom, Anna
    Stockholm Univ, Dept Archaeol & Class Studies, Osteoarchaeol Res Lab, S-10691 Stockholm, Sweden.
    Strobl, Christina
    Med Univ Innsbruck, Inst Legal Med, A-6020 Innsbruck, Austria.
    Zimmermann, Bettina
    Med Univ Innsbruck, Inst Legal Med, A-6020 Innsbruck, Austria.