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  • 1.
    Ahmadian, Afshin
    et al.
    KTH, Superseded Departments, Biotechnology.
    Lundeberg, Joakim
    KTH, Superseded Departments, Biotechnology.
    A brief history of genetic variation analysis2002In: BioTechniques, ISSN 0736-6205, E-ISSN 1940-9818, Vol. 32, no 5, p. 1122-+Article, review/survey (Refereed)
    Abstract [en]

    As the human genome sequence is determined, there is an emerging need for the analysis of human sequence variations as genetic markers in diagnosis, linkage and association studies, cancer research, and pharmacogenomics. There are several different techniques and approaches for detecting these genetic variations, and here we review some of these techniques and their application fields. However, all the techniques have advantages and disadvantages, and factors such as laboratory instrumentation, personnel experience, required accuracy, required throughput, and cost often have to be taken into account before selecting a method.

  • 2.
    Ahmadian, Afshin
    et al.
    KTH, Superseded Departments, Biotechnology.
    Lundeberg, Joakim
    KTH, Superseded Departments, Biotechnology.
    Nyrén, Pål
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Uhlén, Mathias
    KTH, Superseded Departments, Biotechnology.
    Ronaghi, M.
    Analysis of the p53 tumor suppressor gene by pyrosequencing2000In: BioTechniques, ISSN 0736-6205, E-ISSN 1940-9818, Vol. 28, no 1, p. 140-+Article in journal (Refereed)
    Abstract [en]

    Tumor suppressor genes are implicated in cell cycle progression. Inactivation of these genes predominantly occurs through mutations and/or allelic loss that involves both alleles. With inactivation by multiple mutations in a single gene, cloning of the amplified gene is necessary to determine whether the mutations reside on one ol both alleles. Using pyrosequencing, a recently developed approach based on sequencing-by-synthesis, we studied genetic variability in the p53 tumor suppressor gene and could quantify the ratio between the mutated and wild-type amplified fragments. Further-more, this sequencing technique also allows allelic determination of adjacent mutations with no cloning of amplified fragments.

  • 3.
    Ahmadian, Afshin
    et al.
    KTH, Superseded Departments, Biotechnology.
    Russom, Aman
    KTH, Superseded Departments, Biotechnology.
    Andersson, Helene
    KTH, Superseded Departments, Biotechnology.
    Uhlén, Mathias
    KTH, Superseded Departments, Biotechnology.
    Stemme, Göran
    KTH, Superseded Departments, Biotechnology.
    Nilsson, Peter
    KTH, Superseded Departments, Biotechnology.
    SNP analysis by allele-specific extension in a micromachined filter chamber2002In: BioTechniques, ISSN 0736-6205, E-ISSN 1940-9818, Vol. 32, no 4, p. 748-754Article in journal (Refereed)
  • 4.
    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

  • 5.
    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.

  • 6. Andersson, T.
    et al.
    Unneberg, P.
    Nilsson, Peter
    KTH, Superseded Departments, Biotechnology.
    Odeberg, Jacob
    KTH, Superseded Departments, Biotechnology.
    Quackenbush, J.
    Lundeberg, Joakim
    KTH, Superseded Departments, Biotechnology.
    Monitoring of representational difference analysis subtraction procedures by global microarrays2002In: BioTechniques, ISSN 0736-6205, E-ISSN 1940-9818, Vol. 32, no 6, p. 1348-+Article in journal (Refereed)
    Abstract [en]

    Various approaches to the study of differential gene expression are applied to compare cell lines and tissue samples in a wide range of biological contexts. The compromise between focusing on only the important genes in certain cellular processes and achieving a complete picture is critical for the selection of strategy. We demonstrate how global microarray technology can be used for the exploration of the differentially expressed genes extracted through representational difference analysis (RDA). The subtraction of ubiquitous gene fragments from the two samples was demonstrated using cDNA microarrays including more than 32 000 spotted, PCR-amplified human clones. Hybridizations indicated the expression of 9100 of the microarray elements in a macrophage/foam cell atherosclerosis model system, of which many were removed during the RDA process. The stepwise subtraction procedure was demonstrated to yield an efficient enrichment of gene fragments overrepresented in either sample (18% in the representations, 86% after the first subtraction, and 88% after the second subtraction), many of which were impossible to detect in the starting material. Interestingly, the method allowed for the observation of the differential expression of several members of the low-abundant nuclear receptor gene family. We also observed a certain background level in the difference products of nondifferentially expressed gene fragments, warranting a verification strategy for selected candidate genes. The differential expression of several genes was verified by real-time PCR.

  • 7.
    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.

  • 8.
    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.

  • 9. Barken, K. B.
    et al.
    Gabig-Ciminska, Magdalena
    KTH, Superseded Departments, Biotechnology.
    Holmgren, Anders
    KTH, Superseded Departments, Biotechnology.
    Molin, S.
    Effect of unlabeled helper probes on detection of an RNA target by bead-based sandwich hybridization2004In: BioTechniques, ISSN 0736-6205, E-ISSN 1940-9818, Vol. 36, no 1, p. 124-+Article in journal (Refereed)
    Abstract [en]

    Unlabeled helper oligonucleotides assisting a bead-based sandwich hybridization assay were tested for the optimal placement of the capture and detection probes. The target used was a full-length in vitro synthesized mRNA molecule. Helper probes complementary to regions adjacent to the binding site of the 5' end attached capture probe were found much more effective than helper probes targeting positions adjacent to the detection probe binding site. The difference is believed to be caused by a disruption of the RNA secondary structure in the area where the capture probe binds, thereby reducing structural interference from the bead. The use of additional helpers showed an additive effect. Using helpers, at both sides of the capture and detection probes showed a 15- to 40-fold increase in hybridization efficiency depending on the target, thereby increasing the sensitivity of the hybridization assays. Using an electrical chip linked to the detection probe for the detection of p-ominophenol, which is produced by alkaline phosphatase, a detection limit of 2 x 10(-13) M mRNA molecules was reached without the use of a nucleic acid amplification step.

  • 10. Beisvåg, Vidar
    et al.
    Kauffmann, Audrey
    Malone, James
    Foy, Carole
    Salit, Marc
    Schimmel, Heinz
    Bongcam-Rudloff, Erik
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, The Linnaeus Centre for Bioinformatics. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Landegren, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Parkinson, Helen
    Huber, Wolfgang
    Brazma, Alvis
    Sandvik, Arne K.
    Kuiper, Martin
    Contributions of the EMERALD project to assessing and improving microarray data quality2011In: BioTechniques, ISSN 0736-6205, E-ISSN 1940-9818, Vol. 50, no 1, p. 27-31Article in journal (Refereed)
    Abstract [en]

    While minimum information about a microarray experiment (MIAME) standards have helped to increase the value of the microarray data deposited into public databases like ArrayExpress and Gene Expression Omnibus (GEO), limited means have been available to assess the quality of this data or to identify the procedures used to normalize and transform raw data. The EMERALD FP6 Coordination Action was designed to deliver approaches to assess and enhance the overall quality of microarray data and to disseminate these approaches to the microarray community through an extensive series of workshops, tutorials, and symposia. Tools were developed for assessing data quality and used to demonstrate how the removal of poor-quality data could improve the power of statistical analyses and facilitate analysis of multiple joint microarray data sets. These quality metrics tools have been disseminated through publications and through the software package arrayQualityMetrics. Within the framework provided by the Ontology of Biomedical Investigations, ontology was developed to describe data transformations, and software ontology was developed for gene expression analysis software. In addition, the consortium has advocated for the development and use of external reference standards in microarray hybridizations and created the Molecular Methods (MolMeth) database, which provides a central source for methods and protocols focusing on microarray-based technologies.

  • 11.
    Belikov, Sergey
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Lackmann, Fredrik
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Wieslander, Lars
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Incorrect assignment of affected nucleotides in footprinting/probing experiments2017In: BioTechniques, ISSN 0736-6205, E-ISSN 1940-9818, Vol. 63, no 3, p. 105-106Article in journal (Refereed)
  • 12. Blomstergren, A.
    et al.
    O'Meara, D.
    Lukacs, M.
    Uhlén, Mathias
    KTH, Superseded Departments, Biotechnology.
    Lundeberg, Joakim
    KTH, Superseded Departments, Biotechnology.
    Cooperative oligonucleotides in purification of cycle sequencing products2000In: BioTechniques, ISSN 0736-6205, E-ISSN 1940-9818, Vol. 29, no 2, p. 352-+Article in journal (Refereed)
    Abstract [en]

    Nucleic acid hybridization is an essential component in many of today's standard molecular biology techniques. In a recent study, we investigated whether nucleic acid capture could be improved by taking advantage of stacking hybridization, which refers to the stabilizing effect that exists between oligonucleotides when they hybridize in a contiguous tandem fashion. Here, we describe a specific approach for purification of sequencing products using cooperative probes that hybridize to single-strand targets where one of the probes has been coupled to a magnetic bead. This approach has been developed for standard sequencing primers and has been applied to shotgun plasmid libraries. The cooperative probes have been designed to anneal within the common vector sequence and to avoid copurification of non extended sequencing primers and misprimed sequencing products. The reuse of magnetic beads, together with salt independent elution, makes the approach suitable for high-capacity capillary electrophoresis instruments.

  • 13. Chen, Xingqi
    et al.
    Shi, Chengxi
    Yammine, Samer
    Göndör, Anita
    Rönnlund, Daniel
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Fernandez-Woodbridge, Alejandro
    Sumida, Noriyuki
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Ohlsson, Rolf
    Chromatin in situ proximity (ChrISP): Single-cell analysis of chromatin proximities at a high resolution2014In: BioTechniques, ISSN 0736-6205, E-ISSN 1940-9818, Vol. 56, no 3, p. 117-124Article in journal (Refereed)
    Abstract [en]

    Current techniques for analyzing chromatin structures are hampered by either poor resolution at the individual cell level or the need for a large number of cells to obtain higher resolution. This is a major problem as it hampers our understanding of chromatin conformation in single cells and how these respond to environmental cues. Here we describe a new method, chromatin in situ proximity (ChrISP), which reproducibly scores for proximities between two different chromatin fibers in 3-D with a resolution of similar to 170 angstrom in single cells. The technique is based on the in situ proximity ligation assay (ISPLA), but ChrISP omits the rolling circle amplification step (RCA). Instead, the proximities between chromatin fibers are visualized by a fluorescent connector oligonucleotide DNA, here termed splinter, forming a circular DNA.with another circle-forming oligonucleotide, here termed backbone, upon ligation. In contrast to the regular ISPLA technique, our modification enables detection of chromatin fiber proximities independent of steric hindrances from nuclear structures. We use this method to identify higher order structures of individual chromosomes in relation to structural hallmarks of interphase nuclei and beyond the resolution of the light microscope.

  • 14.
    Darmanis, Spyros
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Kähler, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Spångberg, Lena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Kamali-Moghaddam, Masood
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Landegren, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Schallmeiner, Edith
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Self-assembly of proximity probes for flexible and modular proximity ligation assays2007In: BioTechniques, ISSN 0736-6205, E-ISSN 1940-9818, Vol. 43, no 4, p. 443-450Article in journal (Refereed)
    Abstract [en]

    Proximity ligation assay (PLA) is a recently developed strategy for protein analysis in which antibody-based detection of a target protein via a DNA ligation reaction of oligonucleotides linked to the antibodies results in the formation of an amplifiable DNA strand suitable for analysis. Here we describe a faster and more cost-effective strategy to construct the antibody-based proximity ligation probes used in PLA that is based on the noncovalent interaction of biotinylated oligonucleotides with streptavidin followed by the interaction of this complex with biotinylated antibodies.

  • 15.
    Dizdar (Dizdar Segrell), Nil
    et al.
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Clinical Chemistry. Östergötlands Läns Landsting, Centre for Laboratory Medicine, Department of Clinical Chemistry.
    Årstrand, Kerstin
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Clinical Chemistry.
    Kågedal, Bertil
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Clinical Chemistry. Östergötlands Läns Landsting, Centre for Laboratory Medicine, Department of Clinical Chemistry.
    Analysis of L-dopa in human serum2002In: BioTechniques, ISSN 0736-6205, E-ISSN 1940-9818, Vol. 33, no 5, p. 1000-1002Article in journal (Refereed)
  • 16. Gidlöf, Olof
    et al.
    Burvall, Sofia
    Edvinsson, Lars
    Montelius, Maria
    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.
    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.
    Molin, Magnus
    Complete discrimination of six individuals based on high-resolution melting of hypervariable regions I and II of the mitochondrial genome2009In: BioTechniques, ISSN 0736-6205, E-ISSN 1940-9818, Vol. 47, no 2, p. 671-678Article in journal (Refereed)
    Abstract [en]

    Analysis of mitochondrial DNA in forensic samples is routinely carried out by direct sequencing of hypervariable regions within the non-coding displacement loop. Although the accuracy and sensitivity of this method cannot be questioned, it is both time-consuming and labor intensive. Finding a way to rapidly pre-screen forensic samples-prior to sequencing, to reduce the number of samples that need to be sequenced-would greatly benefit forensic laboratories. Herein, we describe an assay for discrimination of DNA from different individuals based on high-resolution melting analysis of the two hypervariable regions HVI and HVII of the mitochondrial genome. By clearly distinguishing the DNA melting curves of six different individuals, we show that this assay has the potential to function as a rapid and inexpensive pre-screening method for forensic samples prior to DNA sequencing.

  • 17. Hanke, J
    et al.
    Sanchez, DO
    Henriksson, Jan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Åslund, Lena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Pettersson, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Frasch, ACC
    Hoheisel, JD
    Mapping the Trypanosoma cruzi genome: Analyses of representative cosmid libraries1996In: BioTechniques, ISSN 0736-6205, E-ISSN 1940-9818, Vol. 21, no 4, p. 686-688Article in journal (Refereed)
    Abstract [en]

    In order to generate contiguous cosmid coverage of the genome of the protozoan parasite Trypanosoma cruzi for large-scale sequence analysis, a cosmid library of 36864 individual, primary clones was generated. Total genomic DNA of the reference strain CL Brener was fragmented both by partial digestion with MboI and by physical shearing. For cloning, a modified cosmid vector was used that simplifies analyses such as restriction mapping. The library's representation is about 25 genome equivalents, assuming a size of 55 Mb per haploid genome. No chimerism of inserts in the clones could be detected. The colinearity between cosmid inserts and genomic DNA was verified. Also, hybridizations to the gel-separated karyotype of the organism were carried out as a quality check. Gridded onto two nylon filters, the library was analyzed with a variety of probes. Apart from being used for combined physical and transcriptional mapping of the genome, library filters and clones are also available to interested parties.

  • 18. Ignatov, Konstantin B.
    et al.
    Barsova, Ekaterina V.
    Fradkov, Arkady F.
    Blagodatskikh, Konstantin A.
    Kramarova, Tatiana V.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Kramarov, Vladimir M.
    A strong strand displacement activity of thermostable DNA polymerase markedly improves the results of DNA amplification2014In: BioTechniques, ISSN 0736-6205, E-ISSN 1940-9818, Vol. 57, no 2, p. 81-87Article in journal (Refereed)
    Abstract [en]

    The sensitivity and robustness of various DNA detection and amplification techniques are to a large extent determined by the properties of the DNA polymerase used. We have compared the performance of conventional Taq and Bst DNA polymerases to a novel Tag DNA polymerase mutant (SD DNA polymerase), which has a strong strand displacement activity, in PCR (including amplification of GC-rich and complex secondary structure templates), long-range PCR (LR PCR), loop-mediated amplification (LAMP), and polymerase chain displacement reaction (PCDR). Our results demonstrate that the strand displacement activity of SD DNA polymerase, in combination with the robust polymerase activity, provides a notable improvement in the sensitivity and efficiency of all these methods.

  • 19. Ihalainen, J
    et al.
    Siitari, H
    Laine, S
    Syvänen, Ann-Christine
    Palotie, A
    Towards automatic detection of point mutations: use of scintillating microplates in solid-phase minisequencing1994In: BioTechniques, ISSN 0736-6205, E-ISSN 1940-9818, Vol. 16, no 5, p. 938-943Article in journal (Refereed)
    Abstract [en]

    Simplification of molecular genetic techniques is one of the main features of large-scale clinical applications of mutation analysis. The solid-phase minisequencing method, which is based on single-nucleotide primer extension by a DNA polymerase on a solid support, is an easy way of detecting point mutations of previously known locations. Here the procedure was further simplified by the use of microplates made of scintillating plastics, a microplate format scintillation counter and an automatic microplate washer. DNA samples from patients with either a hereditary aspartylglucosaminidase (AGA) gene point mutation or an acquired N-ras gene mutation were analyzed by three different minisequencing detection procedures utilizing tritiated nucleotides. The new counting method with scintillating plates was compared to traditional liquid scintillation counting in scintillation vials or to another microplate format procedure, which requires addition of scintillation liquid. In all three methods, normal individuals, heterozygous carriers of the AGA mutation and homozygous patients could be unequivocally discriminated. The N-ras mutation in leukemic blasts could also be detected with high resolution. The coefficients of variation and reproducibility of the scintillating microplate method were almost identical to those of the traditional liquid scintillation assay, which was used as a reference method. The technical innovations adopted here for performing minisequencing assays reduce significantly the labor required without affecting the quality of the results.

  • 20.
    Jarvius, Jonas
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Landegren, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    DNA Skyline: fonts to facilitate visual inspection of nucleic acid sequences2006In: BioTechniques, ISSN 0736-6205, E-ISSN 1940-9818, Vol. 40, no 6, p. 740-Article in journal (Other academic)
  • 21.
    Jobs, Magnus
    Dalarna University, School of Health and Social Studies, Medical Science.
    Creating Arrays by Centrifugation2002In: BioTechniques, ISSN 0736-6205, E-ISSN 1940-9818, Vol. 32, no 6, p. 1322-1329Article in journal (Refereed)
  • 22.
    Kulytè, Agnè
    et al.
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Navakauskiene, R.
    Institute of Biochemistry, Vilnius, Lithuanuia.
    Treigyte, G.
    Institute of Biochemistry, Vilnius, Lithuanuia.
    Gineitis, A.
    Institute of Biochemistry, Vilnius, Lithuanuia.
    Magnusson, Karl-Eric
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Parallel assessment of tyrosine phosphorylation and nuclear targeting of proteins2001In: BioTechniques, ISSN 0736-6205, E-ISSN 1940-9818, Vol. 31, no 3, p. 508-517Article in journal (Refereed)
    Abstract [en]

    Phosphotyrosine signaling plays a vital role in cell regulation - from receptor activation, through stimulation of signal networks and nuclear targeting, to final cellular responses. Here, we propose a new approach to monitor the spatial and temporal aspects of tyrosine phosphorylation and dephosphorylation. The method can be used to determine whether protein tyrosine phosphorylations and dephosphorylations occur in the cytosol or the nucleus and to ascertain whether such modifications are associated with nuclear traffic. Promyelocytic leukemia (HL-60) cells are used as the experimental model. Biotinylated cytosolic proteins from donor cells are used to trace nuclear transport in permeabilized recipient cells. Thereafter, 2-D gel electrophoresis is applied to fractionate the cytosolic and nuclear proteins of the recipient cells, which are subsequently blotted onto polyvinylidene difluoride membranes. The membranes are developed with streptavidin and then reprobed with anti-phosphotyrosine antibodies. The major advantages of the protocol are that it is simple to perform, and reproducible results are obtained by overlaying the patterns of biotinylated and/or tyrosine-phosphorylated proteins. Moreover, several hundred cytosolic and nuclear proteins can be analyzed in parallel. Thus, by comparing the 2-D gel electrophoresis maps of biotinylated and tyrosine-phosphorylated proteins, it is possible to determine the involvement of trafficking of the latter proteins in cell signaling.

  • 23.
    Lindskog, Mats
    et al.
    KTH, School of Biotechnology (BIO).
    Rockberg, Johan
    KTH, School of Biotechnology (BIO).
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO).
    Sterky, Fredrik
    KTH, School of Biotechnology (BIO).
    Selection of protein epitopes for antibody production2005In: BioTechniques, ISSN 0736-6205, E-ISSN 1940-9818, Vol. 38, no 5, p. 723-727Article in journal (Refereed)
    Abstract [en]

    Protein functional analysis in the post-genomic era is a huge task that has to be approached by different methods in parallel. The use of protein-specific antibodies in conjunction with tissue microarrays has proven to be one important technology. In this study, we present a strategy for the optimized design of protein subfragments for subsequent antibody production. The fragments are selected based on a principle of lowest sequence similarity to other human proteins, optimally to generate antibodies with high selectivity. Furthermore, the fragments should have properties optimized for efficient protein production in Escherichia coli. The strategy has been implemented in Bishop, which is a Java-based software enabling the high-throughput production of protein fragments. Bishop allows for the avoidance of certain restriction enzyme sites, transmembrane regions, and signal peptides. A Basic Local Alignment Search Tool (BLAST) scanning procedure permits the selection of fragments of a selected size with a minimal sequence similarity to other proteins. The software and the strategy were evaluated on a human test data set and verified to fulfill the requested criteria.

  • 24.
    Nestor, Colm
    et al.
    Western General Hospital, Edinburgh, Scotland, UK.
    Ruzov, Alexey
    Western General Hospital, Edinburgh, Scotland, UK.
    Meehan, Richard
    Western General Hospital, Edinburgh, Scotland, UK.
    Dunican, Donncha
    Western General Hospital, Edinburgh, Scotland, UK.
    Enzymatic approaches and bisulfite sequencing cannot distinguish between 5-methylcytosine and 5-hydroxymethylcytosine in DNA2010In: BioTechniques, ISSN 0736-6205, E-ISSN 1940-9818, Vol. 48, no 4, p. 317-319Article in journal (Refereed)
    Abstract [en]

    DNA cytosine methylation (5mC) is highly abundant in mammalian cells and is associated with transcriptional repression. Recently, hydroxymethylcytosine (hmC) has been detected at high levels in certain human cell types; however, its roles are unknown. Due to the structural similarity between 5mC and hmC, it is unclear whether 5mC analyses can discriminate between these nucleotides. Here we show that 5mC and hmC are experimentally indistinguishable using established 5mC mapping methods, thereby implying that existing 5mC data sets will require careful re-evaluation in the context of the possible presence of hmC. Potential differential enrichment of 5mC and hmC DNA sequences may be facilitated using a 5mC monoclonal antibody.

  • 25.
    Nilsson, Peter
    et al.
    KTH, Superseded Departments, Biotechnology.
    Larsson, A
    Lundeberg, Joakim
    KTH, Superseded Departments, Biotechnology.
    Uhlen, Mathias
    KTH, Superseded Departments, Biotechnology.
    Nygren, Per-Åke
    KTH, Superseded Departments, Biotechnology.
    Mutational scanning of PCR products by subtractive oligonucleotide hybridization analysis1999In: BioTechniques, ISSN 0736-6205, E-ISSN 1940-9818, Vol. 26, no 2, p. 308-+Article in journal (Refereed)
    Abstract [en]

    Here, we describe a new approach for mutational scanning of PCR products through hybridization analysis between complementary oligonucleotides. Sets of overlapping probe oligonucleotides complementary to wild-type (WT) sequence are hybridized to microbead-immobilized PCR products under solution-like conditions. Mismatch-hybridization situations between a mutant sample and probe oligonucleotides result in higher remaining concentrations in solution of involved probe oligonucleotides. Post-hybridization supernatants are subsequently analyzed for their probe oligonucleotide compositions using surface plasmon resonance-based biosensor technology Relative remaining probe oligonucleotide concentrations are monitored in real-time through hybridization analysis between probe oligonucleotides and their corresponding sensor-chip immobilized complementary counterparts. This allows for the construction of composition diagrams revealing the existence and approximate location of a mutation within an investigated sample DNA sequence. Applied on PCR products derived from clinical samples of microdissected tumor biopsies, single mutations in exons 6 and 7 of the human p53 tumor-suppressor gene were successfully detected and approximately localized.

  • 26.
    Odeberg, Jacob
    et al.
    KTH, Superseded Departments, Biotechnology.
    Holmberg, K.
    Eriksson, P.
    Uhlén, Mathias
    KTH, Superseded Departments, Biotechnology.
    Molecular haplotyping by pyrosequencing (TM)2002In: BioTechniques, ISSN 0736-6205, E-ISSN 1940-9818, Vol. 33, no 5, p. 1104-+Article in journal (Refereed)
  • 27.
    Pavankumar, Asalapuram Ramachand
    et al.
    KTH, School of Biotechnology (BIO), Environmental Microbiology (closed September 2009).
    Ayyappasamy, S. P.
    Sankaran, K.
    Small RNA fragments in complex culture media cause alterations in protein profiles of three species of bacteria2012In: BioTechniques, ISSN 0736-6205, E-ISSN 1940-9818, Vol. 52, no 3, p. 167-172Article in journal (Refereed)
    Abstract [en]

    Efforts to delineate the basis for variations in protein profiles of different membrane fractions from various bacterial pathogens led to the finding that even the same medium [e.g., Luria Bertani (LB) broth] purchased from different commercial sources generates remarkably dissimilar protein profiles despite similar growth characteristics. Given the pervasive roles small RNAs play in regulating gene expression, we inquired if these source-specific differences due to media arise from disparities in the presence of small RNAs. Indeed, LB media components from two different commercial suppliers contained varying, yet significant, amounts of 10-80 bp small RNAs. Removal of small RNA from LB using RNaseA during media preparation resulted in significant changes in bacterial protein expression profiles. Our studies underscore the fact that seemingly identical growth media can lead to dramatic alterations in protein expression patterns, highlighting the importance of utilizing media free of small RNA during bacteriological studies. Finally, these results raise the intriguing possibility that similar pools of small RNAs in the environment can influence bacterial adaptation.

  • 28. Røsok, O
    et al.
    Odeberg, Jacob
    KTH, Superseded Departments, Biotechnology.
    Rode, M
    Stokke, T
    Funderud, S
    Smeland, E
    Lundeberg, Joakim
    KTH, Superseded Departments, Biotechnology.
    Solid-phase method for differential display of genes expressed in hematopoietic stem cells.1996In: BioTechniques, ISSN 0736-6205, E-ISSN 1940-9818, Vol. 21, no 1, p. 114-21Article in journal (Refereed)
    Abstract [en]

    A solid-phase differential display method was designed to analyze differential gene expression in samples with low amounts of mRNA. The principle was based on using a biotinylated probe to capture the mRNA and priming both the first-strand synthesis and the subsequent polymerase chain reaction step. Coupling the mRNA to a solid phase during the procedure simplified the purification steps, limited sample loss and enabled rapid handling of mRNA. DNA contamination was also minimized when the mRNA was bound to a solid phase. Optimization of the differential display method was achieved by analyzing both the enzymatic conditions and the required cell amounts. The approach was used for the characterization of genes expressed in the most immature hematopoietic progenitor cells (CD34+CD38-). The majority of the differentially expressed fragments represented previously uncharacterized sequences.

  • 29.
    Täpp, Ida
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Malmberg, Lovisa
    Rennel, Emma
    Majstin, Wik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Syvänen, Ann-Christine
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Molecular Medicine.
    Homogeneous scoring of single nucleotide polymorphisms: The 5’-nuclease ”TaqMan” assay versus Molecular beacon probes2000In: BioTechniques, ISSN 0736-6205, E-ISSN 1940-9818, Vol. 28, no 4, p. 732-738Article in journal (Refereed)
    Abstract [en]

    Homogeneous assays based on real-time fluorescence monitoring during PCR are relevant alternatives for large-scale genotyping of single-nucleotide polymorphisms (SNPs). We compared the performance of the homogeneous TaqMan 5'-nuclease assay and the Molecular Beacon assay using three SNPs in the human estrogen receptor gene as targets. When analyzing a panel of 90 DNA samples, both assays yielded a comparable power of discrimination between the genotypes of a C-to-T transition in codon 10 and a G-to-A transition in codon 594 of the estrogen receptor gene. The Molecular Beacon probes distinguished better than the TaqMan probes between homozygous and heterozygous genotypes of a C-to-G transversion in codon 325. The sensitivity of detecting one allele, present as a minority in a mixed sample, varied between the SNPs and was similar for both assays. With the Molecular Beacon assay, the measured signal ratios were proportional to the amount of the minor allele over a wider range than with the TaqMan assay at all three SNPs.

  • 30.
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics.
    Affinity as a tool in life science2008In: BioTechniques, ISSN 0736-6205, E-ISSN 1940-9818, Vol. 44, no 5, p. 649-654Article, review/survey (Refereed)
    Abstract [en]

    The use of affinity-based tools has become invaluable as a platform for basic research and in the development of drugs and diagnostics. Applications include affinity chromatography and affinity tag fusions for efficient purification of proteins as well as methods to probe the protein network interactions on a whole-proteome level. A variety of selection systems has been described for in vitro evolution of affinity reagents using combinatorial libraries, which make it possible to create high-affinity reagents to virtually all biomolecules, as exemplified by generation of therapeutic antibodies and new protein scaffold binders. The strategies for high-throughput generation of affinity reagents have also opened up the possibility of generating specific protein probes on a whole-proteome level. Recently, such affinity proteomics have allowed the detailed analysis of human protein expression in a comprehensive manner both in normal and disease tissue using tissue microarrays and confocal microscopy.

  • 31.
    Vennström, Lina
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Bysell, Camilla
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Björkelund, Hanna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Lundqvist, Hans
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Andersson, Karl
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Real-time viability assay based on 51Cr retention in adherent cells2008In: BioTechniques, ISSN 0736-6205, E-ISSN 1940-9818, Vol. 44, no 2, p. 237-240Article in journal (Refereed)
    Abstract [en]

    The chromium (51Cr) release assay has been widely used for viability measurements, even though it has major disadvantages such as high manual workload and poor time resolution. By the use of LigandTracer 51Cr release viability measurements on adherent cells can be significantly simplified and improved. LigandTracer enables a time-resolved detection of 5SCr in target cells, with the result that the effect of toxic material is updated continuously throughout the experiment. Here we explain the principle behind this novel real-time viability assay and show viability curves for known toxic compounds on A431 and U343MGaCl2:6 cell lines.

  • 32.
    Xia, Hongyan
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine. Swedish Univ Agr Sci, Dept Biomed Sci & Vet Publ Hlth, S-75007 Uppsala, Sweden..
    Gravelsina, Sabine
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine. Riga Stradins Univ, August Kirchenstein Inst Microbiol & Virol, Riga, Latvia..
    Öhrmalm, Christina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine, Clinical Virology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology.
    Ottoson, Jakob
    Swedish Univ Agr Sci, Dept Biomed Sci & Vet Publ Hlth, S-75007 Uppsala, Sweden.;Natl Food Adm Toxicol Lab, Dept Risk & Benefit Assessment, Uppsala, Sweden..
    Blomberg, Jonas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine, Clinical Virology.
    Development of single-tube nested real-time PCR assays with long internally quenched probes for detection of norovirus genogroup II2016In: BioTechniques, ISSN 0736-6205, E-ISSN 1940-9818, Vol. 60, no 1, p. 28-34Article in journal (Refereed)
    Abstract [en]

    The high sequence variation of RNA viruses necessitates use of degenerate primers and probes or multiple primers and probes in molecular diagnostic assays. We showed previously that PCR amplification in two rounds, first with long target-specific primers and then with short generic primers, followed by detection using long probes, can tolerate sequence variation. Here we demonstrate that long primers and probes of up to 56 nucleotides can also be applied in real-time PCR for the detection of norovirus genogroup II with improved sensitivity. Probe design (method of incorporating quenchers, use of Zen internal quencher or traditional quenchers) greatly affects the sensitivity of the real-time PCR assays.

1 - 32 of 32
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