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  • 1.
    Ahmadian, Afshin
    et al.
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Gharizadeh, B.
    Gustafsson, A. C.
    Sterky, Fredrik
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Nyrén, Pål
    KTH, Tidigare Institutioner                               , Biokemi och biokemisk teknologi.
    Uhlén, Mathias
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Lundeberg, Joakim
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Single-nucleotide polymorphism analysis by pyrosequencing2000Inngår i: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 280, nr 1, s. 103-110Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    There is a growing demand for high-throughput methods for analysis of single-nucleotide polymorphic (SNP) positions. Here, we have evaluated a novel sequencing approach, pyrosequencing, for such purposes. Pyrosequencing is a sequencing-by-synthesis method in which a cascade of enzymatic reactions yields detectable light, which is proportional to incorporated nucleotides. One feature of typing SNPs with pyrosequencing is that each allelic variant will give a unique sequence compared to the two other variants. These variants can easily be distinguished by a pattern recognition software. The software displays the allelic: alternatives and allows for direct comparison with the pyrosequencing raw data. For optimal determination of SNPs, various protocols of nucleotide dispensing order were investigated. Here, we demonstrate that typing of SNPs can efficiently be performed by pyrosequencing using an automated system for parallel analysis of 96 samples in approximately 5 min, suitable for large-scale screening and typing of SNPs.

  • 2. Andersson, R. M.
    et al.
    Carlsson, Kjell
    KTH, Tidigare Institutioner                               , Fysik.
    Liljeborg, A.
    Brismar, Hjalmar
    Characterization of probe binding and comparison of its influence on fluorescence lifetime of two pH-sensitive benzo c xanthene dyes using intensity-modulated multiple-wavelength scanning technique2000Inngår i: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 283, nr 1, s. 104-110Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Quantitative pH imaging using the carboxy semi-naphthofluorescein dyes SNAFL-1 and SNAFL-2 can be performed by measurement of intensity ratios or fluorescence lifetimes. However, there is a controversy as to whether the latter method has the practical advantage of a straightforward pH calibration in buffers compared to a cumbersome and time-consuming procedure in cells. In this study we have undertaken a systematic study of the potential factors influencing the fluorescence lifetime of the probes at different pH using confocal microscopy. In vitro results demonstrate that factors such as lipid and protein concentrations have a substantial influence on pH measurements based on fluorescence lifetime. The pH could be overestimated by more than 2 pH units. Studies in permeabilized COS-7 cells demonstrate the same trends as observed in the in vitro studies.

  • 3.
    Ehn, Maria
    et al.
    KTH, Tidigare Institutioner, Bioteknologi.
    Nourizad, Nader
    KTH, Tidigare Institutioner, Bioteknologi.
    Bergstrom, Kristina
    KTH, Tidigare Institutioner, Bioteknologi.
    Ahmadian, Afshin
    KTH, Tidigare Institutioner, Bioteknologi.
    Nyrén, Pål
    KTH, Tidigare Institutioner, Biokemi och biokemisk teknologi.
    Lundeberg, Joakim
    KTH, Tidigare Institutioner, Bioteknologi.
    Hober, Sophia
    KTH, Tidigare Institutioner, Bioteknologi.
    Toward pyrosequencing on surface-attached genetic material by use of DNA-binding luciferase fusion proteins2004Inngår i: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 329, nr 1, s. 11-20Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Mutation detection and single-nucleotide polymorphisin genotyping require screening of large samples of materials and therefore the importance of high-throughput DNA analysis techniques is significant. Pyrosequencing is a four-enzyme bioluminometric DNA sequencing technology based on the sequencing-by-synthesis principle. Currently, the technique is limited to simultaneous analysis of 96 or 384 samples. Earlier, attempts to increase the sample capacity were made using micromachined filter chamber arrays where parallel analyses of nanoliter samples could be monitored in real time. We have developed a strategy for specific immobilization of the light-producing enzyme luciferase to the DNA template within a reaction chamber. By this approach, luciferase is genetically fused to a DNA-binding protein (Klenow polymerase or Escherichia coli single-stranded DNA-binding (SSB) protein) and to a purification handle (Z(basic)). The proteins are produced in E. coli and purified using cation and anion exchange chromatography with removal of Z(basic). The produced proteins have been analyzed using an assay for complete primer extension of DNA templates immobilized on magnetic beads detected by pyrosequencing chemistry. Results from these experiments show that the proteins bind selectively to the immobilized DNA and that their enzymatic domains were active. Z(basic)-SSB-luciferase produced the highest signal in this assay and was further exploited as enzymatic reagent for DNA sequencing.

  • 4. Eriksson, J.
    et al.
    Karamohamed, S.
    Nyrén, Pål
    KTH, Tidigare Institutioner                               , Biokemi och biokemisk teknologi.
    Method for real-time detection of inorganic pyrophosphatase activity2001Inngår i: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 293, nr 1, s. 67-70Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    A sensitive and simple method for real-time detection of inorganic pyrophosphatase (PPase) (EC 3.6.1.1) activity has been developed. The method is based on PPase-induced activation of the firefly luciferase activity in the presence of inorganic pyrophosphate (PPi). PPi inhibits the luciferase activity, but in the presence of PPase the luciferase activity is restored and the luminescence output increases. The assay yields linear responses between 8 and 500 mU. The detection limit was found to be 8 mU PPase. The method was used to detect the hydrolytic activity of PPases from Saccharomyces cerevisiae, Escherichia coli, and Bacillus stearothermophilus. As substrate for the luciferase, adenosine 5'-phosphosulfate can replace ATP, which is an advantage for detection of PPase activity in crude extracts containing ATP-hydrolyzing activities. The method can be used for kinetic and inhibition studies as well as for detection of PPase activity during different purification procedures.

  • 5.
    Gabig-Ciminska, Magdalena
    et al.
    KTH, Skolan för bioteknologi (BIO), Bioprocessteknik.
    Liu, Yanling
    KTH, Skolan för bioteknologi (BIO), Bioprocessteknik.
    Enfors, Sven-Olof
    KTH, Skolan för bioteknologi (BIO), Bioprocessteknik.
    Gene-based identification of bacterial colonies with an electric chip2005Inngår i: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 345, nr 2, s. 270-276Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    A method for the identification of bacterial colonies based on their content of specific genes is presented. This method does not depend on DNA separation or DNA amplification. Bacillus cereus carrying one of the genes (hblC) coding for the enterotoxin hemolysin was identified with this method. It is based on target DNA hybridization to a capturing probe immobilized on magnetic beads, followed by enzymatic labeling and measurement of the enzyme product with a silicon-based chip. An hblC-positive colony containing 10(7) cells could be assayed in 30 min after ultrasonication and centrifugation. The importance of optimizing the ultrasonication is illustrated by analysis of cell disruption kinetics and DNA fragmentation. An early endpoint PCR analysis was used to characterize the DNA fragmentation as a function of ultrasonication time. The first minutes of sonication increased the signal due to both increased DNA release and increased DNA fragmentation. The latter is assumed to increase the signal due to improved diffusion and faster hybridization of the target DNA. Too long sonication decreased the signal, presumably due to loss of hybridization sites on the targets caused by extensive DNA fragmentation. The results form a basis for rational design of an ultrasound cell disruption system integrated with analysis on chip that will move nucleic acid-based detection through real-time analysis closer to reality.

  • 6.
    Gabig-Ciminska, Magdalena
    et al.
    KTH, Tidigare Institutioner, Bioteknologi.
    Los, M.
    Holmgren, Anders
    KTH, Tidigare Institutioner, Fiber- och polymerteknologi.
    Albers, J.
    Czyz, A.
    Hintsche, R.
    Wegrzyn, G.
    Enfors, Sven-Olof
    KTH, Tidigare Institutioner, Bioteknologi.
    Detection of bacteriophage infection and prophage induction in bacterial cultures by means of electric DNA chips2004Inngår i: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 324, nr 1, s. 84-91Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Infections of bacterial cultures by bacteriophages are common and serious problems in many biotechnological laboratories and factories. A method for specific, quantitative, and quick detection of phage contamination, based on the use of electric DNA chip is described here. Different phages of Escherichia coli and Bacillus subtilis were analyzed. Phage DNA was isolated from bacterial culture samples and detected by combination of bead-based sandwich hybridization with enzyme-labeled probes and detection of the enzymatic product using silicon chips. The assay resulted in specific signals from all four tested phages without significant background. Although high sensitivity was achieved in 4h assay time, a useful level of sensitivity (10(7)-10(8) phages) is achievable within 25 min. A multiplex DNA chip technique involving a mixture of probes allows for detection of various types of phages in one sample.. These analyses confirmed the specificity of the assay.

  • 7. Gharizadeh, B.
    et al.
    Eriksson, J.
    Nourizad, N.
    Nordstrom, Tommy
    KTH, Tidigare Institutioner, Bioteknologi.
    Nyrén, Pål
    KTH, Tidigare Institutioner, Biokemi och biokemisk teknologi.
    Improvements in pyrosequencing technology by employing sequenase polymerase2004Inngår i: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 330, nr 2, s. 272-280Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Pyrosequencing is a DNA sequencing technique based on the bioluminometric detection of inorganic pyrophosphate, which is released when nucleotides are incorporated into a target DNA. Since the technique is based on an enzymatic cascade, the choice of enzymes is a critical factor for efficient performance of the sequencing reaction. In this study we have analyzed the performance of an alternative DNA polymerase, Sequenase, on the sequencing performance of the Pyrosequencing technology. Compared to the Klenow fragment of DNA polymerase I, Sequenase could read through homopolymeric regions with more than five T bases. In addition, Sequenase reduces remarkably interference from primer-dimers and loop structures that give rise to false sequence signals. By using Sequenase, synchronized extensions and longer reads can be obtained on challenging templates, thereby opening new avenues for applications of Pyrosequencing technology.

  • 8. Gharizadeh, B.
    et al.
    Nordstrom, T.
    Ahmadian, Afshin
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Ronaghi, M.
    Nyrén, Pål
    KTH, Tidigare Institutioner                               , Biokemi och biokemisk teknologi.
    Long-read pyrosequencing using pure 2 '-deoxyadenosine-5 '-O '-(1-thiotriphosphate) Sp-isomer2002Inngår i: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 301, nr 1, s. 82-90Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Pyrosequencing, a nonelectrophoretic DNA sequencing method that uses a luciferase-based enzymatic system to monitor DNA synthesis in real time, has so far been limited to sequencing of short stretches of DNA. To increase the signal-to-noise ratio in pyrosequencing the natural dATP was replaced by dATPalphaS (M. Ronaghi et al., 1996, Anal. Biochem. 242, 84-89). The applied dATPaS was a mixture of two isomers (Sp and Rp). We show here that by the introduction of pure 2'-deoxyadenosine-5'-O'-(1-thiotriphosphate) Sp-isomer in pyrosequencing substantial longer reads could be obtained. The pure Sp-isomer allowed lower nucleotide concentration to be used and improved the possibility to read through poly(T) regions. In general, a doubling of the read length could be obtained by the use of pure Sp-isomer. Pyrosequencing data for 50 to 100 bases could be generated on different types of template. The longer read will enable numerous new applications, such as identification and typing of medically important microorganisms as well as resequencing of DNA fragments for mutation screening and clone checking.

  • 9.
    Gutierrez Arenas, Omar
    et al.
    Department of Biochemistry and Organic Chemistry, Uppsala University.
    Danielson, U Helena
    Detection of competitive enzyme inhibition with end point progress curve data2006Inngår i: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 358, nr 1, s. 11-19Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    A model for a dimensionless factor, the inhibition detection limit (IDL), which describes the limit of detection of competitive inhibition for end point assays as a function of the proportion of substrate converted into product, has been developed. For a given end point enzymatic assay, the IDL function has a maximum that is dependent on the error structure parameters (four parameters) of the assay, the value of [S]o/K(ms), and the extent of product inhibition (K(ms)/K(mp)). Accordingly, the substrate conversion level that maximized the ability to detect samples with high Ki/[I] ratios was predicted for each member of a population of simulated assays. Furthermore, we identified a consensus substrate conversion level where the probability of a near-optimal robustness and detection limit for all the members of the assay population is maximal. Unlike the optimal substrate conversion level for individual assays, this consensus substrate conversion level was dependent only on [S]o/K(m), K(ms)/K(mp), and whether the signal increases or decreases during the course of the reaction. Consensus substrate conversion levels were beyond the initial velocity range for almost all the analyzed assay populations. It was shown that the IDL factor was a more informative indicator of assay quality than the popular Z' factor.

  • 10.
    Gutierrez Arenas, Omar
    et al.
    Department of Biochemistry and Organic Chemistry, Uppsala University.
    Danielson, U Helena
    Sensitivity analysis and error structure of progress curves2006Inngår i: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 358, nr 1, s. 1-10Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Both the sensitivity of the monitored signal in progress curves to variations in enzyme concentration and the standard deviation of this signal were analyzed as a function of the proportion of transformed substrate. Three enzymes catalyzing essentially irreversible reactions were used as model systems: HIV-1 protease, glutathione reductase, and glutathione transferase. For all enzymes analyzed, the sensitivity was maximal when 60-80% of the substrate had been transformed. The standard deviation of reaction progress curve data replicates was also maximal at these substrate conversion levels, a result that was attributed to the influence of the sensitivity to random dispersion of the enzyme concentration. On this basis, we developed a model for the standard deviation of reaction progress curves that gave a good description of the experimental data and efficiently reduced the heteroscedasticity of residuals in a weighted fit of progress curves. This standard deviation model can be used for obtaining more efficient parameter estimates, to simulate noise in Monte Carlo procedures, and to delineate detection limits of enzyme inhibition. The transient increases in the sensitivity and in the standard deviation in progress curves are proposed to be features common to most enzymatic assays.

  • 11.
    Hamberg, Anders
    et al.
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Kempka, Martin
    KTH, Skolan för kemivetenskap (CHE), Kemi, Analytisk kemi.
    Sjödahl, Johan
    KTH, Skolan för kemivetenskap (CHE), Kemi, Analytisk kemi.
    Roeraade, Johan
    KTH, Skolan för kemivetenskap (CHE), Kemi, Analytisk kemi.
    Hult, Karl
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    C-terminal ladder sequencing of peptides using an alternative nucleophile in carboxypeptidase Y digests2006Inngår i: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 357, nr 2, s. 167-172Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

     A method for improved sequence coverage in C-terminal sequencing of peptides, based on carboxypeptidase digestion, is described. In conventional carboxypeptidase digestions, the peptide substrate is usually extensively degraded and a full amino acid sequence cannot be obtained due to the lack of a complete peptide ladder. In the presented method, a protecting group is introduced at the C terminus of a fraction of the peptide fragments formed in the digest, and thereby further degradation of the C-terminally modified peptides are slowed down. The protecting group was attached to the C-terminal amino acid through a carboxypeptidase-catalyzed reaction with an alternative nucleophile, 2-pyridylmethylamine, added to the aqueous digestion buffer. Six peptides were digested by carboxypeptidase Y with and without 2-pyridylmethylamine present in the digest buffer, and the resulting fragments subsequently were analyzed with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Comparison of the two digestion methods showed that the probability of successful ladder sequencing increased, by more than 50% using 2-pyridylmethylamine as a competing nucleophile in carboxypeptidase Y digests.

  • 12.
    Jacksén, Johan
    et al.
    KTH, Skolan för kemivetenskap (CHE), Kemi.
    Emmer, Åsa
    KTH, Skolan för kemivetenskap (CHE), Kemi.
    Evaluation of 2,6-dihydroxyacetophenone as matrix-assisted laser desorption/ionization matrix for analysis of hydrophobic proteins and peptides2012Inngår i: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 425, nr 1, s. 18-20Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is widely used for analysis of macromolecules like peptides and proteins. The analysis procedure is generally simple but must be adapted to the characteristics of the analytes. Therefore, specific matrices suitable for, e.g., hydrophobic proteins and peptides that are difficult to analyze would be preferable in order to optimize the outcome. In the present work, 2,6-dihydroxyacetophenone (DHAP) was shown to be beneficial in comparison to DHB for intact bacteriorhodopsin (BR) as well as for chemically digested BR.

  • 13. Karlstrom, A.
    et al.
    Nygren, Per-Åke
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Dual labeling of a binding protein allows for specific fluorescence detection of native protein2001Inngår i: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 295, nr 1, s. 22-30Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Fluorescence resonance energy transfer has been investigated in the context of specific detection of unlabeled proteins. A model system based on the staphylococcal. protein A (SPA)-IgG interaction was designed, in which a single domain was engineered to facilitate site-specific incorporation of fluorophores. An Asn(23)Cys mutant of the B domain from SPA was expressed in Escherichia coli and subsequently labeled at the introduced unique thiol and at an amino group, using N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (1,5-LAEDANS) and succinimidyl 6-(N-(7-nitrobenz-2-oxa- 1,3-diazol-4-yl) amino) hexanoate (NBD-X, SE), respectively. Biosensor analysis of Purified doubly labeled protein showed that high-affinity binding to the Fe region of IgG was retained. The fluorescence emission spectrum of the doubly labeled protein showed a shift in the relative emission of the two fluorophores in the presence of FC3(1)) fragments, which bind specifically to the B domain. In addition, the fluorescence emission ratio 480/525 nm was shown to increase with increasing concentration of Fc(3(1)), whereas the presence of a control protein did not affect the emission ratio over the same concentration range.

  • 14.
    Liu, Yanling
    et al.
    KTH, Skolan för bioteknologi (BIO), Bioprocessteknik.
    Elsholz, Bruno
    Fraunhofer Institute for Silicon Technology, Itzehoe, Germany.
    Enfors, Sven-Olof
    KTH, Skolan för bioteknologi (BIO), Bioprocessteknik.
    Gabig-Ciminska, Magdalena
    KTH, Skolan för bioteknologi (BIO), Bioprocessteknik.
    Critical factors for the performance of chip array-based electrical detection of DNA for analysis of pathogenic bacteria2008Inngår i: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 382, nr 2, s. 77-86Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Different factors influencing chip array-based electrical detection of DNA for analysis of pathogenic bacteria were examined. Both rehydration of capture probe layer of functionalized chip arrays and efficient hybridization of targets irrespective of their length resulted in signal enhancement when high-ionic phosphate-buffered saline (i.e., 600 mM sodium chloride and 40 mM disodium hydrogen phosphate) was used. Similarly, placement of two adjacent capture and detection probe-binding sites at a terminal part of the target strand resulted in significant signal increase. Moreover, 10-min ultrasonic fragmentation of targets amplified the signals Lip to twofold for longer DNA strands (i.e., >300 bp). No obvious effects on signals were visible for shorter than 400-bp PCR amplicons subjected to ultrasonication. For DNA strands of all sizes, more than 10 min ultrasonication diminished the specific electrical responses. Our results also demonstrate that target analytes are detected with discrimination against mismatches even for single nucleotide sequence alteration. The mismatch detection appeared in order of ease Of recognition as follows: triple random > quintuple middle > triple middle > single middle mismatch. Among the three variants of one-base mismatches, a sequence variation was most remarkable for adenine. On the other hand, no benefits in assay sensitivity were recognized by the use of longer capture probe linkers as the 6-C linker.

  • 15. Lundbäck, Anna-Karin
    et al.
    van den Berg, Susanne
    Hebert, Hans
    KTH, Skolan för teknik och hälsa (STH), Strukturell bioteknik.
    Berglund, Helena
    Eshaghi, Said
    Exploring the activity of tobacco etch virus protease in detergent solutions2008Inngår i: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 382, nr 1, s. 69-71Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Tobacco etch virus (TEV) protease is generally used to remove affinity tags from target proteins. It has been reported that some detergents inhibit the activity of this protease, and therefore should be avoided when removing affinity tags from membrane proteins. The aim of this study was to explore and evaluate this further. Hence, affinity tag removal with TEV protease was tested from three membrane proteins (a Pgp synthase and two CorA homologs) in the presence of 16 different detergents commonly used in membrane protein purification and crystallization. We observed that in the presence of the same detergent (Triton X-100), TEV protease could remove the affinity tag completely from one protein (CorA) but not from another protein (Pgp synthase). There was also a large variation in yield of cleaved membrane protein in different detergents, which probably depends on features of the protein-detergent complex. These observations show that, contrary to an earlier report, detergents do not inhibit the enzymatic activity of the TEV protease.

  • 16.
    Nilsson, Peter
    et al.
    KTH, Tidigare Institutioner                               , Bioteknologi.
    O'Meara, Deirdre
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Edebratt, Fredrik
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Persson, Björn
    Uhlén, Mattias
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Lundeberg, Joakim
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Nygren, Per-Åke
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Quantitative Investigation of the Modular Primer Effect for DNA and Peptide Nucleic Acid Hexamers1999Inngår i: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 269, nr 1, s. 155-161Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The effect on oligonucleotide–template duplex stability upon cohybridization of adjacently annealing oligonucleotides, the modular primer effect, was studied with biosensor technology. DNA and peptide nucleic acid (PNA) hexamer modules and sensor chip-immobilized template DNA strands were designed for analysis of nick, overlap, and gap modular hybridization situations. The fast hybridization kinetics for such hexamer modules allowed for the determination of apparent duplex affinities from equilibrium responses. The results showed that the hybridizational stability of modular hexamer pairs is strongly dependent on the positioning, concentration, and inherent affinity of the adjacently annealing hexamer module. Up to 80-fold increases in apparent affinities could be observed for adjacent modular oligonucleotide pairs compared to affinities determined for single hexamer oligonucleotide hybridizations. Interestingly, also for coinjections of different module combinations where DNA hexamer modules were replaced by their PNA counterparts, a modular primer effect was observed. The introduction of a single base gap between two hexamer modules significantly reduced the stabilization effect, whereas a gap of two bases resulted in a complete loss of the effect. The results suggest that the described biosensor-based methodology should be useful for the selection of appropriate modules and working concentrations for use in different modular hybridization applications.

  • 17.
    NILSSON, PETER
    et al.
    KTH, Tidigare Institutioner, Bioteknologi.
    PERSSON, BJÖRN
    UHLEN, MATHIAS
    KTH, Tidigare Institutioner, Bioteknologi.
    NYGREN, PER-ÅKE
    KTH, Tidigare Institutioner, Bioteknologi.
    REAL-TIME MONITORING OF DNA MANIPULATIONS USING BIOSENSOR TECHNOLOGY1995Inngår i: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 224, nr 1, s. 400-408Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The potential of real-time biospecific interaction analysis technology for applications in molecular biology is described. DNA fragments are immobilized onto a biosensor surface using the high-affinity streptavidin-biotin system and subsequently used to monitor different unit operations in molecular biology, e.g., DNA strand separation, DNA hybridization kinetics, and enzymatic modifications. A model system comprising six oligonucleotides was used, which can be assembled into a 69-bp double-stranded DNA fragment. Using this system, the biosensor approach was employed to analyze multistep solid-phase gene assembly and the performance of different enzymes routinely used for the synthesis and manipulation of DNA. In addition, a concept for the determination of single-point mutations in DNA samples is described.

  • 18. Nordstrom, T.
    et al.
    Gharizadeh, B.
    Pourmand, N.
    Nyrén, Pål
    KTH, Tidigare Institutioner                               , Biokemi och biokemisk teknologi.
    Ronaghi, M.
    Method enabling fast partial sequencing of cDNA clones2001Inngår i: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 292, nr 2, s. 266-271Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Pyrosequencing is a nonelectrophoretic single-tube DNA sequencing method that takes advantage of cooperativity between four enzymes to monitor DNA synthesis. To investigate the feasibility of the recently developed technique for tag sequencing, 64 colonies of a selected cDNA library from human were sequenced by both pyrosequencing and Sanger DNA sequencing. To determine the needed length for finding a unique DNA sequence, 100 sequence tags from human were retrieved from the database and different lengths from each sequence were randomly analyzed. An homology search based on 20 and 30 nucleotides produced 97 and 98% unique hits, respectively. An homology search based on 100 nucleotides could identify all searched genes. Pyrosequencing was employed to produce sequence data for 30 nucleotides. A similar search using BLAST revealed 16 different genes. Forty-six percent of the sequences shared homology with one gene at different positions. Two of the 64 clones had unique sequences. The search results from pyrosequencing were in 100% agreement with conventional DNA sequencing methods. The possibility of using a fully automated pyrosequencer machine for future high-throughput tag sequencing is discussed.

  • 19. Nordstrom, T.
    et al.
    Nourizad, K.
    Ronaghi, M.
    Nyrén, Pål
    KTH, Tidigare Institutioner                               , Biokemi och biokemisk teknologi.
    Method enabling pyrosequencing on double-stranded DNA2000Inngår i: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 282, nr 2, s. 186-193Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Pyrosequencing is a new nonelectrophoretic, single-tube DNA sequencing method that takes advantage of co-operativity between four enzymes to monitor DNA synthesis (M. Ronaghi, M. Uhlen, and P. Nyren, Science 281, 363-365). Pyrosequencing has so far only been performed on single-stranded DNA, In this paper different enzymatic strategies for template preparation enabling pyrosequencing on double-stranded DNA were studied. High quality data were obtained with several different enzyme combinations: (i) shrimp alkaline phosphatase and exonuclease I, (ii) calf intestine alkaline phosphatase and exonuclease I, (iii) apyrase and inorganic pyrophosphatase together with exonuclease I, and (iv) apyrase and ATP sulfurylase together with exonuclease I. In many cases, when the polymerase chain reaction was efficient exonuclease I could be omitted. In certain cases, additives such as dimethyl sulfoxide, single-stranded DNA-binding protein, and Klenow DNA polymerase improved the sequence quality. Apyrase was the fastest and most efficient of the three different nucleotide degrading enzymes tested. The data quality obtained on double-stranded DNA was comparable with that on single-stranded DNA. Pyrosequencing data for more than 30 bases could be generated on both long and short templates, as well as on templates with high GC content.

  • 20. Nygren, M.
    et al.
    Ronaghi, M.
    Nyrén, Pål
    KTH, Tidigare Institutioner                               , Biokemi och biokemisk teknologi.
    Albert, J.
    Lundeberg, Joakim
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Quantification of HIV-1 using multiple quantitative polymerase chain reaction standards and bioluminometric detection2001Inngår i: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 288, nr 1, s. 28-38Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    A non-gel-based quantification assay based on competitive PCR and bioluminometric detection has been developed. Samples containing human immunodeficiency virus type 1 (HIV-1) DNA and three quantitative standards at discrete concentrations were coamplified by PCR with primers annealing in the polymerase gene region. The quantitative standards contained the same primer binding sequences and had the same amplicon length as the wild-type DNA, but differed in an internal homopolymeric stretch (A, C, or T) over three base pairs. The PCR products were captured onto a solid support and treated with NaOH to separate the strands. Discrimination between the wild-type DNA and the three quantitative standard amplicons was achieved on the solid support by four parallel extension reactions with 3'-end specific primers. Inorganic pyrophosphate (PPi) released as a result of successful extension was converted to ATP by ATP sulfurylase and the level of ATP was sensed by firefly luciferase, generating a proportional amount of visible light which was detected by a luminometer. Here, we show that the obtained calibration curves, using the signal intensities of the three quantitative standards, enabled determination of the amount of target HIV-1 DNA.

  • 21.
    O'Meara, Deirdre
    et al.
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Nilsson, Peter
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Nygren, Per-Åke
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Uhlen, Mathias
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Lundeberg, Joakim
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Capture of single-stranded DNA assisted by oligonucleotide modules1998Inngår i: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 255, nr 2, s. 195-203Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Real-time biospecific interaction analysis was employed to monitor direct capture of a hepatitis C virus (HCV) derived polymerase chain reaction (PCR) product by nucleic acid hybridization. Different formats for hybridization were used to study the interaction between a single-stranded HCV PCR product and capture oligonucleotides immobilized on a sensor chip via streptavidin-biotin chemistry. By employing a prehybridization step in solution with nonbiotin oligonucleotides complementary to the single-stranded target and adjacent to the immobilized probe, a significant capture was achieved in comparison to the low capture efficiency obtained using single immobilized probes (9-36 mer). High capture efficiencies were also observed when shorter immobilized probes were used in combination with strings of adjacently positioned prehybridized probes (i.e., modules). Interestingly, the introduction of single nucleotide gaps between prehybridized and/or immobilized probes dramatically reduced the capture efficiency. These results suggest that flexible systems for capture could be designed from libraries of short oligonucleotides (9 mers) used in module fashion, taking advantage of stacking interactions between the oligonucleotides. The potential applications of such oligonucleotide-assisted capture systems are discussed.

  • 22. Persson, A. E.
    et al.
    Ling, G.
    Williams, C.
    Backvall, H.
    Ponten, J.
    Ponten, F.
    Lundeberg, Joakim
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Analysis of p53 mutations in single cells obtained from histological tissue sections2000Inngår i: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 287, nr 1, s. 25-31Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    We have previously reported on direct sequence analysis of the p53 gene in laser-dissected single cells from tissue sections, where each allele of two fragments (exons 7 and 8) could be accurately analyzed in only 14% of the cells due to the high frequency of exon and allele dropout. Here in an effort to minimize this problem, we have investigated various approaches for sample preparation and gene amplification. By pinpointing some critical steps in the procedure, we could increase the number of investigated exons and substantially improve the genetic analysis of single cells obtained from histochemically stained frozen tissue sections. The biggest improvement was achieved by minimizing DNA degradation using EDTA as a nuclease inhibitor in all sample preparation steps. Efforts to increase primer annealing, by increasing the concentration of template and primers, in addition to prolonging the annealing and extension times, also improved the amplification efficiency. With these measures we can now amplify six individual exons of the p53 gene (exons 4-9) in 70% of the cells and in 50% of these cells both alleles are amplified. This allows application of the method in various investigations such as within the held of tumor pathology.

  • 23. Persson, A E
    et al.
    Ling, G
    Williams, Cecilia
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Bäckvall, H
    Pontén, J
    Pontén, F
    Lundeberg, J
    Analysis of p53 mutations in single cells obtained from histological tissue sections.2000Inngår i: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 287, nr 1Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    We have previously reported on direct sequence analysis of the p53 gene in laser-dissected single cells from tissue sections, where each allele of two fragments (exons 7 and 8) could be accurately analyzed in only 14% of the cells due to the high frequency of exon and allele dropout. Here in an effort to minimize this problem, we have investigated various approaches for sample preparation and gene amplification. By pinpointing some critical steps in the procedure, we could increase the number of investigated exons and substantially improve the genetic analysis of single cells obtained from histochemically stained frozen tissue sections. The biggest improvement was achieved by minimizing DNA degradation using EDTA as a nuclease inhibitor in all sample preparation steps. Efforts to increase primer annealing, by increasing the concentration of template and primers, in addition to prolonging the annealing and extension times, also improved the amplification efficiency. With these measures we can now amplify six individual exons of the p53 gene (exons 4-9) in 70% of the cells and in 50% of these cells both alleles are amplified. This allows application of the method in various investigations such as within the field of tumor pathology.

  • 24. Persson, Björn
    et al.
    Stenhag, K
    Nilsson, Peter
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Larsson, A
    Uhlen, Mathias
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Nygren, Per-Åke
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Analysis of oligonucleotide probe affinities using surface plasmon resonance: A means for mutational scanning1997Inngår i: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 246, nr 1, s. 34-44Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    A novel strategy for real-time analysis of oligonucleotide probe hybridization based on detection by surface plasmon resonance is described. The design of the analysis, exploiting the rapid dissociation kinetics of short oligonucleotides from their hybridization templates, allows monitoring in genuine sensor mode of equilibrium hybridization responses, circumventing the need for regeneration between sample cycles. Applied to a model system comprising oligonucleotide probes and different immobilized hybridization targets the effects of temperature, probe length, and nucleotide substitutions in template were investigated. The procedure described was observed to have an efficient discriminatory power with respect to end-mismatch situations. Affinity determinations of octamer probes showed good correlation between calculated T-m-values and probe affinities. From affinity data collected at different temperatures thermodynamic parameters were determined, which correlated well with data obtained from theoretical calculations. The technique, modified to a simplified form, allowed detection of single nucleotide substitutions in a target template, suggesting that procedures for confirmatory DNA sequencing can be envisioned.

  • 25.
    Renberg, Björn
    et al.
    KTH, Tidigare Institutioner, Bioteknologi.
    Nygren, Per-Åke
    KTH, Tidigare Institutioner, Bioteknologi.
    Eklund, Malin
    KTH, Tidigare Institutioner, Bioteknologi.
    Eriksson Karlström, Amelie
    KTH, Tidigare Institutioner, Bioteknologi.
    Fluorescence resonance energy transfer-based detection of analytes using antiidiotypic affinity protein pairs2004Inngår i: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 334, nr 1, s. 72-80Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    A new method for specific detection of proteins based on fluorescence resonance energy transfer (FRET) using affinity proteins (affibodies) derived from combinatorial engineering of Staphylococcal protein A has been developed. Antiidiotypic affibody pairs were used in a homogeneous competitive binding assay, where the idiotypic, target-specific affibody was labeled with fluorescein and the antiidiotypic affibody was labeled with tetramethylrhodamine. Intermolecular FRET between the two fluorescent probes was observed in the antiidiotypic affibody complex, but upon addition of target protein the antiidiotypic affibody was displaced, which was monitored by a shift in the relative emission of the donor and acceptor fluorophores. The feasibility of the system was demonstrated by the detection of IgA and Taq DNA polymerase with high specificity, using two different antiidiotypic affibody pairs. Detection of Taq DNA polymerase in 25% human plasma was successfully carried out, demonstrating that the method can be used for analysis of proteins in samples of complex composition.

  • 26.
    Renberg, Björn
    et al.
    KTH, Skolan för bioteknologi (BIO).
    Shiroyama, Ikue
    KTH, Skolan för bioteknologi (BIO).
    Engfeldt, Torun
    KTH, Skolan för bioteknologi (BIO).
    Nygren, Per-Åke
    KTH, Skolan för bioteknologi (BIO).
    Eriksson Karlström, Amelie
    KTH, Skolan för bioteknologi (BIO).
    Affibody protein capture microarrays: synthesis and evaluation of random and directed immobilization of affibody molecules2005Inngår i: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 341, nr 2, s. 334-343Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Affibody molecules, 58-amino acid three-helix bundle proteins directed to different targets by combinatorial engineering of staphylococcal protein A, were used as capture ligands on protein microarrays. An evaluation of slide types and immobilization strategies was performed to find suitable conditions for microarray production. Two affibody molecules, ZTaq and ZIgA, binding Taq DNA polymerase and human IgA, respectively, were synthesized by solid phase peptide synthesis using an orthogonal protection scheme, allowing incorporation of selective immobilization handles. The resulting affibody variants were used for random surface immobilization (through amino groups) or oriented surface immobilization (through cysteine or biotin coupled to the side chain of Lys58). Evaluation of the immobilization techniques was carried out using both a real-time surface plasmon resonance biosensor system and a microarray system using fluorescent detection of Cy3-labeled target protein. The results from the biosensor analyses showed that directed immobilization strategies significantly improved the specific binding activity of affibody molecules. However, in the microarray system, random immobilization onto carboxymethyl dextran slides and oriented immobilization onto thiol dextran slides resulted in equally good signal intensities, whereas biotin-mediated immobilization onto streptavidin-coated slides produced slides with lower signal intensities and higher background staining. For the best slides, the limit of detection was 3 pM for IgA and 30 pM for Taq DNA polymerase.

  • 27.
    Rokhas, Maria Khihon
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Kemi, Tillämpad fysikalisk kemi.
    Ronn, Johanna Liljestrand
    Uppsala Univ, Dept Ecol & Genet Anim Ecol, Uppsala, Sweden..
    Wiklund, Christer
    Stockholm Univ, Dept Zool, Ecol, Stockholm, Sweden..
    Emmer, Åsa
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Kemi, Tillämpad fysikalisk kemi.
    Analysis of butterfly reproductive proteins using capillary electrophoresis and mass spectrometry2019Inngår i: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 566, s. 23-26Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    A method for analysis of proteins from spermatophores transferred from male to female Pieris napi butterflies during mating has been developed. The proteins were solubilized from the dissected spermatophores using different solubilization agents (water, methanol, acetonitrile and hexafluoroisopropanol). Capillary electrophoresis (CE) analysis was performed using an acidic background electrolyte containing a fluorosurfactant to avoid protein-wall adsorption, and to increase separation performance. The samples were also analyzed with matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS), in a lower m/z range (1000-6000) and a higher m/z range (6000-12000). Solubilization with different solvents and the use of alternative matrices gave partly complementary profiles.

  • 28.
    Wiklund, Martin
    et al.
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Biomedicinsk fysik och röntgenfysik.
    Nord, O.
    Gothall, R.
    Chernyshev, A. V.
    Nygren, Per-Åke
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Hertz, Hans M.
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Biomedicinsk fysik och röntgenfysik.
    Fluorescence-microscopy-based image analysis for analyte-dependent particle doublet detection in a single-step immuno agglutination assay2005Inngår i: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 338, nr 1, s. 90-101Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    A novel fluorescence-microscopy-based image analysis method for classification of singlet and doublet latex particles is demonstrated and applied to a particle-based immunoagglutination assay for quantification of biomolecules in microliter-volume bulk samples. The image analysis method, verified by flow cytometric agglutination analysis, is based on a pattern recognition algorithm employing Gaussian-base-function fitting which allows robust identification and counting of singlets, doublets, and higher agglomerates of fluorescent microparticles. The immunoagglutination assay is experimentally modeled by a biotin-streptavidin interaction, with the goal of both theoretically and experimentally investigating the performance of a general immunoagglutination-based assay. For this purpose a theoretical model of the initial agglutination kinetics, based on particle diffusion combined with a steric factor determined by the level of specific and nonspecific agglutination, was developed. The theoretical model combined with the experimental data can be used to optimize an agglutination-based assay with regard to sensitivity and dynamic range and to estimate the affinity, receptor surface density, molecular and binding site sizes, and level of nonspecific binding that is present in the assay. The experimental results are in good agreement with the theoretical model, indicating the usefulness of the model for immunoagglutination assay optimization.

  • 29.
    Zamani, Leila
    et al.
    Department of Analytical Chemistry, Stockholm University.
    Andersson, Fredrik O.
    Edebrink, Per
    Yang, Yang
    Jacobsson, Sven P.
    Conformational studies of a monoclonal antibody, IgG1, by chemical oxidation: Structural analysis by ultrahigh-pressure LC-electrospray ionization time-of-flight MS and multivariate data analysis2008Inngår i: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 380, nr 2, s. 155-163Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    We describe the development of a method in which protein oxidation by H2O2 followed by ultrahigh-pressure liquid chromatography (UHPLC) coupled with electrospray ionization time-of-flight mass spectrometry (ESI-ToFMS) and multivariate analysis are used to detect alterations in conformational states of proteins. In the study reported here, an IgG1 monoclonal antibody in native and denatured conformational states was oxidized by treatment with hydrogen peroxide. Peptide fragments generated by tryptic digestion were then analyzed by UHPLC-ESI-ToFMS. After reducing noise and extracting peaks from the LC-MS data using MzExplorer, software developed in-house and based on Matlab, we were able to distinguish peptides arising from the native and denatured states of the oxidized protein by principal component analysis. Peptides containing residues, which are inclined to undergo oxidation, such as methionine, are founded to be particularly important in this approach. We believe that the methodology could facilitate attempts to characterize the conformational states of recombinant monoclonal antibodies and other proteins.

1 - 29 of 29
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