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  • 1.
    Ahmadian, Afshin
    et al.
    KTH, Superseded Departments, Biotechnology.
    Gharizadeh, B.
    Gustafsson, A. C.
    Sterky, Fredrik
    KTH, Superseded Departments, Biotechnology.
    Nyrén, Pål
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Uhlén, Mathias
    KTH, Superseded Departments, Biotechnology.
    Lundeberg, Joakim
    KTH, Superseded Departments, Biotechnology.
    Single-nucleotide polymorphism analysis by pyrosequencing2000In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 280, no 1, p. 103-110Article in journal (Refereed)
    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.
    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    KTH, Superseded Departments, Biotechnology.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    KTH, Superseded Departments, Biotechnology.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    KTH, Superseded Departments, Biochemistry and Biotechnology.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    KTH, Superseded Departments, Biotechnology.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.
    Akhras, Michael
    et al.
    KTH, School of Biotechnology (BIO).
    Thiyagarajan, Sreedevi
    Stanford Univ, Stanford Genome Technol Ctr.
    Villablanca, Andrea C.
    Stanford Univ, Stanford Genome Technol Ctr.
    Davis, Ronald W.
    Stanford Univ, Stanford Genome Technol Ctr.
    Nyrén, Pål
    KTH, School of Biotechnology (BIO).
    Pourmand, Nader
    Stanford Univ, Stanford Genome Technol Ctr.
    PathogenMip Assay: A Multiplex Pathogen Detection Assay2007In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 2, no 2, p. e223-Article in journal (Refereed)
    Abstract [en]

    The Molecular Inversion Probe (MIP) assay has been previously applied to a large-scale human SNP detection. Here we describe the PathogenMip Assay, a complete protocol for probe production and applied approaches to pathogen detection. We have demonstrated the utility of this assay with an initial set of 24 probes targeting the most clinically relevant HPV genotypes associated with cervical cancer progression. Probe construction was based on a novel, cost-effective, ligase-based protocol. The assay was validated by performing pyrosequencing and Microarray chip detection in parallel experiments. HPV plasmids were used to validate sensitivity and selectivity of the assay. In addition, 20 genomic DNA extracts from primary tumors were genotyped with the PathogenMip Assay results and were in 100% agreement with conventional sequencing using an L1-based HPV genotyping protocol. The PathogenMip Assay is a widely accessible protocol for producing and using highly discriminating probes, with experimentally validated results in pathogen genotyping, which could potentially be applied to the detection and characterization of any microbe.

  • 4.
    Akhras, Michael
    et al.
    KTH, School of Biotechnology (BIO).
    Unemo, Magnus
    Örebro Univ Hosp, Dept Clin Microbiol.
    Thiyagarajan, Sreedevi
    Stanford Univ, Stanford Genome Technol Ctr.
    Nyrén, Pål
    KTH, School of Biotechnology (BIO).
    Davis, Ronald W.
    Stanford Univ, Stanford Genome Technol Ctr.
    Fire, Andrew Z.
    Stanford Univ.
    Pourmand, Nader
    Univ Calif Santa Cruz.
    Connector Inversion Probe Technology: A Powerful One- Primer Multiplex DNA Amplification System for Numerous Scientific Applications2007In: PLoS ONE, ISSN 1932-6203, Vol. 2, no 9, p. e915-Article in journal (Refereed)
    Abstract [en]

    We combined components of a previous assay referred to as Molecular Inversion Probe (MIP) with a complete gap filling strategy, creating a versatile powerful one-primer multiplex amplification system. As a proof-of-concept, this novel method, which employs a Connector Inversion Probe (CIPer), was tested as a genetic tool for pathogen diagnosis, typing, and antibiotic resistance screening with two distinct systems: i) a conserved sequence primer system for genotyping Human Papillomavirus (HPV), a cancer-associated viral agent and ii) screening for antibiotic resistance mutations in the bacterial pathogen Neisseria gonorrhoeae. We also discuss future applications and advances of the CIPer technology such as integration with digital amplification and next-generation sequencing methods. Furthermore, we introduce the concept of two-dimension informational barcodes, i. e. "multiplex multiplexing padlocks'' (MMPs). For the readers' convenience, we also provide an on-line tutorial with user-interface software application CIP creator 1.0.1, for custom probe generation from virtually any new or established primer-pairs.

  • 5. Andersson, Anders F.
    et al.
    Lindberg, Mathilda
    Jakobsson, Hedvig
    Bäckhed, Fredrik
    Nyrén, Pål
    KTH, School of Biotechnology (BIO).
    Engstrand, Lars
    Comparative Analysis of Human Gut Microbiota by Barcoded Pyrosequencing2008In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 3, no 7, p. e2836-Article in journal (Refereed)
    Abstract [en]

    Humans host complex microbial communities believed to contribute to health maintenance and, when in imbalance, to the development of diseases. Determining the microbial composition in patients and healthy controls may thus provide novel therapeutic targets. For this purpose, high-throughput, cost-effective methods for microbiota characterization are needed. We have employed 454-pyrosequencing of a hyper-variable region of the 16S rRNA gene in combination with sample-specific barcode sequences which enables parallel in-depth analysis of hundreds of samples with limited sample processing. In silico modeling demonstrated that the method correctly describes microbial communities down to phylotypes below the genus level. Here we applied the technique to analyze microbial communities in throat, stomach and fecal samples. Our results demonstrate the applicability of barcoded pyrosequencing as a high-throughput method for comparative microbial ecology.

  • 6. Babrzadeh, F.
    et al.
    Varghese, V.
    Pacold, M.
    Liu, T. F.
    Nyrén, Pål
    KTH, School of Biotechnology (BIO), Biochemistry.
    Schiffer, C.
    Fessel, W. J.
    Shafer, R. W.
    Collinearity of protease mutations in HIV-1 samples with high-level protease inhibitor class resistance2013In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 68, no 2, p. 414-418Article in journal (Refereed)
    Abstract [en]

    Objectives: To determine whether pan-protease inhibitor (PI)-resistant virus populations are composed predominantly of viruses with resistance to all PIs or of diverse virus populations with resistance to different subsets of PIs. Methods: We performed deep sequencing of plasma virus samples from nine patients with high-level genotypic and/or phenotypic resistance to all licensed PIs. The nine virus samples had a median of 12 PI resistance mutations by direct PCR Sanger sequencing. Results: For each of the nine virus samples, deep sequencing showed that each of the individual viruses within a sample contained nearly all of the mutations detected by Sanger sequencing. Indeed, a median of 94.9% of deep sequence reads had each of the PI resistance mutations present as a single chromatographic peak in the Sanger sequence. A median of 5.0% of reads had all but one of the Sanger mutations that were not part of an electrophoretic mixture. Conclusions: The collinearity of PI resistance mutations in the nine virus samples demonstrated that pan-PI-resistant viruses are able to replicate in vivo despite their highly mutated protease enzymes. We hypothesize that the marked collinearity of PI resistance mutations in pan-PI-resistant virus populations results from the unique requirements for multi-PI resistance and the extensive cross-resistance conferred by many of the accessory PI resistance mutations.

  • 7. Babrzadeh, Farbod
    et al.
    Jalili, Roxana
    Wang, Chunlin
    Shokralla, Shadi
    Pierce, Sarah
    Robinson-Mosher, Avi
    Nyrén, Pål
    KTH, School of Biotechnology (BIO), Biochemistry.
    Shafer, Robert W.
    Basso, Luiz C.
    de Amorim, Henrique V.
    de Oliveira, Antonio J.
    Davis, Ronald W.
    Ronaghi, Mostafa
    Gharizadeh, Baback
    Stambuk, Boris U.
    Whole-genome sequencing of the efficient industrial fuel-ethanol fermentative Saccharomyces cerevisiae strain CAT-12012In: Molecular Genetics and Genomics, ISSN 1617-4615, E-ISSN 1617-4623, Vol. 287, no 6, p. 485-494Article in journal (Refereed)
    Abstract [en]

    The Saccharomyces cerevisiae strains widely used for industrial fuel-ethanol production have been developed by selection, but their underlying beneficial genetic polymorphisms remain unknown. Here, we report the draft whole-genome sequence of the S. cerevisiae strain CAT-1, which is a dominant fuel-ethanol fermentative strain from the sugarcane industry in Brazil. Our results indicate that strain CAT-1 is a highly heterozygous diploid yeast strain, and the similar to 12-Mb genome of CAT-1, when compared with the reference S228c genome, contains similar to 36,000 homozygous and similar to 30,000 heterozygous single nucleotide polymorphisms, exhibiting an uneven distribution among chromosomes due to large genomic regions of loss of heterozygosity (LOH). In total, 58 % of the 6,652 predicted protein-coding genes of the CAT-1 genome constitute different alleles when compared with the genes present in the reference S288c genome. The CAT-1 genome contains a reduced number of transposable elements, as well as several gene deletions and duplications, especially at telomeric regions, some correlated with several of the physiological characteristics of this industrial fuel-ethanol strain. Phylogenetic analyses revealed that some genes were likely associated with traits important for bioethanol production. Identifying and characterizing the allelic variations controlling traits relevant to industrial fermentation should provide the basis for a forward genetics approach for developing better fermenting yeast strains.

  • 8.
    Ehn, Maria
    et al.
    KTH, Superseded Departments, Biotechnology.
    Nourizad, Nader
    KTH, Superseded Departments, Biotechnology.
    Bergstrom, Kristina
    KTH, Superseded Departments, Biotechnology.
    Ahmadian, Afshin
    KTH, Superseded Departments, Biotechnology.
    Nyrén, Pål
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Lundeberg, Joakim
    KTH, Superseded Departments, Biotechnology.
    Hober, Sophia
    KTH, Superseded Departments, Biotechnology.
    Toward pyrosequencing on surface-attached genetic material by use of DNA-binding luciferase fusion proteins2004In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 329, no 1, p. 11-20Article in journal (Refereed)
    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.

  • 9. Eriksson, J.
    et al.
    Karamohamed, S.
    Nyrén, Pål
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Method for real-time detection of inorganic pyrophosphatase activity2001In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 293, no 1, p. 67-70Article in journal (Refereed)
    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.

  • 10.
    Eriksson, Jonas
    et al.
    KTH, Superseded Departments, Biotechnology.
    Gharizadeh, B.
    Nourizad, Nader
    KTH, Superseded Departments, Biotechnology.
    Nyrén, Pål
    KTH, Superseded Departments, Biotechnology.
    7-deaza-2 '-deoxyadenosine-5 '-triphosphate as an alternative nucleotide for the pyrosequencing technology2004In: Nucleosides, Nucleotides & Nucleic Acids, ISSN 1525-7770, E-ISSN 1532-2335, Vol. 23, no 10, p. 1583-1594Article in journal (Refereed)
    Abstract [en]

    A new adenosine nucleotide analog suitable for the Pyrosequencing method is presented. The new analog, 7-deaza-2'-deoxyadenosine-5'-triphosphate (c(7)dATP), has virtually the. same low substrate specificity for luciferase as the currently used analog, 2'-deoxyadenosine-5'-O-(1-thiotriphosphate) (dATPalphaS). The inhibitory effect dATPalphaS displays on the nucleotide degrading activity of apyrase was reduced significantly by substituting the c(7)dATP for the dATPalphaS. Both analogs show high stability after long time storage at +8degreesC. Furthermore, with the new nucleotide a read length of up to 100 bases was obtained for several templates from fungi, bacteria and viruses.

  • 11.
    Eriksson, Jonas
    et al.
    KTH, Superseded Departments, Biotechnology.
    Gharizadeh, Baback
    KTH, Superseded Departments, Biotechnology.
    Nordström, Tommy
    KTH, Superseded Departments, Biotechnology.
    Nyrén, Pål
    KTH, Superseded Departments, Biotechnology.
    Pyrosequencing (TM) technology at elevated temperature2004In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 25, no 1, p. 20-27Article in journal (Refereed)
    Abstract [en]

    To date, the Pyrosequencing(TM) technology has been performed at 28degreesC due to the low thermostability of the firefly luciferase. In this study, firefly luciferase was stabilized in the presence of glycine betaine, allowing DNA sequencing at 37degreesC. By increasing the temperature to 37degreesC, false signals due to primer-dimers and loop-structures were decreased significantly. In addition, a combination of (i) replacing the natural dGTP with 7'deaza-dGTP in the polymerase chain reaction (PCR), (ii) 1.6 m glycine betaine, and (iii) an increase of the temperature to 37degreesC enabled us to sequence a DNA template with the initial sequence 3'-ATGGCCCGGGGGGGAGCTCCA . . . 5'. Furthermore, we describe a method to analyze if a primer forms a primer-dimer with extendable 3'-ends.

  • 12. Gambelunghe, G.
    et al.
    Ghaderi, M.
    Brozzetti, A.
    Del Sindaco, P.
    Gharizadeh, B.
    Nyrén, Pål
    KTH, Superseded Departments (pre-2005), Biochemistry and Biotechnology.
    Hjelmstrom, P.
    Nikitina-Zake, L.
    Sanjeevi, C. B.
    Falorni, A.
    Umbria Type 1 Diabetes, Registry
    Lack of association of CCR2-64I and CCR5-Delta 32 with type 1 diabetes and latent autoimmune diabetes in adults2003In: Human Immunology, ISSN 0198-8859, E-ISSN 1879-1166, Vol. 64, no 6, p. 629-632Article in journal (Refereed)
    Abstract [en]

    It is well known that type I diabetes mellitus (T1DM) is a complex genetic disease resulting from the autoimmune destruction of pancreatic beta cells. Several genes have been associated with susceptibility and/or protection for T1DM, but the disease risk is mostly influenced by genes located in the class II region of the major histocompatibility complex. The attraction of leukocytes to tissues is essential for inflammation and the beginning of autoimmune reaction. The process is controlled by chemokines, which are chemotactic cytolines. Some studies have shown that CCR2-64I and CCR5-Delta32 might be important for protection of susceptibility to some immunologically-mediated disorders. In the present study, we demonstrate the lack of association between CCR2-64I and CCR5-Delta32 gene polymorphism and TIDM and we desrcibe a new method for a simple and more precise genotyping of the CCR2 gene.

  • 13. Gambelunghe, G.
    et al.
    Ghaderi, M.
    Gharizadeh, Baback
    KTH, Superseded Departments, Biotechnology.
    Brozzetti, A.
    Tortoioli, C.
    Del Sindaco, P.
    Sanjeevi, C. B.
    Hjelmstrom, P.
    Sirsjo, A.
    Nyrén, Pål
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Santeusanio, F.
    Falorni, A.
    Lack of association of human chemokine receptor gene polymorphisms CCR2-64I and CCR5-Delta 32 with autoimmune Addison's disease2004In: European journal of immunogenetics, ISSN 0960-7420, E-ISSN 1365-2370, Vol. 31, no 2, p. 73-76Article in journal (Refereed)
    Abstract [en]

    The attraction of leukocytes to tissues is essential for inflammation and the initiation of the autoimmune reaction. The process is controlled by chemokines, which are chemotactic cytokines. We investigated whether human chemokine receptor gene polymorphisms, namely CCR5-Delta32 and CCR2-64I, are associated with susceptibility to autoimmune Addison's disease. Genotyping was performed in 56 patients and 127 healthy controls by a new method using pyrosequencing for CCR2-64I and by polymerase chain reaction and detecting gel for CCR5-Delta32. None of the CCR2 or CCR5 alleles was found to be associated, either positively or negatively, with disease risk. Our results indicate that the CCR2-64I and CCR5-Delta32 gene polymorphisms do not play a major role in conferring genetic risk for, and/or protection against, autoimmune Addison's disease.

  • 14. Garcia, C. A.
    et al.
    Ahmadian, Afshin
    KTH, Superseded Departments, Biotechnology.
    Gharizadeh, B.
    Lundeberg, Joakim
    KTH, Superseded Departments, Biotechnology.
    Ronaghi, M.
    Nyrén, Pål
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Mutation detection by pyrosequencing: sequencing of exons 5-8 of the p53 tumor suppressor gene2000In: Gene, ISSN 0378-1119, E-ISSN 1879-0038, Vol. 253, no 2, p. 249-257Article in journal (Refereed)
    Abstract [en]

    The ability to sequence a large number of DNA samples rapidly and accurately for detection of all possible mutations is a critical goal for the future application of DNA sequencing in routine medical diagnostics. Pyrosequencing(TM) is a non-electrophoretic real-time DNA sequencing method that uses the luciferase-luciferin light release as the detection signal for nucleotide incorporation into target DNA. For pyrosequencing of the human p53 gene, a nested multiplex PCR method for amplification of exons 5-8 was prepared. In order to investigate the use of pyrosequencing in mutation detection, DNA samples from skin-cancer patients were used. Two forms of nucleotide dispensation strategy were used, cyclic and programmed. Bi-directional pyrosequencing was performed and the overlapping sequence data produced were assembled to determine the sequence of the gene. Reliable sequencing data were obtained with both dispensation strategies, but some advantages were obtained using the programmed nucleotide dispensation approach, such as longer and faster reads, and fewer out-of-phase problems. The accuracy of pyrosequencing for detection of p53 mutations and allele distribution was demonstrated.

  • 15. Gharizadeh, B.
    et al.
    Eriksson, J.
    Nourizad, N.
    Nordstrom, Tommy
    KTH, Superseded Departments, Biotechnology.
    Nyrén, Pål
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Improvements in pyrosequencing technology by employing sequenase polymerase2004In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 330, no 2, p. 272-280Article in journal (Refereed)
    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.

  • 16. Gharizadeh, B.
    et al.
    Ghaderi, M.
    Donnelly, D.
    Amini, B.
    Wallin, K. L.
    Nyrén, Pål
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Multiple-primer DNA sequencing method2003In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 24, no 08-jul, p. 1145-1151Article in journal (Refereed)
    Abstract [en]

    A multiple-primer DNA sequencing approach suitable for genotyping, detection and identification of microorganisms and viruses has been developed. In this new method two or m ore sequencing primers, combined in a pool, are added to a DNA sample of interest. The oligonucleotide that hybridizes to the DNA sample will function as a primer during the subsequent DNA sequencing procedure. This strategy is suited for selective detection and genotyping of relevant microorganisms and samples harboring different DNA targets such as,multiple variant/infected samples as well as unspecific amplification products. This method is used here in a model system for detection and typing of high-risk oncogenic human papilloma viruses (HPVs) in samples containing multiple infections/variants or unspecific amplification products. Type-specific sequencing primers were designed for four of the most oncogenic (high-risk) HPV types (HPV-16, HPV-18, HPV-33, and HPV-45). The primers were combined and added to a sample containing a mixture of one high-risk (16, 18, 33, or 45) and one or two low-risk types. The DNA samples were sequenced by the Pyrosequencing(TM) technology and the Sanger dideoxy sequencing method. Correct genotyping was achieved in all tested combinations. This multiple-sequencing primer approach also improved the sequence data quality for samples containing unspecific amplification products. The new strategy is highly suitable for diagnostic typing of relevant species/genotypes of microorganisms.

  • 17. Gharizadeh, B.
    et al.
    Kalantari, M.
    Garcia, C. A.
    Johansson, B.
    Nyrén, Pål
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Typing of human papillomavirus by pyrosequencing2001In: Laboratory Investigation, ISSN 0023-6837, E-ISSN 1530-0307, Vol. 81, no 5, p. 673-679Article in journal (Refereed)
    Abstract [en]

    The possibility of using a new bioluminometric UNA sequencing technique, called pyrosequencing, for typing of human papillomaviruses (HPV) was investigated. A blinded pyrosequencing test was performed on an HPV test panel of 67 GP5+/GP6+ PCR-derived amplification products. The 67 clinical DNA samples were sequenced up to 25 bases and sequences were searched using BLAST. All of the samples were correctly genotyped by pyrosequencing and the results were unequivocally in accordance with the results obtained from conventional DNA sequencing. Pyrosequencing was found to be a fast and efficient tool for identifying individual HPV types. Furthermore, pyrosequencing has the capability of determining novel HPV types as well as HPV sequence variants harboring mutation(s). The method is robust and well suited for large-scale programs.

  • 18. Gharizadeh, B.
    et al.
    Nordstrom, T.
    Ahmadian, Afshin
    KTH, Superseded Departments, Biotechnology.
    Ronaghi, M.
    Nyrén, Pål
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Long-read pyrosequencing using pure 2 '-deoxyadenosine-5 '-O '-(1-thiotriphosphate) Sp-isomer2002In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 301, no 1, p. 82-90Article in journal (Refereed)
    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.

  • 19. Gharizadeh, B.
    et al.
    Oggionni, M.
    Zheng, B. Y.
    Akom, E.
    Pourmand, N.
    Ahmadian, Afshin
    KTH, School of Biotechnology (BIO), Gene Technology.
    Wallin, K. L.
    Nyrén, Pål
    KTH, School of Biotechnology (BIO), Biochemistry.
    Type-specific multiple sequencing primers - A novel strategy for reliable and rapid genotyping of human papilloma viruses by pyrosequencing technology2005In: Journal of Molecular Diagnostics, ISSN 1525-1578, E-ISSN 1943-7811, Vol. 7, no 2, p. 198-205Article in journal (Refereed)
    Abstract [en]

    DNA sequencing is the gold standard method for accurate microbial and viral typing. However, DNA sequencing techniques have been facing limitations in typing of human papillomaviruses when the specimen harbors multiple genotypes and yields nonspecific amplification products, resulting in nonspecific and noninterpretable sequence data. To address these limitations we have developed a type-specific multiple sequencing primer DNA-sequencing method. This new strategy is suitable for sequencing and typing of samples harboring different genotypes (co-infections with multiple genotypes) and yielding nonspecific amplifications, thus eliminating the need for nested polymerase chain reaction (PCR), stringent PCR conditions, and cloning. The new approach has also proved useful for amplicons containing low PCR yield or subdominant types, avoiding reperforming of amplifications. We have applied the multiple sequencing primer method for genotyping of clinically relevant human papillomaviruses in a clinical test panel by using a combined pool of seven type-specific sequencing primers for HPV-6, -11, -16, -18, -31, -33, and -45. Furthermore, we introduced a sequence pattern recognition approach when there was a plurality of genotypes in the sample to facilitate typing of more than one target DNA in the sample. The multiple sequencing primer method has proved to be a multifaceted approach for typing of human papillomaviruses by DNA sequencing technologies.

  • 20. Gharizadeh, B.
    et al.
    Ohlin, A.
    Molling, P.
    Backman, A.
    Amini, B.
    Olcen, P.
    Nyrén, Pål
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Multiple group-specific sequencing primers for reliable and rapid DNA sequencing2003In: Molecular and Cellular Probes, ISSN 1044-7431, E-ISSN 1095-9327, Vol. 17, no 4, p. 203-210Article in journal (Refereed)
    Abstract [en]

    Pyrosequencing(TM) technology is a bioluminometric DNA sequencing method that employs a cascade of four enzymes to deliver sequence signals. To date this technology has been limited to the sequencing of short stretches of DNA. As an improvement to this technique, we have introduced a bacterial group-specific, multiple sequencing primer approach that circumvents sequencing of less informative semi-conservative regions of the 16S rRNA gene. This new approach is suitable for challenging templates, improving sequence data quality, avoiding sequencing of non-specific amplification products, lessening sequencing time, and moreover, this strategy should open the way for many new applications in the future. The group-specific, multiple sequencing primers can be applied in the Sanger dideoxy sequencing method as well. In addition, we have improved the chemistry of the Pyrosequencing system enabling sequencing of longer stretches of DNA, which allows numerous new applications.

  • 21.
    Gharizadeh, Baback
    et al.
    Stanford Univ, Stanford Genome Technol Ctr.
    Akhras, Michael
    KTH, School of Biotechnology (BIO), Biochemistry.
    Nourizad, Nader
    KTH, School of Biotechnology (BIO), Biochemistry.
    Ghaderi, Mehran
    Univ Örebro, Dept Caring Sci, Div Biomed.
    Yasuda, Kenji
    Univ Tokyo, Grad Sch Arts & Sci, Dept Life Sci.
    Nyrén, Pål
    KTH, School of Biotechnology (BIO), Biochemistry.
    Pourmand, Nader
    Stanford Univ, Stanford Genome Technol Ctr.
    Methodological improvements of pyrosequencing technology2006In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 124, no 3, p. 504-511Article in journal (Refereed)
    Abstract [en]

    Pyrosequencing technology is a rather novel DNA sequencing method based on the sequencing-by-synthesis principle. This bioluminometric, real-time DNA sequencing technique employs a cascade of four enzymatic reactions producing sequence peak signals. The method has been proven highly suitable for single nucleotide polymorphism analysis and sequencing of short stretches of DNA. Although the pyrosequencing procedure is relatively straightforward, users may face challenges due to varying parameters in PCR and sequencing primer design, sample preparation and nucleotide dispensation; such challenges are labor and cost intensive. In this study, these issues have been addressed to increase signal quality and assure sequence accuracy.

  • 22.
    Gharizadeh, Baback
    et al.
    Stanford Univ, Stanford Genome Technol Ctr.
    Akhras, Michael
    KTH, School of Biotechnology (BIO).
    Unemo, Magnus
    Örebro Univ Hosp, Dept Clin Microbiol.
    Wretlind, Bengt
    Karolinska Inst, Dept Lab Med.
    Nyrén, Pål
    KTH, School of Biotechnology (BIO).
    Pourmand, Nader
    Stanford Univ, Stanford Genome Technol Ctr.
    Detection of gyrA mutations associated with ciprofloxacin resistance in Neisseria gonorrhoeae by rapid and reliable pre-programmed short DNA sequencing2005In: International Journal of Antimicrobial Agents, ISSN 0924-8579, E-ISSN 1872-7913, Vol. 26, no 6, p. 486-490Article in journal (Refereed)
    Abstract [en]

    Quinolone resistance is rapidly increasing in Neisseria gonorrhoeae and is posing a significant public health threat that requires constant surveillance. A rapid and reliable mutation detection assay has been developed. The assay is based on pre-programmed short DNA sequencing and is designed to detect point mutations in the gyrA gene that are highly related to ciprofloxacin resistance, i.e. in codons 91 and 95. By developing an assay based on pyrosequencing and exploiting the pre-programmed nucleotide dispensation capability of this technology, the sequence comprising the mutations will be analysed and promptly reveal whether the N. gonorrhoeae pathogen carries resistance to ciprofloxacin. A panel of 40 N. gonorrhoeae clinical isolates, of which 27 phenotypically displayed decreased susceptibility or resistance to ciprofloxacin, was used in the present study. All point mutations in the short stretch of the N. gonorrhoeae gyrA gene were easily discriminated, and the genotypic results obtained by pre-programmed sequencing were mainly in agreement with the phenotypically identified decreased susceptibility or resistance to ciprofloxacin. The new method used in the present study has the potential for rapid and reliable identification of known as well as previously unknown drug resistance mutations.

  • 23.
    Gharizadeh, Baback
    et al.
    KTH, Superseded Departments, Biotechnology.
    Norberg, E.
    Löffler, J.
    Jalal, S.
    Tollemar, J.
    Einsele, H.
    Klingspor, L.
    Nyrén, Pål
    KTH, Superseded Departments, Biotechnology.
    Identification of medically important fungi by the Pyrosequencing (TM) technology2004In: Mycoses (Berlin), ISSN 0933-7407, E-ISSN 1439-0507, Vol. 47, no 1-2, p. 29-33Article in journal (Refereed)
    Abstract [en]

    The Pyrosequencing(TM) technology was used for identification of different clinically relevant fungi. The tests were performed on amplicons derived from the 18S rRNA gene using polymerase chain reaction (PCR) universal primers for amplification. Sequencing was performed up to 40 bases in a variable region with a designed general sequencing primer and the Pyrosequence data were analyzed by BLAST sequence search in the GenBank database. DNA from a total of 21 fungal specimens consisting of nine strains of clinically relevant fungi and 12 clinical specimens from patients suffering from proven invasive fungal infections were PCR-amplified and analyzed by gel electrophoresis, PCR-enzyme-linked immunosorbent assay (ELISA) and the Pyrosequencing technology. All data obtained by the Pyrosequencing technology were in agreement with the results obtained by PCR-ELISA using species/genus-specific oligonucleotides and were as well in accordance with the culture results. The results demonstrate that the Pyrosequencing method is a reproducible and reliable technique for identification of fungal pathogens.

  • 24.
    Gharizadeh, Baback
    et al.
    Stanford Univ, Stanford Genome Technol Ctr.
    Zheng, Biying
    Karolinska Inst, Dept Mol Med.
    Akhras, Michael
    KTH, School of Biotechnology (BIO).
    Ghaderi, Mehran
    Karolinska Univ Hosp, Clinical Pathology/Cytology.
    Jejelowo, Olufisayo
    Texas So Univ.
    Strander, Björn
    Gothenburg Univ, Sahlgrens Acad, Ctr Oncol.
    Nyrén, Pål
    KTH, School of Biotechnology (BIO).
    Wallin, Keng-Ling
    Karolinska Inst, Dept Mol Med.
    Pourmand, Nader
    Stanford Univ, Stanford Genome Technol Ctr.
    Sentinel-base DNA genotyping using multiple sequencing primers for high-risk human papillomaviruses2006In: Molecular and Cellular Probes, ISSN 1044-7431, E-ISSN 1095-9327, Vol. 20, no 3-4, p. 230-238Article in journal (Refereed)
    Abstract [en]

    Despite the various technologies in place for genotyping human papillomaviruses (HPV), clinical use and clinical research demand a method that is fast, more reliable and cost-effective. The technology described here represents a breakthrough development in that direction. By combining the method of multiple sequencing primers with DNA sequencing, we have developed a rapid assay for genotyping HPV that relies on the identification of a single, type-specific 'sentinel' base. As described here, the prototype assay has been developed to recognize the 12 most high-risk HPV types (HPV-16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58 and 59) and is capable of recognizing and simultaneously genotyping multiple HPV co-infections. By providing sequence information on multiple HPV infections, this method eliminates the need for labor- and cost-intensive PCR cloning. These proof-of-concept studies establish the assay to be accurate, reliable, rapid, flexible, and cost-effective, providing evidence of the feasibility this technique for use in clinical settings.

  • 25. Javanmard, Mehdi
    et al.
    Babrzadeh, Farbod
    KTH, School of Biotechnology (BIO), Biochemistry.
    Nyrén, Pål
    KTH, School of Biotechnology (BIO), Biochemistry.
    Davis, Ronald W.
    Improvement in cell capture throughput using parallel bioactivated microfluidic channels2012In: Biomedical microdevices (Print), ISSN 1387-2176, E-ISSN 1572-8781, Vol. 14, no 4, p. 625-629Article in journal (Refereed)
    Abstract [en]

    Optimization of targeted cell capture with microfluidic devices continues to be a challenge. On the one hand, microfluidics allow working with microliter volumes of liquids, whereas various applications in the real world require detection of target analyte in large volumes, such as capture of rare cell types in several ml of blood. This contrast of volumes (microliter vs. ml) has prevented the emergence of microfluidic cell capture sensors in the clinical setting. Here, we study the improvement in cell capture and throughput achieved using parallel bioactivated microfluidic channels. The device consists of channels in parallel with each other tied to a single channel. We discuss fabrication and testing of our devices, and show the ability for an improvement in throughput detection of target cells.

  • 26. Laytragoon-Lewin, Nongnit
    et al.
    Nilsson, Per J.
    Castro, Juan
    Gharizadeh, Baback
    Nyrén, Pål
    KTH, School of Biotechnology (BIO), Biochemistry.
    Glimelius, Bengt
    Elmberger, Goran
    Turesson, Ingela
    Svensson, Christer
    Human papillomavirus (HPV), DNA aberrations and cell cycle progression in anal squamous cell carcinoma patients2007In: Anticancer Research, ISSN 0250-7005, E-ISSN 1791-7530, Vol. 27, no 6C, p. 4473-4479Article in journal (Refereed)
    Abstract [en]

    Human papillomavirus (HP) infections of the genital tract are sexually transmitted and prevalent worldwide. In this study, the role of HPV in 72 patients with anal squamous cell carcinoma was investigated. Patients and Methods: Polymerase chain reaction (PCR) in combination with in situ hybridization was used to identify HPV-DNA in the patients biopsies. The HPV typing was conducted by pyrosequencing. Cell cycle and DNA content were analysed by cytometry. Results: Ninety percent of the carcinoma biopsies carried high-risk oncogenic HPV in their malignant cells. Eighty-one percent of these demonstrated a single infection with HPV16, 18 or 33 and 19% were double infected with HPV16 and HPV18 Accumulations of viral genes were seen at the necrotic area of the tumours. The HPV genome in the tumour cell influenced significant the host cell cycle progression, but not DNA aberrations. Within these patients, HPV status in the malignant cells was not found to be associated with patient survival time. Conclusion: High-risk oncogenic HPV may play an important role in the initiation of host cell proliferation in anal squamous cell carcinoma. However, infection with HPV may not have any direct influence itself on the clinical outcome of these patients considering the treatments currently available.

  • 27. Lindback, E.
    et al.
    Gharizadeh, B.
    Ataker, F.
    Airell, A.
    Jalal, S.
    Nyrén, Pål
    KTH, School of Biotechnology (BIO), Biochemistry.
    Wretlind, B.
    DNA gyrase gene in Neisseria gonorrhoeae as indicator for resistance to ciprofloxacin and species verification2005In: International Journal of STD and AIDS (London), ISSN 0956-4624, E-ISSN 1758-1052, Vol. 16, no 2, p. 142-147Article in journal (Refereed)
    Abstract [en]

    We have identified a unique region of eight amino acids in the quinolone resistance-determining region in the gyrA gene in Neisseria gonorrhoeae as an indicator of resistance to fluoroquinolones. We sequenced that region by the Pyrosequencing(TM) technology in 46 N. gonorrhoeae strains and 11 urine samples positive in AMPLICOR(TM) N. gonorrhoeae polymerase chain reaction (Roche Diagnostics), with corresponding isolates of N. gonorrhoeae. The results showed that 28 samples with minimum inhibitory concentration (MIC) of ciprofloxacin > 1 mg/L had mutations in positions 91 and 95. Fifteen samples with MIC 0.125-1.0 mg/L had either one or both of the mutations. The 14 susceptible samples had no mutations. The target region also discriminates N. gonorrhoeae from other species of Neisseria. Our conclusion is that gyrA is an indicator of resistance to ciprofloxacin in N. gonorrhoeae and sequencing by Pyrosequencing technology is a suitable tool for analysis of DNA in urine samples.

  • 28. Lindback, E.
    et al.
    Gharizadeh, B.
    Ataker, F.
    Airell, A.
    Jalal, S.
    Nyrén, Pål
    KTH, School of Biotechnology (BIO), Biochemistry (closed 20130101).
    Wretlind, B.
    Quinolone-resistant Neisseria gonorrhoeae - response2005In: International Journal of STD and AIDS (London), ISSN 0956-4624, E-ISSN 1758-1052, Vol. 16, no 12, p. 835-836Article in journal (Refereed)
  • 29. Nordstrom, T.
    et al.
    Alderborn, A.
    Nyrén, Pål
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Method for one-step preparation of double-stranded DNA template applicable for use with Pyrosequencing (TM) technology2002In: Journal of Biochemical and Biophysical Methods, ISSN 0165-022X, E-ISSN 1872-857X, Vol. 52, no 2, p. 71-82Article in journal (Refereed)
    Abstract [en]

    A new one-step method for fast and efficient preparation of double-stranded DNA template, suitable for use with Pyrosequencing(TM) technology, has been developed. In the new method, two different types of oligonucleotides were used to prevent reannealing of remaining PCR primers to the template: oligonucleotides complementary to the PCR primers and 3'-end modified oligonucleotides with the same sequence as the PCR primers. Advantages with the new strategy are: (i) faster and simpler template preparation procedure (one-step); (ii) no need for exonuclease I treatment; and (iii) less problem with unspecific priming from loop structures and dimers. By careful oligonucleotide design, and/or by addition of single-stranded DNA-binding protein, problems with unspecific sequence signals due to mispriming can be reduced. The new method was used for analysis of genotype variations within the renin-angiotensin-aldosterone system.

  • 30. Nordstrom, T.
    et al.
    Gharizadeh, B.
    Pourmand, N.
    Nyrén, Pål
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Ronaghi, M.
    Method enabling fast partial sequencing of cDNA clones2001In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 292, no 2, p. 266-271Article in journal (Refereed)
    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.

  • 31. Nordstrom, T.
    et al.
    Nourizad, K.
    Ronaghi, M.
    Nyrén, Pål
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Method enabling pyrosequencing on double-stranded DNA2000In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 282, no 2, p. 186-193Article in journal (Refereed)
    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.

  • 32. Nordstrom, T.
    et al.
    Ronaghi, M.
    Forsberg, L.
    de Faire, U.
    Morgenstern, R.
    Nyrén, Pål
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Direct analysis of single-nucleotide polymorphism on double-stranded DNA by pyrosequencing2000In: Biotechnology and applied biochemistry, ISSN 0885-4513, E-ISSN 1470-8744, Vol. 31, p. 107-112Article in journal (Refereed)
    Abstract [en]

    Pyrosequencing, a new method for DNA sequencing, is gaining widespread use for many different types of DNA analysis. The method takes advantage of four coupled enzymes in a single tube assay to monitor DNA synthesis in real time using a luminometric detection system. Here, we demonstrate the use of pyrosequencing for direct analysis of single-nucleotide polymorphism on double-stranded PCR product. Pyrosequencing data on the human glutathione peroxidase gene (GPXI) from several individuals were analysed and three different allelic variants were determined and confirmed. The possibility of further simplifying the sequencing and template-preparation steps is discussed.

  • 33. Nourizad, N.
    et al.
    Ehn, M.
    Gharizadeh, B.
    Hober, Sophia
    KTH, Superseded Departments, Biotechnology.
    Nyrén, Pål
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Methylotrophic yeast Pichia pastoris as a host for production of ATP-diphosphohydrolase (apyrase) from potato tubers (Solanum tuberosum)2003In: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 27, no 2, p. 229-237Article in journal (Refereed)
    Abstract [en]

    ATP-diphosphohydrolase (apyrase) catalyzes the hydrolysis of phosphoanhydride bonds of nucleoside tri- and di-phosphates in the presence of divalent cations. This enzyme has broad substrate specificity for nucleotides, which makes it an ideal enzyme for different biotechnical applications, such as DNA sequencing and platelet-aggregation inhibition. The only commercially available apyrase is isolated from potato tubers. To avoid batch-to-batch variations in activity and quality, we decided to produce a recombinant enzyme. The methylotrophic yeast Pichia pastoris was chosen as an eukaryotic expression host. The coding sequence of potato apyrase, without the signal peptide, was cloned into the YpDC541 vector to create a fusion with the alpha-mating secretion signal of Saccharomyces cerevisiae. The gene was placed under the control of the methanol-inducible alcohol oxidase promoter. The YpDC541-apyrase construct was integrated into P. pastoris strain SMD1168. Methanol induction resulted in secretion of apyrase to a level of 1 mg/L. The biologically active recombinant apyrase was purified by hydrophobic interaction and ion exchange chromatography. According to SDS-PAGE and Western blot analysis, the purified enzyme showed to be hyperglycosylated. By enzymatic removal of N-glycans, a single band corresponding to a molecular mass of 48 kDa was detected. The recombinant apyrase was found to function well when it was used in combination with the Pyrosequencing technology.

  • 34. Nourizad, N.
    et al.
    Gharizadeh, B.
    Nyrén, Pål
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Method for clone checking2003In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 24, no 11, p. 1712-1715Article in journal (Refereed)
    Abstract [en]

    A new method for simple and fast clone checking is described. We combined the Pyrosequencing(TM) technology with a preprogrammed nucleoticle dispensation strategy for fast analysis of DNA constructs. To test this method, the N-terminus region of plasmids constructed for the production of recombinant apyrase was analyzed. Of the ten plasmids tested, seven constructs were correct, two constructs showed one base deletion, and one construct showed deletion of a 195 bp fragment. The preprogrammed nucleoticle dispensation strategy allowed the identification of the sequence downstream of the deletions. Thus, this method determines both the location and nature of possible artifacts.

  • 35. Nygren, M.
    et al.
    Ronaghi, M.
    Nyrén, Pål
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Albert, J.
    Lundeberg, Joakim
    KTH, Superseded Departments, Biotechnology.
    Quantification of HIV-1 using multiple quantitative polymerase chain reaction standards and bioluminometric detection2001In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 288, no 1, p. 28-38Article in journal (Refereed)
    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.

  • 36.
    Nyrén, Pål
    KTH, School of Biotechnology (BIO).
    Method enabling detection of single-stranded DNA ligation activity2017In: Archives of industrial biotechnology, Vol. 1, no 1, p. 35-40Article in journal (Refereed)
    Abstract [en]

    The enzyme DNA ligase is an essential tool in many different applications in the field of biotechnology. For these applications the DNA quality can be crucial for the final result. In this paper we present a new method that can be used for specific studies of DNA ligases and for simultaneous analyzes of DNA quality. More specifically, a simple, fast and sensitive method for detection of single-stranded DNA (ssDNA) ligase activity is presented. The new method is based on real-time detection of nucleotide incorporation over the ligation site catalyzed by a DNA polymerase. The ratio between the incorporation signal after and before the ligation site gives an estimate of the ligation efficiency. To further increase the robustness of the assay the exact amount of unligated product can be easily estimated in a separate assay using a 3’-end blocked oligonucleotide functioning as template. Produced circular DNA can be cleaned from unligated DNA by exonuclease I treatment and the quality of the circle can be easily analyzed by a simple sequencing procedure. If the ssDNA used for the ligation reaction contains truncated fragments this will be observed by uneven signals during the analysis. The new method can be a useful tool for researchers working with both basic and applied projects in the field of biotechnology.

  • 37.
    Nyrén, Pål
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    The History of Pyrosequencing®2015In: Methods in Molecular Biology, ISSN 1064-3745, E-ISSN 1940-6029, Vol. 1315, p. 3-15Article in journal (Refereed)
    Abstract [en]

    One late afternoon in the beginning of January 1986, bicycling from the lab over the hill to the small village of Fulbourn, the idea for an alternative DNA sequencing technique came to my mind. The basic concept was to follow the activity of DNA polymerase during nucleotide incorporation into a DNA strand by analyzing the pyrophosphate released during the process. Today, the technique is used in multidisciplinary fields in academic, clinical, and industrial settings all over the word. This technique can be used for both single-base sequencing and whole-genome sequencing, depending on the format used.In this chapter, I give my personal account of the development of Pyrosequencing()-beginning on a winter day in 1986, when I first envisioned the method-until today, nearly 30 years later.

  • 38.
    Nyrén, Pål
    KTH, School of Biotechnology (BIO).
    Use of osmolytes to heat stabilise luciferase and/or apyrase2002Patent (Other (popular science, discussion, etc.))
    Abstract [en]

    The present invention refers to the use of at least one osmolyte to heat stabilise at least one enzyme, such as luciferase and/or apyrase, that is used in an in-vitro bio-luminometric assay, such as a sequencing-by-synthesis reaction, especially Pyrose-quencingTM, during the enzymatic reaction. Moreover, the invention refers to a composition and a kit for use in the bioluminometric assay. Hereby, the temperature sensitive enzyme is heat stabilised, whereby the bioluminometric assay may be per-formed at a temperature that is higher and therefore more suitable for the other en-zymes of the assay.

  • 39.
    Ronaghi, Martin
    et al.
    KTH, Superseded Departments (pre-2005), Biochemistry and Biotechnology.
    Uhlén, Mathias
    KTH, Superseded Departments (pre-2005), Biochemistry and Biotechnology.
    Nyrén, Pål
    KTH, Superseded Departments (pre-2005), Biochemistry and Biotechnology.
    A sequencing method based on real-time pyrophosphate1998In: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 281, no 5375, p. 363-365Article in journal (Refereed)
  • 40. Sakthivel, Priya
    et al.
    Wang, Xiongbiao
    Gharizadeh, Baback
    Giscombe, Ricardo
    Pirskanen, Ritva
    Nyrén, Pål
    KTH, School of Biotechnology (BIO), Biochemistry (closed 20130101).
    Lefvert, Ann Kari
    Single-nucleotide polymorphisms in the B7H3 gene are not associated with human autoimmune myasthenia gravis2006In: Journal of Genetics, ISSN 0022-1333, E-ISSN 0973-7731, Vol. 85, no 3, p. 217-220Article in journal (Refereed)
  • 41.
    Svantesson, Anna
    et al.
    KTH, School of Computer Science and Communication (CSC), Numerical Analysis and Computer Science, NADA.
    Nordström, Tommy
    KTH, School of Biotechnology (BIO).
    Hellgren Kotaleski, Jeanette
    KTH, School of Computer Science and Communication (CSC), Numerical Analysis and Computer Science, NADA.
    Nyrén, Pål
    KTH, School of Biotechnology (BIO).
    An Investigation of the Apyrase Stepin the Pyrosequencing Model:Combining Experiments and Simulations.2005Report (Other academic)
  • 42.
    Svantesson, Anna
    et al.
    KTH, Superseded Departments, Numerical Analysis and Computer Science, NADA.
    Westermark, Pål. O.
    KTH, Superseded Departments, Numerical Analysis and Computer Science, NADA.
    Hellgren Kotaleski, Jeanette
    KTH, Superseded Departments, Numerical Analysis and Computer Science, NADA.
    Gharizadeh, Baback
    KTH, Superseded Departments, Biotechnology.
    Lansner, Anders
    KTH, Superseded Departments, Numerical Analysis and Computer Science, NADA.
    Nyrén, Pål
    KTH, Superseded Departments, Biotechnology.
    A mathematical model of the Pyrosequencing reaction system2004In: Biophysical Chemistry, ISSN 0301-4622, E-ISSN 1873-4200, Vol. 110, no 02-jan, p. 129-145Article in journal (Refereed)
    Abstract [en]

    The Pyrosequencing(TM) technology is a newly developed DNA sequencing method that monitors DNA nucleotide incorporation in real-time. A set of coupled enzymatic reactions, together with bioluminescence, detects incorporated nucleotides in the form of light pulses, yielding a characteristic light profile. In this study, a biochemical model of the Pyrosequencing reaction system is suggested and implemented. The model is constructed utilizing an assumption of irreversible Michaelis-Menten rate equations and a constant incorporation efficiency. The kinetic parameters are studied and values are chosen to obtain as reliable simulation results as possible. The results presented here show strong resemblance with real experiments. The model is able to capture the dynamics of a single light pulse with great accuracy, as well as the overall characteristics of a whole pyrogram(TM). The plus- and minus-shift effects observed in experiments are successfully reconstructed by two constant efficiency factors. Furthermore, pulse broadening can partly be explained by apyrase inhibition and successive dilution.

  • 43.
    Svantesson, Anna
    et al.
    KTH, School of Computer Science and Communication (CSC), Numerical Analysis and Computer Science, NADA.
    Westermark, Pål O.
    KTH, School of Computer Science and Communication (CSC), Numerical Analysis and Computer Science, NADA.
    Nyrén, Pål
    KTH, School of Biotechnology (BIO).
    Hellgren Kotaleski, Jeanette
    KTH, School of Computer Science and Communication (CSC), Numerical Analysis and Computer Science, NADA.
    Towards an Improved Polymerase Descriptionin the Pyrosequencing Model.2005Report (Other academic)
  • 44. Zhao, X. Y.
    et al.
    Gharizadeh, B.
    Hjelmstrom, P.
    Pirskanen, R.
    Nyrén, Pål
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Lefvert, A. K.
    Ghaderi, M.
    Genotypes of CCR2 and CCR5 chemokine receptors in human myasthenia gravis2003In: International Journal of Molecular Medicine, ISSN 1107-3756, E-ISSN 1791-244X, Vol. 12, no 5, p. 749-753Article in journal (Refereed)
    Abstract [en]

    The aim of this study was to examine the association of human autoimmune myasthenia gravis (MG) with two DNA polymorphisms of the chemokine receptors CCR5-Delta32 and CCR2-64I. CCR2 and CCR5 interact primarily with the human CC family ligands CCL2 (formerly called monocyte chemoattractant protein; MCP-1), CCL3 and CCL4 (macrophage inflammatory protein-1alpha and -1beta; MIP-1alpha/beta), and their main function is to recruit leukocytes from circulation into the tissues, thus playing an important role in human inflammatory disorders. A PCR-based genotyping method was used to determine the genetic variation at the CCR5 gene and an automated real-time Pyrosequencing technology was employed for the analysis of G-->A point mutation at the CCR2 gene. Results obtained from 158 patients and 272 healthy controls demonstrate no evidence of association between genetic variants of CCR2 and CCR5 with MG and its clinical manifestations. CCR2-64I and CCR5-Delta32 genotypes are thus unlikely to be involved in protection or predisposition to MG.

1 - 44 of 44
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