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  • 1.
    Ahmadian, Afshin
    KTH, Superseded Departments, Biotechnology.
    Approaches for analysis of mutations and genetic variations2001Doctoral thesis, monograph (Other academic)
    Abstract [en]

    Detecting mutations and genomic variations is fundamental indiagnosis, isolating disease genes, association studies,functional genomics and pharmacogenomics. The objective hasbeen to use and further develop a variety of tools andtechnologies to analyze these genetic alterations andvariations.

    The p53 tumor suppressor gene and short arm of chromosome 9have been used as genetic markers to investigate fundamentalquestions concerning early events preceding non-melanoma skincancers, clonal progression and timing of different mutationsand deletions. Conventional gel based DNA sequencing andfragment analysis of microsatellite markers were utilized forthis purpose. In addition, a sequence-specific PCR-mediatedartifact is discussed.

    Pyrosequencing, a bioluminometric technique based onsequencing-by-synthesis, has been utilized to determinemutation ratios in the p53 gene. In addition, in the case ofmultiple mutations, pyrosequencing was adopted to determineallelic distribution of mutations without the use of cloningprocedures. Exons 5 to 8 of the p53 gene were also sequenced bythis method.

    The possibility of typing single base variations bypyrosequencing has been evaluated. Two different nucleotidedispensation orders were investigated and data were comparedwith the predicted pattern for each alternative of the variableposition. Analysis of loss of heterozygosity was possible byutilizing single nucleotide polymorphisms.

    A modified allele-specific extension strategy for genotypingof single nucleotide polymorphisms has been developed. Throughthe use of a real-time bioluminometric assay, it has beendemonstrated that reaction kinetics for a mismatchedprimer-template is slower than the matched configuration,butthe end-point signals are comparable. By introduction ofapyrase, the problems associated with mismatch extensions havebeen circumvented and accurate data has been obtained.

    Keywords:fragment analysis, microsatellite, loss ofheterozygosity, DNA sequencing, pyrosequencing, cancer,mutation, variation, single nucleotide polymorphism,allele-specific extension, bioluminescence, apyrase.

  • 2.
    Ahmadian, Afshin
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    AnderssonSvahn, Helene
    KTH, School of Biotechnology (BIO), Nano Biotechnology (closed 20130101). KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Massively parallel sequencing platforms using lab on a chip technologies2011In: Lab on a Chip, ISSN 1473-0197, E-ISSN 1473-0189, Vol. 11, no 16, p. 2653-2655Article in journal (Refereed)
  • 3.
    Ahmadian, Afshin
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology.
    Ehn, M.
    Hober, Sophia
    KTH, School of Biotechnology (BIO), Proteomics.
    Pyrosequencing: History, biochemistry and future2006In: Clinica Chimica Acta, ISSN 0009-8981, E-ISSN 1873-3492, Vol. 363, no 02-jan, p. 83-94Article, review/survey (Refereed)
    Abstract [en]

    Background: Pyrosequencing is a DNA sequencing technology based on the sequencing-by-synthesis principle. Methods: The technique is built on a 4-enzyme real-time monitoring of DNA synthesis by bioluminescence using a cascade that upon nucleotide incorporation ends in a detectable light signal (bioluminescence). The detection system is based on the pyrophosphate released when a nucleotide is introduced in the DNA-strand. Thereby, the signal can be quantitatively connected to the number of bases added. Currently, the technique is limited to analysis of short DNA sequences exemplified by single-nucleotide polymorphism analysis and genotyping. 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. In order to expand the field for pyrosequencing, the read length needs to be improved. Conclusions: Th pyrosequencing system is based on an enzymatic system. There are different current and future applications of this technique.

  • 4.
    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.

  • 5.
    Ahmadian, Afshin
    et al.
    KTH, Superseded Departments, Biotechnology.
    Gharizadeh, B.
    O'Meara, D.
    Odeberg, Jacob
    KTH, Superseded Departments, Biotechnology.
    Lundeberg, Joakim
    KTH, Superseded Departments, Biotechnology.
    Genotyping by apyrase-mediated allele-specific extension2001In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 29, no 24Article in journal (Refereed)
    Abstract [en]

    This report describes a single-step extension approach suitable for high-throughput single-nucleotide polymorphism typing applications. The method relies on extension of paired allele-specific primers and we demonstrate that the reaction kinetics were slower for mismatched configurations compared with matched configurations. In our approach we employ apyrase, a nucleotide degrading enzyme, to allow accurate discrimination between matched and mismatched primer-template configurations. This apyrase-mediated allele-specific extension (AMASE) protocol allows incorporation of nucleotides when the reaction kinetics are fast (matched 3'-end primer) but degrades the nucleotides before extension when the reaction kinetics are slow (mismatched 3'-end primer). Thus, AMASE circumvents the major limitation of previous allele-specific extension assays in which slow reaction kinetics will still give rise to extension products from mismatched 3'-end primers, hindering proper discrimination. It thus represents a significant improvement of the allele-extension method. AMASE was evaluated by a bioluminometric assay in which successful incorporation of unmodified nucleotides is monitored in real-time using an enzymatic cascade.

  • 6.
    Ahmadian, Afshin
    et al.
    KTH, Superseded Departments, Biotechnology.
    Lundeberg, Joakim
    KTH, Superseded Departments, Biotechnology.
    A brief history of genetic variation analysis2002In: BioTechniques, ISSN 0736-6205, E-ISSN 1940-9818, Vol. 32, no 5, p. 1122-+Article, review/survey (Refereed)
    Abstract [en]

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

  • 7.
    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.

  • 8.
    Ahmadian, Afshin
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology.
    Ren, Z P
    Williams, Cecilia
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Pontén, F
    Odeberg, Jacob
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Pontén, J
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology.
    Genetic instability in the 9q22.3 region is a late event in the development of squamous cell carcinoma.1998In: Oncogene, ISSN 0950-9232, E-ISSN 1476-5594, Vol. 17, no 14Article in journal (Refereed)
    Abstract [en]

    Squamous cell carcinoma (SCC) of the skin represents a group of neoplasms which is associated with exposure to UV light. Recently, we obtained data suggesting that invasive skin cancer and its precursors derive from one original neoplastic clone. Here, the analysis were extended by loss of heterozygosity (LOH) analysis in the chromosome 9q22.3 region. A total of 85 samples, taken from twenty-two sections of sun-exposed sites, corresponding to normal epidermis, morphological normal cells with positive immuno-staining for the p53 protein (p53 patches), dysplasias, cancer in situ (CIS) and squamous cell carcinomas (SCC) of the skin were analysed. Overall, about 70% of p53 patches had mutations in the p53 gene but not LOH in the p53 gene or 9q22.3 region. Approximately 70% of the dysplasias showed p53 mutations of which about 40% had LOH in the p53 region but not in the 9q22.3 region. In contrast, about 65% of SCC and CIS displayed LOH in the 9q22.3 region, as well as frequent (80%) mutations and/or LOH in the p53 gene. These findings strongly suggest that alterations in the p53 gene is an early event in the progression towards SCC, whereas malignant development involves LOH and alterations in at least one (or several) tumor suppressor genes located in chromosome 9q22.3.

  • 9.
    Ahmadian, Afshin
    et al.
    KTH, Superseded Departments, Biotechnology.
    Russom, Aman
    KTH, Superseded Departments, Biotechnology.
    Andersson, Helene
    KTH, Superseded Departments, Biotechnology.
    Uhlén, Mathias
    KTH, Superseded Departments, Biotechnology.
    Stemme, Göran
    KTH, Superseded Departments, Biotechnology.
    Nilsson, Peter
    KTH, Superseded Departments, Biotechnology.
    SNP analysis by allele-specific extension in a micromachined filter chamber2002In: BioTechniques, ISSN 0736-6205, E-ISSN 1940-9818, Vol. 32, no 4, p. 748-754Article in journal (Refereed)
  • 10.
    Ardalan, Arman
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology.
    Oskarsson, Mattias
    KTH, School of Biotechnology (BIO), Gene Technology.
    Natanaelsson, Christian
    KTH, School of Biotechnology (BIO), Gene Technology.
    Wilton, Alan N.
    Ahmadian, Afshin
    KTH, School of Biotechnology (BIO), Gene Technology.
    Savolainen, Peter
    KTH, School of Biotechnology (BIO), Gene Technology.
    Narrow genetic basis for the Australian dingo confirmed through analysis of paternal ancestry2012In: Genetica, ISSN 0016-6707, E-ISSN 1573-6857, Vol. 140, no 1-3, p. 65-73Article in journal (Refereed)
    Abstract [en]

    The dingo (Canis lupus dingo) is an iconic animal in the native culture of Australia, but archaeological and molecular records indicate a relatively recent history on the continent. Studies of mitochondrial DNA (mtDNA) imply that the current dingo population was founded by a small population of already tamed dogs from Southeast Asia. However, the maternal genetic data might give a unilateral picture, and the gene pool has yet to be screened for paternal ancestry. We sequenced 14,437 bp of the Y-chromosome (Y-chr) from two dingoes and one New Guinea Singing Dog (NGSD). This positioned dingo and NGSD within the domestic dog Y-chr phylogeny, and produced one haplotype not detected before. With this data, we characterized 47 male dingoes in 30 Y-chr single-nucleotide polymorphism sites using protease-mediated allele-specific extension technology. Only two haplotypes, H3 and H60, were found among the dingoes, at frequencies of 68.1 and 31.9 %, respectively, compared to 27 haplotypes previously established in the domestic dog. While H3 is common among Southeast Asian dogs, H60 was specifically found in dingoes and the NGSD, but was related to Southeast Asian dog Y-chr haplotypes. H3 and H60 were observed exclusively in the western and eastern parts of Australia, respectively, but had a common range in Southeast. Thus, the Y-chr diversity was very low, similar to previous observations for d-loop mtDNA. Overall genetic evidence suggests a very restricted introduction of the first dingoes into Australia, possibly from New Guinea. This study further confirms the dingo as an isolated feral dog.

  • 11. Asplund, A.
    et al.
    Sivertsson, A.
    Backvall, H.
    Ahmadian, Afshin
    KTH, School of Biotechnology (BIO), Gene Technology.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology.
    Ponten, F.
    Genetic mosaicism in basal cell carcinoma2005In: Experimental dermatology, ISSN 0906-6705, E-ISSN 1600-0625, Vol. 14, no 8, p. 593-600Article in journal (Refereed)
    Abstract [en]

    Human basal cell cancer (BCC) shows unique growth characteristics, including a virtual inability to metastasize, absence of a precursor stage and lack of tumour progression. The clonal nature of BCC has long been a subject for debate because of the tumour growth pattern. Despite a morphologically multifocal appearance, genetic analysis and three-dimensional reconstructions of tumours have favoured a unicellular origin. We have utilized the X-chromosome inactivation assay in order to examine clonality in 13 cases of BCC. Four parts of each individual tumour plus isolated samples of stroma were analysed following laser-assisted microdissection. In 12/13 tumours, the epithelial component of the tumour showed a monoclonal pattern suggesting a unicellular origin. Surprisingly, one tumour showed evidence of being composed of at least two non-related monoclonal clones. This finding was supported by the analysis of the ptch and p53 gene. Clonality analysis of tumour stroma showed both mono- and polyclonal patterns. A prerequisite for this assay is that the extent of skewing is determined and compensated for in each case. Owing to the mosaic pattern of normal human epidermis, accurate coefficients are difficult to obtain; we, therefore, performed all analyses both with and without considering skewing. This study concludes that BCC are monoclonal neoplastic growths of epithelial cells, embedded in a connective tissue stroma at least in part of polyclonal origin. The study results show that what appears to be one tumour may occasionally constitute two or more independent tumours intermingled or adjacent to each other, possibly reflecting a local predisposition to malignant transformation.

  • 12.
    Borgström, Erik
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Redin, David
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Lundin, Sverker
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Berglund, Emelie
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Andersson, Anders F.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Ahmadian, Afshin
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Phasing of single DNA molecules by massively parallel barcoding2015In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 6, article id 7173Article in journal (Refereed)
    Abstract [en]

    High-throughput sequencing platforms mainly produce short-read data, resulting in a loss of phasing information for many of the genetic variants analysed. For certain applications, it is vital to know which variant alleles are connected to each individual DNA molecule. Here we demonstrate a method for massively parallel barcoding and phasing of single DNA molecules. First, a primer library with millions of uniquely barcoded beads is generated. When compartmentalized with single DNA molecules, the beads can be used to amplify and tag any target sequences of interest, enabling coupling of the biological information from multiple loci. We apply the assay to bacterial 16S sequencing and up to 94% of the hypothesized phasing events are shown to originate from single molecules. The method enables use of widely available short-read-sequencing platforms to study long single molecules within a complex sample, without losing phase information.

  • 13.
    Dezfouli, Mahya
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology.
    Magnusson, M.
    Arvestad, L.
    Lohi, H.
    Van Asch, D.
    Fain, S.
    Kennedy, L. J.
    Ahmadian, Afshin
    KTH, School of Biotechnology (BIO), Gene Technology.
    Savolainen, Peter
    KTH, School of Biotechnology (BIO), Gene Technology.
    Massively Parallel MHC-Typing by Sequencing RevealedNovel Variants of Canine Leukocyte AntigenManuscript (preprint) (Other academic)
  • 14.
    Dezfouli, Mahya
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology.
    Redin, David
    KTH, School of Biotechnology (BIO), Protein Technology.
    Borgström, Erik
    KTH, School of Biotechnology (BIO), Gene Technology.
    Edfors, Fredrik
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Schwenk, Jochen M.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Ahmadian, Afshin
    KTH, School of Biotechnology (BIO), Gene Technology.
    Droplet-based Immuno-Sequencing to Deconvolute Affinity Recognition EventsManuscript (preprint) (Other academic)
  • 15.
    Dezfouli, Mahya
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Vickovic, Sanja
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Iglesias, Maria Jesus
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Nilsson, Peter
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Schwenk, Jochen M.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Ahmadian, Afshin
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Magnetic bead assisted labeling of antibodies at nanogram scale2014In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 14, no 1, p. 14-18Article in journal (Refereed)
    Abstract [en]

    There are currently several initiatives that aim to produce binding reagents for proteome-wide analysis. To enable protein detection, visualization, and target quantification, covalent coupling of reporter molecules to antibodies is essential. However, current labeling protocols recommend considerable amount of antibodies, require antibody purity and are not designed for automation. Given that small amounts of antibodies are often sufficient for downstream analysis, we developed a labeling protocol that combines purification and modification of antibodies at submicrogram quantities. With the support of magnetic microspheres, automated labeling of antibodies in parallel using biotin or fluorescent dyes was achieved.

  • 16.
    Dezfouli, Mahya
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Vickovic, Sanja
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Iglesias, Maria Jesus
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Schwenk, Jochen M.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Ahmadian, Afshin
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Parallel barcoding of antibodies for DNA-assisted proteomics2014In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 14, no 21-22, p. 2432-2436Article in journal (Refereed)
    Abstract [en]

    DNA-assisted proteomics technologies enable ultra-sensitive measurements in multiplex format using DNA-barcoded affinity reagents. Although numerous antibodies are available, nowadays targeting nearly the complete human proteome, the majority is not accessible at the quantity, concentration, or purity recommended for most bio-conjugation protocols. Here, we introduce a magnetic bead-assisted DNA-barcoding approach, applicable for several antibodies in parallel, as well as reducing required reagents quantities up to a thousand-fold. The success of DNA-barcoding and retained functionality of antibodies were demonstrated in sandwich immunoassays and standard quantitative Immuno-PCR assays. Specific DNA-barcoding of antibodies for multiplex applications was presented on suspension bead arrays with read-out on a massively parallel sequencing platform in a procedure denoted Immuno-Sequencing. Conclusively, human plasma samples were analyzed to indicate the functionality of barcoded antibodies in intended proteomics applications.

  • 17. Ehn, M.
    et al.
    Ahmadian, Afshin
    KTH, Superseded Departments, Biotechnology.
    Nilsson, Peter
    KTH, Superseded Departments, Biotechnology.
    Lundeberg, Joakim
    KTH, Superseded Departments, Biotechnology.
    Hober, Sophia
    KTH, Superseded Departments, Biotechnology.
    Escherichia coli single-stranded DNA-binding a molecular tool for improved sequence protein quality in pyrosequencing2002In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 23, no 19, p. 3289-3299Article in journal (Refereed)
    Abstract [en]

    Pyrosequencing is a four-enzyme bioluminometric DNA sequencing technique based on a DNA sequencing by synthesis principle. Currently, the technique is limited to analysis of short DNA sequences exemplified by single-nucleotide polymorphism analysis. In order to expand the field for pyrosequencing, the read length needs to be improved and efforts have been made to purify reaction components as well as add single-stranded DNA-binding protein (SSB) to the pyrosequencing reaction. In this study, we have performed a systematic effort to analyze the effects of SSB by comparing the pyrosequencing result of 103 independent complementary DNA (cDNA) clones. More detailed information about the cause of low quality sequences on templates with different characteristics was achieved by thorough analysis of the pyrograms. Also, real-time biosensor analysis was performed on individual cDNA clones for investigation of primer annealing and SSB binding on these templates. Results from these studies indicate that templates with high performance in pyrosequencing without SSB possess efficient primer annealing and low SSB affinity. Alternative strategies to improve the performance in pyrosequencing by increasing the primer-annealing efficiency have also been evaluated.

  • 18.
    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.

  • 19. Ericsson, O.
    et al.
    Sivertsson, A.
    Lundeberg, Joakim
    KTH, Superseded Departments, Biotechnology.
    Ahmadian, Afshin
    KTH, Superseded Departments, Biotechnology.
    Microarray-based resequencing by apyrase-mediated allele-specific extension2003In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 24, no 19-20, p. 3330-3338Article in journal (Refereed)
    Abstract [en]

    We have developed an array-based resequencing method to determine genetic alterations in putative cancer genes. The method relies on that the specificity of DNA polymerase in allele-specific extensions can be enhanced by terminating the extension reactions with apyrase and that a tiling set of primers are synthesized covering the investigated gene sequence. We report on such apyrase-mediated allele-specific primer extension (AMASE) assay as a method suitable for high-throughout resequencing and mutation detection in tumor suppressor genes and oncogenes. In the experimental setup, primers complementary to codons 12, 13 and codon 61 of the N-ras proto-oncogene were spotted onto glass slides. Overlapping sense and anti-sense primers were designed so that complementary primers for all possible mutations in each base position were investigated. The extension reactions were performed in a single step following hybridization of target DNA to the immobilized primers on the array surface. Mutation detection limits and the possibility of quantifying the mutations were investigated using synthetic oligonucleotides. In addition, 64 clinical samples were sequenced and 16 of these showed mutations in the N-ras gene.

  • 20. 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.

  • 21. 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.

  • 22. 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.

  • 23.
    Gharizadeh, Baback
    et al.
    KTH, Superseded Departments, Biotechnology.
    Käller, Max
    KTH, Superseded Departments, Biotechnology.
    Nyrén, Pål
    KTH, Superseded Departments, Biotechnology.
    Andersson,, Anders F.
    KTH, Superseded Departments, Biotechnology.
    Uhlén, Mathias
    KTH, Superseded Departments, Biotechnology.
    Lundeberg, Joakim
    KTH, Superseded Departments, Biotechnology.
    Ahmadian, Afshin
    KTH, Superseded Departments, Biotechnology.
    Viral and microbial genotyping by a combination of multiplex competitive hybridization and specific extension followed by hybridization to generic tag arrays2003In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 31, no 22, p. e146-Article in journal (Refereed)
    Abstract [en]

     Detection and identification of microbial pathogens are important for disease diagnosis, treatment and prophylaxis measurements. By introducing an innovative technique, we show a robust, reliable and accurate microarray-based method for identification of microbial pathogens. The technique utilizes a unique combination of multiplex competitive hybridization, which enhances hybridization accuracy of oligonucleotides to the specific target, and apyrase-mediated allele-specific extension, which improves specific extension. As a model system, different clinically relevant human papillomaviruses were selected for this study. The method generated accurate results and proves to be promising for specific and correct microbial and viral typing.

  • 24. Gustafsson, A. C.
    et al.
    Guo, Z. M.
    Hu, X. R.
    Ahmadian, Afshin
    KTH, Superseded Departments, Biotechnology.
    Brodin, B.
    Nilsson, A.
    Ponten, J.
    Ponten, F.
    Lundeberg, Joakim
    KTH, Superseded Departments, Biotechnology.
    HPV-related cancer susceptibility and p53 codon 72 polymorphism2001In: Acta Dermato-Venereologica, ISSN 0001-5555, E-ISSN 1651-2057, Vol. 81, no 2, p. 125-129Article in journal (Refereed)
    Abstract [en]

    Conflicting results regarding the association of a polymorphism at codon 72 of the p53 tumour suppressor gene and susceptibility to develop human papilloma virus (HPV)-associated cervical cancer have been published over the last year, implicating differences in ethnic background, sample origin, sample size and/or detection assay, The material for this study was collected in the identical geographical region as for 2 previous reports with contradictory results regarding the association of codon 72 genotype with squamous cell cancer (SCC), We have used an alternative detection assay, based on pyrosequencing technology, that interrogates the variable position by the accuracy of DNA polymerase. In addition to cervical clinical specimens from SCC, HPV16- and HPV18-infected adenocarcinoma cases as well as cervical intraepithelial neoplasia (CM) were investigated. No significant association was found between p53 codon 72 genotype and the risk to develop adenocarcinoma, SCC or CIN in the Swedish population.

  • 25. Hoiom, Veronica
    et al.
    Tuominen, Rainer
    Käller, Max
    KTH, School of Biotechnology (BIO), Gene Technology.
    Linden, Diana
    Ahmadian, Afshin
    KTH, School of Biotechnology (BIO), Gene Technology.
    Mansson-Brahme, Eva
    Egyhazi, Suzanne
    Sjöberg, Klas
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology.
    Hansson, Johan
    MC1R variation and melanoma risk in the Swedish population in relation to clinical and pathological parameters2009Article in journal (Refereed)
    Abstract [en]

    The genetic background of cutaneous malignant melanoma (CMM) includes both germ line aberrations in high-penetrance genes, like CDKN2A, and allelic variation in low-penetrance genes like the melanocortin-1 receptor gene, MC1R. Red-hair colour associated MC1R alleles (RHC) have been associated with red hair, fair skin and risk of CMM. We investigated MC1R and CDKN2A variation in relation to phenotype, clinical factors and CMM risk in the Swedish population. The study cohort consisted of sporadic primary melanoma patients, familial melanoma patients and a control group. An allele-dose dependent increase in melanoma risk for carriers of variant MC1R alleles (after adjusting for phenotype), with an elevated risk among familial CMM patients, was observed. This elevated risk was found to be significantly associated with an increased frequency of dysplastic nevi (DN) among familial patients compared to sporadic patients. MC1R variation was found to be less frequent among acral lentiginous melanomas (ALM) and dependent on tumour localisation. No association was found between CDKN2A gene variants and general melanoma risk. Two new variants in the POMC gene were identified in red haired individuals without RHC alleles.

  • 26.
    Hultin, Emelie
    et al.
    KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Käller, Max
    KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Ahmadian, Afshin
    KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Competitive enzymatic reaction to control allele-specific extensions2005In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 33, no 5, p. e48:1-e48:10Article in journal (Refereed)
    Abstract [en]

    Here, we present a novel method for SNP genotyping based on protease-mediated allele-specific primer extension (PrASE), where the two allele-specific extension primers only differ in their 3'-positions. As reported previously [Ahmadian, A., Gharizadeh, B., O'Meara, D., Odeberg, J. and Lundeberg, J. (2001), Nucleic Acids Res., 29, e121], the kinetics of perfectly matched primer extension is faster than mismatched primer extension. In this study, we have utilized this difference in kinetics by adding protease, a protein-degrading enzyme, to discriminate between the extension reactions. The competition between the polymerase activity and the enzymatic degradation yields extension of the perfectly matched primer, while the slower extension of mismatched primer is eliminated. To allow multiplex and simultaneous detection of the investigated single nucleotide polymorphisms (SNPs), each extension primer was given a unique signature tag sequence on its 50 end, complementary to a tag on a generic array. A multiplex nested PCR with 13 SNPs was performed in a total of 36 individuals and their alleles were scored. To demonstrate the improvements in scoring SNPs by PrASE, we also genotyped the individuals without inclusion of protease in the extension. We conclude that the developed assay is highly allele-specific, with excellent multiplex SNP capabilities.

  • 27.
    Hultin, Emilie
    et al.
    KTH, School of Biotechnology (BIO).
    Asplund, Anna
    KTH, School of Biotechnology (BIO).
    Berggren, Lovisa
    KTH, School of Biotechnology (BIO).
    Edlund, Karolina
    KTH, School of Biotechnology (BIO).
    Ahmadian, Afshin
    KTH, School of Biotechnology (BIO).
    Sundberg, Rolf
    Pontén, Fredrik
    KTH, School of Biotechnology (BIO).
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO).
    Random loss of genetic segments during skin differentiation indicated by analysis of single cellsArticle in journal (Other academic)
  • 28.
    Johansson, Sebastian
    et al.
    Stockholms Universitet.
    Juhos, Szilveszter
    Stockholms Universitet.
    Redin, David
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Gene Technology.
    Ahmadian, Afshin
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Gene Technology.
    Käller, Max
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Gene Technology.
    Comprehensive haplotyping of the HLA gene family using nanopore sequencingManuscript (preprint) (Other academic)
    Abstract [en]

    The HLA gene family is the most polymorphic loci in the human genome; it encodes for the major histocompatibility complexes (MHC) which mediates the immune response in terms of cellular interactions with antigens. Compatibility between HLA alleles is thus of great medical interest for recipients of allogeneic transplantations. Traditional serological techniques to evaluate compatibility are now being replaced by more accurate DNA sequencing-based methods. However, short read sequencing data typically result in collapsed sequences representing a mixture of variants from native haplotypes. In addition, most previous studies have been limited to a few highly polymorphic exons of various HLA genes. Here we present haplotype-resolved full-length sequencing of the six most clinically relevant MHC Class I and Class II genes, to characterize the haplotypes of eight reference individuals, using a single MinION flow cell. The results show that full-length sequencing of single molecules enables haplotypes to be resolved to the highest degree of accuracy (four-field resolution). In this study, a majority of the alleles were classified with four-field resolution and could be verified through previously published genotyping studies. These results support the notion that nanopore sequencing could be a viable solution for highly accurate clinical evaluation of histocompatibility.

  • 29.
    Käller, Max
    et al.
    KTH, Superseded Departments, Biotechnology.
    Ahmadian, Afshin
    KTH, Superseded Departments, Biotechnology.
    Lundeberg, Joakim
    KTH, Superseded Departments, Biotechnology.
    Microarray-based AMASE as a novel approach for mutation detection2004In: Mutation research, ISSN 0027-5107, E-ISSN 1873-135X, Vol. 554, no 1-2, p. 77-88Article in journal (Refereed)
    Abstract [en]

    Alterations in the p53 tumor suppressor gene are important events in many cases of human cancers. We have developed a novel microarray based approach for re-sequencing and mutation detection of the p53 gene. The method facilitates rapid and simple scanning of the target gene sequence and could be expanded to include other candidate cancer genes. The methodology employs the previously described apyrase-mediated allele-specific extension reaction (AMASE). In order to re-sequence the selected region, four extension oligonucleotides with different 3'-termini were used for each base position and they were covalently attached to the glass slide's surface. The amplified single-stranded DNA templates were then hybridized to the array followed by in situ extension with fluorescently labeled dNTPs in the presence of apyrase. The model system used was based on analysis of a 15 bp stretch in exon 5 of the p53 gene. Mutations were scored as allelic fractions calculated as (wt)/(wt + mut) signals. When apyrase was included in the extension reactions of wild type templates, the mean allelic fraction was 0.96. When apyrase was excluded with the same wild type templates, significantly lower allelic fractions were obtained. Two 60-mer synthetic oligonucleotides were used to establish the detectable amount of mutations with AMASE and a clear distinction between all the points could be made. Several samples from different stages of skin malignancies were also analyzed. The results from this study imply the possibility to efficiently and accurately re-sequence the entire p53 gene with AMASE technology.

  • 30.
    Käller, Max
    et al.
    KTH, School of Biotechnology (BIO).
    Hultin, Emelie
    KTH, School of Biotechnology (BIO).
    Zheng, B.
    Department of Molecular Medicine, Karolinska Institutet.
    Gharizadeh, Baback
    KTH, School of Biotechnology (BIO).
    Wallin, K.-L.
    Department of Molecular Medicine, Karolinska Institutet.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO).
    Ahmadian, Afshin
    KTH, School of Biotechnology (BIO).
    Tag-array based HPV genotyping by competitive hybridization and extension2005In: Journal of Virological Methods, ISSN 0166-0934, E-ISSN 1879-0984, Vol. 129, no 2, p. 102-112Article in journal (Refereed)
    Abstract [en]

    A method is described for HPV genotyping based on multiplex competitive hybridization (MUCH) combined with apyrase mediated allele-specific extension (AMASE). Two type-specific oligonucleotides were designed for each of the 23 investigated HPV types and directed towards two highly inter-type heterogeneous regions. The type-specific oligonucleotides were allowed to compete in the hybridization to an immobilized template resulting in a highly specific hybridization process. To increase further the specificity, a second step of type discrimination was used in which specific extension of 3'-termini matched oligonucleotides was performed. The 46 type-specific oligonucleotides each had a unique tag sequence to allow detection via an array of oligonucleotides complementary to the tags. To evaluate the genotyping assay, a total of 92 HPV positive samples were tested in this study. Twelve had double infections and five had three to five coexisting HPV types. The results show that MUCH-AMASE can readily detect multiple infections, whereas conventional dideoxy sequencing resulted in ambiguous sequence. Four samples with three to five genotypes detected were cloned and individual clones were sequenced. The cloning procedure verified the MUCH-AMASE results with indications that we can find minor infections (< 2% relative amounts). We can thus conclude that the developed assay is highly sensitive, with improved throughput and with excellent possibility to detect multiple infections.

  • 31.
    Käller, Max
    et al.
    KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Hultin, Emilie
    KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Holmberg, Kristina
    KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Persson, Marie-Louise
    Clinical Chemistry Laboratory, Blekinge Hospital, Karlskrona.
    Odeberg, Jacob
    KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Ahmadian, Afshin
    KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Comparison of PrASE and Pyrosequencing for SNP Genotyping2006In: BMC Genomics, ISSN 1471-2164, E-ISSN 1471-2164, Vol. 7, p. 291-Article in journal (Refereed)
    Abstract [en]

    Background: There is an imperative need for SNP genotyping technologies that are cost-effective per sample with retained high accuracy, throughput and flexibility. We have developed a microarray-based technique and compared it to Pyrosequencing. In the protease-mediated allele-specific extension (PrASE), the protease constrains the elongation reaction and thus prevents incorrect nucleotide incorporation to mismatched 3'-termini primers.

    Results: The assay is automated for 48 genotyping reactions in parallel followed by a tag-microarray detection system. A script automatically visualizes the results in cluster diagrams and assigns the genotypes. Ten polymorphic positions suggested as prothrombotic genetic variations were analyzed with Pyrosequencing and PrASE technologies in 442 samples and 99.8 % concordance was achieved. In addition to accuracy, the robustness and reproducibility of the technique has been investigated.

    Conclusion: The results of this study strongly indicate that the PrASE technology can offer significant improvements in terms of accuracy and robustness and thereof increased number of typeable SNPs.

  • 32. Käller, Max
    et al.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology.
    Ahmadian, Afshin
    KTH, School of Biotechnology (BIO), Gene Technology.
    Arrayed identification of DNA signatures2007In: Expert Review of Molecular Diagnostics, ISSN 1473-7159, Vol. 7, no 1, p. 65-76Article, review/survey (Refereed)
    Abstract [en]

    Over the last few years, several initiatives have described efforts to combine previously invented techniques in molecular biology with parallel detection principles to sequence or genotype DNA signatures. The Infinium (R) system from Illumina and the Affymetrix GeneChips (R) are two systems suitable for whole-genome scoring of variable positions. However, directed candidate-gene approaches are more cost effective and several academic groups and the private sector provide techniques with moderate typing throughput combined with large sample capacity suiting these needs. Recently, whole-genome sequencing platforms based on the sequencing-by-synthesis principle were presented by 454 Life Sciences and Solexa, showing great potential as alternatives to conventional genotyping approaches. In addition to these sequencing initiatives, many efforts are pursuing novel ideas to facilitate fast and cost-effective whole genome sequencing, such as ligation-based sequencing. Reliable methods for routine resequencing of human genomes as a tool for personalized medicine, however, remain to be developed.

  • 33.
    Käller, Max
    et al.
    KTH, Superseded Departments, Biotechnology.
    Magnusson, Veronica
    Tuominen, Rainer
    Månsson-Brahme, Eva
    Ahmadian, Afshin
    KTH, Superseded Departments, Biotechnology.
    Egyhazi, Suzanne
    Lundeberg, Joakim
    KTH, Superseded Departments, Biotechnology.
    Hansson, Johan
    Genetic variation of the MC1R gene and the risk for cutaneous malignant melanoma in the Swedish populationManuscript (Other academic)
  • 34.
    Käller, Max
    et al.
    KTH, School of Biotechnology (BIO).
    Tuominen, Rainer
    Karolinska Institutet.
    Ahmadian, Afshin
    KTH, School of Biotechnology (BIO).
    Magnusson, Veronica
    Karolinska Institutet.
    Egyhazi, Suzanne
    Karolinska Institutet.
    Hansson, Johan
    Karolinska Institutet.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO).
    Detection of MC1R Polymorphisms with Protease-Mediated Allele-Specific Extension as an Alternative to Direct Sequencing2005In: Clinical Chemistry, ISSN 0009-9147, E-ISSN 1530-8561, Vol. 51, no 12, p. 2388-2391Article in journal (Refereed)
  • 35.
    Lindström, Sara
    et al.
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Hammond, Maria
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Andersson-Svahn, Helene
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Ahmadian, Afshin
    KTH, School of Biotechnology (BIO), Gene Technology.
    PCR amplification and genetic analysis in a microwell cell culturing chip2009In: Lab on a Chip, ISSN 1473-0197, E-ISSN 1473-0189, p. 3465-3471Article in journal (Refereed)
    Abstract [en]

    We have previously described a microwell chip designed for high throughput, long-term single-cell culturing and clonal analysis in individual wells providing a controlled way of studying high numbers of individual adherent or non-adherent cells. Here we present a method for the genetic analysis of cells cultured on-chip by PCR and minisequencing, demonstrated using two human adherent cell lines: one wild type and one with a single-base mutation in the p53 gene. Five wild type or mutated cells were seeded per well (in a defined set of wells, each holding 500 nL of culture medium) in a 672-microwell chip. The cell chip was incubated overnight, or cultured for up to five days, depending on the desired colony size, after which the cells were lysed and subjected to PCR directly in the wells. PCR products were detected, in the wells, using a biotinylated primer and a fluorescently labelled primer, allowing the products to be captured on streptavidin-coated magnetic beads and detected by a fluorescence microscope. In addition, to enable genetic analysis by minisequencing, the double-stranded PCR products were denatured and the immobilized strands were kept in the wells by applying a magnetic field from the bottom of the wells while the wells were washed, a minisequencing reaction mixture was added, and after incubation in appropriate conditions the expected genotypes were detected in the investigated microwells, simultaneously, by an array scanner. We anticipate that the technique could be used in mutation frequency screening, providing the ability to correlate cells' proliferative heterogeneity to their genetic heterogeneity, in hundreds of samples simultaneously. The presented method of single-cell culture and DNA amplification thus offers a potentially powerful alternative to single-cell PCR, with advantageous robustness and sensitivity

  • 36.
    Lindström, Sara
    et al.
    KTH, School of Biotechnology (BIO), Nano Biotechnology (closed 20130101).
    Hammond, Maria
    KTH, School of Biotechnology (BIO), Nano Biotechnology (closed 20130101).
    Gantelius, Jesper
    KTH, School of Biotechnology (BIO), Nano Biotechnology (closed 20130101).
    Ahmadian, Afshin
    KTH, School of Biotechnology (BIO), Gene Technology.
    Andersson-Svahn, Helene
    KTH, School of Biotechnology (BIO), Nano Biotechnology (closed 20130101).
    PCR amplification and genetic analysis in a microwell cell cultivation chip2008In: 12th International Conference on Miniaturized Systems for Chemistry and Life Sciences - The Proceedings of MicroTAS 2008 Conference, Chemical and Biological Microsystems Society , 2008, p. 576-578Conference paper (Refereed)
    Abstract [en]

    We present a method for long-term single cell/clone cultivation followed by cell lysis, DNA amplification and detection of PCR product in a chip containing 672 individual microwells. By performing all steps on-chip in microwells, the proliferation and cell morphology of every single cell or clone can be linked to its genetic information. In this study two mammalian cell lines (mutated A431 vs. wild type U-2 OS) were used as a model system for mutation screening in the p53 gene. The presented method could improve the sensitivity in mutation frequency analysis of heterogeneous tumor samples.

  • 37. Ling, G.
    et al.
    Ahmadian, Afshin
    KTH, Superseded Departments, Biotechnology.
    Persson, A.
    Unden, A. B.
    Afink, G.
    Williams, C.
    Uhlén, Mathias
    KTH, Superseded Departments, Biotechnology.
    Toftgard, R.
    Lundeberg, Joakim
    KTH, Superseded Departments, Biotechnology.
    Ponten, F.
    PATCHED and p53 gene alterations in sporadic and hereditary basal cell cancer2001In: Oncogene, ISSN 0950-9232, E-ISSN 1476-5594, Vol. 20, no 53, p. 7770-7778Article in journal (Refereed)
    Abstract [en]

    It is widely accepted that disruption of the hedgehog-patched pathway is a key event in development of basal cell cancer. In addition to patched gene alterations, p53 gene mutations are also frequent in basal cell cancer. We determined loss of heterozygosity in the patched and p53 loci as well as sequencing the p53 gene in tumors both from sporadic and hereditary cases. A total of 70 microdissected samples from tumor and adjacent skin were subjected to PCR followed by fragment analysis and DNA sequencing. We found allelic loss in the patched locus in 6/8 sporadic basal cell cancer and 17/19 hereditary tumors. All sporadic and 7/20 hereditary tumors showed p53 gene mutations. Loss of heterozygosity in the p53 locus was rare in both groups. The p53 mutations detected in hereditary tumors included rare single nucleotide deletions and unusual double-base substitutions compared to the typical ultraviolet light induced missense mutations found in sporadic tumors. Careful microdissection of individual tumors revealed genetically linked subclones with different p53 and/or patched genotype providing an insight on time sequence of genetic events. The high frequency and co-existence of genetic alterations in the patched and p53 genes suggest that both these genes are important in the development of basal cell cancer.

  • 38.
    Neiman, Mårten
    et al.
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Lundin, Sverker
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Savolainen, Peter
    KTH, School of Biotechnology (BIO), Molecular Biotechnology (closed 20130101).
    Ahmadian, Afshin
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Decoding a substantial set of samples in parallel by massive sequencing2011In: Plos One, ISSN 1932-6203, Vol. 6, no 3Article in journal (Refereed)
    Abstract [en]

    The dramatic increase of throughput seen in the eld of sequenceanalysis during the last years has opened up new possibilities of se-quencing a multitude of samples in parallel. Here we present a novelstrategy where the combination of two tags is used to link reads totheir origins in a pool of samples. The two tags are incorporated intwo steps leading to lowering of sample handling complexity by nearly100 times. The method described here enables accurate identi cationand typing of thousands of samples in parallel and is scalable. In thisstudy the system was designed to test 4992 samples using only 122 tags.

    To proof the concept of two tagging method the highly polymor-phic 2nd exon of DLA-DRB1 in dogs and wolves was sequenced usingthe 454 GS FLX Titanium Chemistry. By requiring a minimum se-quence depth of 20 reads per sample, 94% of the successfully ampli edsamples were genotyped. In addition, the method allowed digital de-tection of chimeric fragments. These results demonstrate that it ispossible to sequence thousands of samples in parallel without complexpooling patterns or primer combinations. Furthermore, the method isscalable and increasing the sample size by 960 samples requires only10 additional tags.

  • 39. Norlén, Hedvig
    et al.
    Pettersson, Erik
    KTH, School of Biotechnology (BIO).
    Ahmadian, Afshin
    KTH, School of Biotechnology (BIO).
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO).
    Sundberg, Rolf
    Classification of SNP genotypes by a Gaussian mixture modelManuscript (Other academic)
  • 40.
    Odeberg, Jacob
    et al.
    KTH, Superseded Departments, Biotechnology.
    Ahmadian, Afshin
    KTH, Superseded Departments, Biotechnology.
    Williams, C
    Pontén, F.
    Uhlén, Mathias
    KTH, Superseded Departments, Biotechnology.
    Lundeberg, Joakim
    KTH, Superseded Departments, Biotechnology.
    Direct sequencing of the ZNF 189 gene promotor reveals artifact hot spot mutations.Manuscript (preprint) (Other academic)
  • 41.
    Odeberg, Jacob
    et al.
    KTH, Superseded Departments (pre-2005), Biotechnology.
    Røsok, O
    Gudmundsson, G H
    Ahmadian, Afshin
    KTH, Superseded Departments (pre-2005), Biotechnology.
    Roshani, L
    Williams, C
    Larsson, C
    Pontén, F
    Uhlén, Mathias
    KTH, Superseded Departments (pre-2005), Biotechnology.
    Asheim, H C
    Lundeberg, Joakim
    KTH, Superseded Departments (pre-2005), Biotechnology.
    Cloning and characterization of ZNF189, a novel human Krüppel-like zinc finger gene localized to chromosome 9q22-q31.1998In: Genomics, ISSN 0888-7543, E-ISSN 1089-8646, Vol. 50, no 2, p. 213-21Article in journal (Refereed)
    Abstract [en]

    A 3-kb-long cDNA encoding a Krüppel-like human zinc finger protein was isolated and mapped to chromosome 9q22-q31. The ZNF189 gene encodes a protein with 16 zinc fingers at its C-terminus and belongs to the Krüppel-associated box (KRAB)-containing group of zinc finger proteins. Four differently spliced cDNA transcripts, differing at the 5' coding region where a KRAB A repressor domain is encoded, were isolated. In addition, Northern blot analysis indicates the presence of two additional unidentified splice variants. Comparison of cDNA and genomic sequences shows that the ZNF189 gene spans approximately 11 kb and is organized into at least four exons, the large 3'-end exon coding for the complete zinc finger domain and the 3' untranslated region. ZNF189 is expressed in all tissues and cell types currently investigated, at varying levels, but with a tissue- or cell-type-restricted expression pattern for the different splice variants. ZNF189 is conserved in the genome of several mammalian species. Direct sequencing of the ZNF189 gene in microdissected tumor biopsies of sporadic basal cell carcinoma and squamous cell carcinoma reveals no mutations in the coding sequence or at exon/intron boundaries.

  • 42. O'Meara, D.
    et al.
    Ahmadian, Afshin
    KTH, Superseded Departments, Biotechnology.
    Odeberg, Jacob
    KTH, Superseded Departments, Biotechnology.
    Lundeberg, Joakim
    KTH, Superseded Departments, Biotechnology.
    SNP typing by apyrase-mediated allele-specific primer extension on DNA microarrays2002In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 30, no 15Article in journal (Refereed)
    Abstract [en]

    This study reports the development of a microarray-based allele-specific extension method for typing of single nucleotide polymorphisms (SNPs). The use of allele-specific primers has been employed previously to identify single base variation but it is acknowledged that certain mismatches are not refractory to extension. Here we have overcome this limitation by introducing apyrase, a nucleotide-degrading enzyme, to the extension reaction. We have show previously that DNA polymerases exhibit slower reaction kinetics when extending a mismatched primer compared with a matched primer. This kinetic difference is exploited in the apyrase-mediated allele-specific extension (AMASE) assay, allowing incorporation of nucleotides when the reaction kinetics are fast but degrading the nucleotides before extension when the reaction kinetics are slow. Here we show that five homozygous variants (14% of the total number of variants) that were incorrectly scored in the absence of apyrase were correctly typed when apyrase was included in the extension reaction. AMASE was performed in situ on the oligonucleotide microarrarys using fluorescent nucleotides to type 10 SNPs and two indels in 17 individuals generating approximately 200 genotypes. Cluster analysis of these data shows three distinct clusters with clear-cut boundaries. We conclude that SNP typing on oligonucleotide microarrays by AMASE is an efficient, rapid and accurate technique for large-scale genotyping.

  • 43.
    Pettersson, Erik
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Ahmadian, Afshin
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Ståhl, Patrik L.
    A Novel Method for Rapid Hybridization of DNA to a Solid Support2013In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 8, p. e70504-Article in journal (Refereed)
    Abstract [en]

    Here we present a novel approach entitled Magnetic Forced Hybridization (MFH) that provides the means for efficient and direct hybridization of target nucleic acids to complementary probes immobilized on a glass surface in less than 15 seconds at ambient temperature. In addition, detection is carried out instantly since the beads become visible on the surface. The concept of MFH was tested for quality control of array manufacturing, and was combined with a multiplex competitive hybridization (MUCH) approach for typing of Human Papilloma Virus (HPV). Magnetic Forced Hybridization of bead-DNA constructs to a surface achieves a significant reduction in diagnostic testing time. In addition, readout of results by visual inspection of the unassisted eye eliminates the need for additional expensive instrumentation. The method uses the same set of beads throughout the whole process of manipulating and washing DNA constructs prior to detection, as in the actual detection step itself.

  • 44.
    Pettersson, Erik
    et al.
    KTH, School of Biotechnology (BIO).
    Lindskog, Mats
    KTH, School of Biotechnology (BIO).
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO).
    Ahmadian, Afshin
    KTH, School of Biotechnology (BIO).
    Tri-nucleotide Threading for parallel amplification of minute amounts of genomic DNA2006In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 34, no 6, p. 9-Article in journal (Refereed)
    Abstract [en]

    Efforts to correlate genetic variations with phenotypic differences are intensifying due to the availability of high-density maps of single nucleotide polymorphisms (SNPs) and the development of high throughput scoring methods. These recent advances have led to an increased interest for improved multiplex preparations of genetic material to facilitate such whole genome analyses. Here we propose a strategy for the parallel amplification of polymorphic loci based on a reduced set of nucleotides. The technique denoted Tri-nucleotide Threading (TnT), allows SNPs to be amplified via controlled linear amplification followed by complete removal of the target material and subsequent amplification with a pair of universal primers. A dedicated software tool was developed for this purpose and variable positions in genes associated with different forms of cancer were analyzed using sub-nanogram amounts of starting material. The amplified fragments were then successfully scored using a microarray-based PrASE technique. The results of this study, in which 75 SNPs were analyzed, show that the TnT technique circumvents potential problems associated with multiplex amplification of SNPs from minute amounts of material. The technique is specific, sensitive and can be readily adapted to equipment and genotyping techniques used in other research laboratories without requiring changes to the preferred typing method.

  • 45.
    Pettersson, Erik
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology.
    Ahmadian, Afshin
    KTH, School of Biotechnology (BIO), Gene Technology.
    Generations of sequencing technologies2009In: Genomics, ISSN 0888-7543, E-ISSN 1089-8646, Vol. 93, no 2, p. 105-111Article, review/survey (Refereed)
    Abstract [en]

    Advancements in the field of DNA sequencing are changing the scientific horizon and promising an era of personalized medicine for elevated human health. Although platforms are improving at the rate of Moore's Law, thereby reducing the sequencing costs by a factor of two or three each year, we find ourselves at a point in history where individual genomes are starting to appear but where the cost is still too high for routine sequencing of whole genomes. These needs will be met by miniaturized and parallelized platforms that allow a lower sample and template consumption thereby increasing speed and reducing costs. Current massively parallel, state-of-the-art systems are providing significantly improved throughput over Sanger systems and future single-molecule approaches will continue the exponential improvements in the field.

  • 46.
    Pettersson, Erik
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology.
    Ståhl, Patrik L.
    KTH, School of Biotechnology (BIO), Gene Technology.
    Mahdessian, Hovsep
    KTH, School of Biotechnology (BIO), Gene Technology.
    Käller, Max
    KTH, School of Biotechnology (BIO), Gene Technology.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology.
    Ahmadian, Afshin
    KTH, School of Biotechnology (BIO), Gene Technology.
    Visual DNA as a diagnostic tool2009In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 30, no 21, p. 3691-3695Article in journal (Refereed)
    Abstract [en]

    We report on the incorporation of the Visual DNA concept in a genotyping assay as a simple and straightforward detection tool. The principle of trapping streptavidin-coated superparamagnetic beads of micrometer size for visualization of genetic variances is used for PrASE-based detection of a panel of mutations in the severe and common genetic disorder of cystic fibrosis. The method allows a final investigation of genotypes by the naked eye and the output is easily documented using a regular hand-held device with an integrated digital camera. A number of samples were run through the assay, showing rapid and accurate detection using superparamagnetic beads and an off-the-shelf neodymium magnet. The assay emphasizes the power of Visual DNA and demonstrates the potential value of the method in future point-of-care tests.

  • 47.
    Pettersson, Erik
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology.
    Zajac, Pawel
    KTH, School of Biotechnology (BIO), Gene Technology.
    Ståhl, Patrik
    KTH, School of Biotechnology (BIO), Gene Technology.
    Jacobsson, Josefin
    Fredriksson, Robert
    Marcus, Claude
    Schiöth, Helgi
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology.
    Ahmadian, Afshin
    KTH, School of Biotechnology (BIO), Gene Technology.
    Allelotyping by Massively Parallel Pyrosequencing of SNP-carrying Trinucleotide Threads2008In: Human Mutation, ISSN 1059-7794, E-ISSN 1098-1004, Vol. 29, no 2, p. 323-329Article in journal (Refereed)
    Abstract [en]

    Here we present an approach for allelotyping combining the multiplexing features of the trinucleotide threading (TnT) method with pooling of genomic DNA and massively parallel pyrosequencing, enabling reliable allele frequency estimation in large cohorts. The approach offers several benefits as compared to array-based methods and allows undertaking highly complex studies without compromising accuracy, while keeping the workload to a minimum. This proof-of-concept study involves formation of trinucleotide threads, targeting a total of 147 single-nucleotide polymorphisms (SNPs) related to obesity and cancer, for multiplex amplification and allele extraction from a pool of 462 genomes, followed by massively parallel pyrosequencing. Approximately 177k reads were approved, identified, and assigned to SNP-carrying threads rendering representative allele frequencies in the cohort.

  • 48.
    Redin, David
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Borgström, Erik
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab. Karolinska Institute (KI), Sweden.
    He, Mengxiao
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Aghelpasand, Hooman
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Käller, Max
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Ahmadian, Afshin
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Droplet Barcode Sequencing for targeted linked-read haplotyping of single DNA molecules2017In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 45, no 13, article id e125Article in journal (Refereed)
    Abstract [en]

    Data produced with short-read sequencing technologies result in ambiguous haplotyping and a limited capacity to investigate the full repertoire of biologically relevant forms of genetic variation. The notion of haplotype-resolved sequencing data has recently gained traction to reduce this unwanted ambiguity and enable exploration of other forms of genetic variation; beyond studies of just nucleotide polymorphisms, such as compound heterozygosity and structural variations. Here we describe Droplet Barcode Sequencing, a novel approach for creating linked-read sequencing libraries by uniquely barcoding the information within single DNA molecules in emulsion droplets, without the aid of specialty reagents or microfluidic devices. Barcode generation and template amplification is performed simultaneously in a single enzymatic reaction, greatly simplifying the workflow and minimizing assay costs compared to alternative approaches. The method has been applied to phase multiple loci targeting all exons of the highly variable Human Leukocyte Antigen A (HLA-A) gene, with DNA from eight individuals present in the same assay. Barcode-based clustering of sequencing reads confirmed analysis of over 2000 independently assayed template molecules, with an average of 753 reads in support of called polymorphisms. Our results show unequivocal characterization of all alleles present, validated by correspondence against confirmed HLA database entries and haplotyping results from previous studies.

  • 49.
    Redin, David
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Gene Technology.
    Frick, Tobias
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Gene Technology.
    Aghelpasand, Hooman
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Gene Technology.
    Theland, Jennifer
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Gene Technology.
    Käller, Max
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Gene Technology.
    Borgström, Erik
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Gene Technology.
    Olsen, Remi-Andre
    Stockholms Universitet.
    Ahmadian, Afshin
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Gene Technology.
    Efficient whole genome haplotyping and single molecule phasing with barcode-linked readsManuscript (preprint) (Other academic)
    Abstract [en]

    The future of human genomics is one that seeks to resolve the entirety of genetic variation through sequencing. The prospect of utilizing genomics for medical purposes require cost-efficient and accurate base calling, long-range haplotyping capability, and reliable calling of structural variants. Short-read sequencing has lead the development towards such a future but has struggled to meet the latter two of these needs. To address this limitation, we developed a technology that preserves the molecular origin of short sequencing reads, with an insignificant increase to sequencing costs. We demonstrate a library preparation method which enables whole genome haplotyping, long-range phasing of single DNA molecules, and de novo genome assembly through barcode-linked reads (BLR). Millions of random barcodes are used to reconstruct megabase-scale phase blocks and call structural variants. We also highlight the versatility of our technology by generating libraries from different organisms using picograms to nanograms of input material.

  • 50.
    Russom, Aman
    et al.
    KTH, Superseded Departments, Signals, Sensors and Systems.
    Ahmadian, Afshin
    KTH, Superseded Departments, Biotechnology.
    Andersson, Helene
    KTH, Superseded Departments, Signals, Sensors and Systems.
    Nilsson, Peter
    KTH, Superseded Departments, Biotechnology.
    Stemme, Göran
    KTH, Superseded Departments, Signals, Sensors and Systems.
    Single-nucleotide polymorphism analysis by allele-specific extension of fluorescently labeled nucleotides in a microfluidic flow-through device2003In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 24, no 1-2, p. 158-161Article in journal (Refereed)
    Abstract [en]

    We describe a microfluidic approach for allele-specific extension of fluorescently labeled nucleotides for scoring of single-nucleotide polymorphism (SNP). The method takes advantage of the fact that the reaction kinetics differs between matched and mismatched configurations of allele-specific primers hybridized to DNA template. A microfluidic flow-through device for biochemical reactions on beads was used to take advantage of the reaction kinetics to increase the sequence specificity of the DNA polymerase, discriminating mismatched configurations from matched. The volume of the reaction chamber was 12.5 nL. All three possible variants of an SNP site at codon 72 of the p53 gene were scored using our approach. This work demonstrates the possibility of scoring SNP by allele-specific extension of fluorescently labeled nucleotides in a microfluidic flow-through device, The sensitive detection system and easy microfabrication of the microfluidic device enable further miniaturization and production of an array format of microfluidic devices for high-throughput SNP analysis.

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