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Toward pyrosequencing on surface-attached genetic material by use of DNA-binding luciferase fusion proteins
KTH, Superseded Departments, Biotechnology.
KTH, Superseded Departments, Biotechnology.
KTH, Superseded Departments, Biotechnology.
KTH, Superseded Departments, Biotechnology.
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2004 (English)In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 329, no 1, 11-20 p.Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
2004. Vol. 329, no 1, 11-20 p.
Keyword [en]
luciferase, SSB, Klenow, pyrosequencing, sample capacity, single-stranded-dna, micromachined filter-chamber, coxsackievirus 3c proteinase, escherichia-coli, cleavage specificity, polymerase, mechanism, strategy, recovery, beads
National Category
Biochemistry and Molecular Biology
URN: urn:nbn:se:kth:diva-23440DOI: 10.1016/j.ab.2004.02.005ISI: 000221602800002ScopusID: 2-s2.0-2342634459OAI: diva2:342138
QC 20100525 QC 20111026Available from: 2010-08-10 Created: 2010-08-10 Last updated: 2012-03-22Bibliographically approved

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Ehn, MariaNourizad, NaderBergstrom, KristinaAhmadian, AfshinNyrén, PålLundeberg, JoakimHober, Sophia
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