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Improvements in pyrosequencing technology by employing sequenase polymerase
KTH, Superseded Departments, Biotechnology.
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2004 (English)In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 330, no 2, 272-280 p.Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
2004. Vol. 330, no 2, 272-280 p.
Keyword [en]
pyrosequencing technology, sequenase, primer-dimers, loop structures, DNA sequencing, double-stranded dna, single-nucleotide polymorphism, real-time pyrophosphate, tumor-suppressor gene, sequencing method, escherichia-coli, primer, identification, mutation
National Category
Biochemistry and Molecular Biology
URN: urn:nbn:se:kth:diva-23553DOI: 10.1016/j.ab.2004.03.018ISI: 000222424800012ScopusID: 2-s2.0-2942525596OAI: diva2:342251
QC 20100525 QC 20110926Available from: 2010-08-10 Created: 2010-08-10 Last updated: 2011-09-26Bibliographically approved

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