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Long-read pyrosequencing using pure 2 '-deoxyadenosine-5 '-O '-(1-thiotriphosphate) Sp-isomer
KTH, Superseded Departments, Biotechnology.
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2002 (English)In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 301, no 1, 82-90 p.Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
2002. Vol. 301, no 1, 82-90 p.
Keyword [en]
pyrosequencing, DNA sequencing, luciferase, apyrase, ATP sulfurylase, DNA polymerase, nucleotides, pure Sp-dATP-alpha-S, luciferin, alkaline phosphatase, long read, single-nucleotide polymorphism, time pyrophosphate, escherichia-coli, enzymatic method, stranded dna, hybridization, triphosphate, mutation
URN: urn:nbn:se:kth:diva-21301DOI: 10.1006/abio.2001.5494ISI: 000173661300011OAI: diva2:339999
QC 20100525Available from: 2010-08-10 Created: 2010-08-10Bibliographically approved

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