Pyrosequencing (TM) technology at elevated temperature
2004 (English)In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 25, no 1, 20-27 p.Article in journal (Refereed) Published
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.
Place, publisher, year, edition, pages
2004. Vol. 25, no 1, 20-27 p.
DNA structures, luciferase, pyrosequencing, stranded-dna-binding, detected magnetic-resonance, escherichia-coli, tryptophan residues, protein, pyrophosphate, triphosphate, complexes
IdentifiersURN: urn:nbn:se:kth:diva-23113DOI: 10.1002/elps.200305708ISI: 000188417100004PubMedID: 14730564OAI: oai:DiVA.org:kth-23113DiVA: diva2:341811
QC 20100525 QC 201111022010-08-102010-08-102011-11-02Bibliographically approved