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Pyrosequencing™ technology at elevated temperature
Author(s) -
Eriksson Jonas,
Gharizadeh Baback,
Nordström Tommy,
Nyrén Pål
Publication year - 2004
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.200305708
Subject(s) - betaine , primer (cosmetics) , luciferase , pyrosequencing , thermostability , glycine , polymerase chain reaction , chemistry , dimer , dna , microbiology and biotechnology , biology , biochemistry , gene , amino acid , enzyme , organic chemistry , transfection
Abstract To date, the Pyrosequencing™ technology has been performed at 28°C 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 37°C. By increasing the temperature to 37°C, 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 37°C enabled us to sequence a DNA template with the initial sequence 3'‐ATGGCCCAGCTCCA … 5'. Furthermore, we describe a method to analyze if a primer forms a primer‐dimer with extendable 3'‐ends.