Pyrosequencing trade mark technology at elevated temperature.
To date, the Pyrosequencing trade mark technology has been performed at 28 degrees 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 degrees C. By increasing the temperature to 37 degrees 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 degrees C enabled us to sequence a DNA template with the initial sequence 3'-ATGGCCCGGGGGGGAGCTCCA em leader 5'. Furthermore, we describe a method to analyze if a primer forms a primer-dimer with extendable 3'-ends.
Eriksson
Jonas
J
Department of Biotechnology, Stockholm Center for Physics, Astronomy and Biotechnology (SCFAB), Stockholm, Sweden.
Gharizadeh
Baback
B
Nordström
Tommy
T
Nyrén
Pål
P
D001622:Betaine; D001709:Biotechnology / Q000379:methods; D017931:DNA Primers; D003848:Deoxyguanine Nucleotides; D019281:Dimerization; D004795:Enzyme Stability; D008156:Luciferases; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA / Q000379:methods* / Q000592:standards; D013696:Temperature; D013698:Templates, Genetic
D017931:DNA Primers; D003848:Deoxyguanine Nucleotides; C065361:2'-deoxy-7-deazaguanosine triphosphate; D001622:Betaine; D008156:Luciferases