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.