Abstract: Pyrosequencing is a method that can be used in almost allDNA sequencing applications. The method uses coupled reactionsof ATP sulfurylase and firefly luciferase to monitor theinorganic pyrophosphate (PPi) released during nucleotideincorporation onto a DNA primer. The objective was to improvepyrosequencing technology in terms of enzyme performance andsupporting assays for further optimization. A solid-phaseapproach for DNA sequencing, three approaches for enzymaticanalysis, and a method for production of one of the detectionenzymes (ATP sulfurylase) are presented here.In the solid-phase pyrosequencing, an immobilizedsingle-strand DNA template was used in repeated cycles ofdeoxynucleotide extension catalyzed by DNA polymerase.Real-time signals proportional to the amount of nucleotidesincorporated were detected simultaneous to the incorporation ofthe complementary bases. An increased signal-to-noise ratio wasobtained by substitution of deoxyadenosine alfa-thiotriphosphate (dATPαS) for the natural deoxyadenosinetriphosphate (dATP). The dATPαS is efficiently used by theDNA polymerase, but is not recognized by the luciferase. As amodel, fifteen bases of a single-stranded PCR product weresequenced using streptavidin-coated paramagnetic beads as asolid support.The detection system used in thesolid-phase approach wasused to detect reverse transcriptase (RT) activity in realtime. Different templates were used as substrates and real-timesignals proportional to the amount of nucleotide incorporatedwere detected. RT efficiently used dATPαS as substrate.Thus, RT can be used as a polymerase in pyrosequencing.A real-time approach for determination of NDP kinaseactivity based on modifications of the detection method used inthe solid-phase approach was developed. In this method, NDPkinase substrates were incubated with firefly luciferase andthe reaction was started by the addition of the NDP kinase. Therate of ATP production was proportional to the NDP kinaseconcentration. This approach was used to detect contaminatingNDP kinase activity in enzyme preparations used inpyrosequencing.As the ATP sulfurylase activity varies from batch to batch,a real-time method for quantification and detection of ATPsulfurylase activity was developed based on PPi detection. Theassay is sensitive and has a linear response between 0.1µU and 50 mU.The gene encoding ATP sulfurylase was cloned into a plasmidvector that direct its expression inE.coli. The soluble active protein was purified andlater used in pyrosequencing. Using this recombinant ATPsulfurylase, the problems associated with enzyme contaminationand batch-to-batch variations were eliminated.Key words:Pyrosequencing, DNA sequencing, polymerase,ATP sulfurylase, luciferase, apyrase, NDP kinase,bioluminescence, real-time assays.
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