We experienced significant problems while developing a PyrosequencingTM (Biotage, Uppsala, Sweden) assay to characterize the rifampin resistance-determining region of Mycobacteriumtuberculosis, a target with high guanosine-cytosine content. This paper describes the successful use of a modified pyrosequencing protocol through partial substitution of deoxyguanosine triphosphate with deoxyinosine triphosphate in the polymerase chain reaction.
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