Rapid RNA sequencing using double-stranded template DNA, SP6 polymerase, and 3'-deoxynucleotide triphosphates.

A simple and efficient nucleic acid sequencing method is described in which RNA transcription by the SP6 polymerase is specifically terminated using 3'-deoxynucleotide triphosphates. Initial difficulties in resolving the RNA ladder were overcome by replacing guanosine triphosphate by inosine triphosphate in the reaction mixture and electrophoresing gels at high temperature (50 degrees C). This method presents advantages over current sequencing techniques: Unprocessed plasmid DNA is the template and preparation of inserts and/or single-stranded templates is unnecessary. Use of the specific promoter for SP6 polymerase removes the need for a primer in sequencing reactions.