Abstract
We present a method for sequencing DNA that relies on the motion of single RNA polymerase molecules. When a given nucleotide species limits the rate of transcription, polymerase molecules pause at positions corresponding to the rare base. An ultrastable optical trapping apparatus capable of base pair resolution was used to monitor transcription under limiting amounts of each of the four nucleotide species. From the aligned patterns of pauses recorded from as few as four molecules, we determined the DNA sequence. This proof of principle demonstrates that the motion of a processive nucleic acid enzyme may be used to extract sequence information directly from DNA.