Sequencing a specific DNA element within a genome or a complex mixture of DNA by the Sanger sequencing method generally involves PCR-mediated amplification of target DNA with forward and reverse primers, followed by a sequencing reaction directed from a single primer. To minimize the contribution of fluorescent signal due to the extension products originating from primers carried over from the amplification step, an intermediate step is routinely incorporated to remove the excess primers before proceeding to the sequencing reaction. We have developed a method called SeqSharp that removes noise in the sequencing data by enzymatically removing chain termination products originating from one or both of the amplification primers. This method substantially improves the quality of sequence information even without an intermediate primer removal step. Importantly, we show that SeqSharp significantly improves the sequence quality from a combined (one-step) amplification/sequencing protocol and provides a more robust method that, unlike previously described one-step sequencing methods, yields high quality sequence data from a single reaction by using equimolar primer concentrations. One-step SeqSharp is generally applicable and produced excellent sequence data from bacterial, fungal, and human DNA.