Improved accuracy in direct automated DNA sequencing of small PCR products by optimizing the template concentration

Data are presented illustrating the optimum concentration range of reverse transcription PCR-generated products under 500 bp for accurate base calling with direct automated DNA sequencing. A 357-bp fragment of the human estrogen receptor, which includes the DNA binding domain of the protein, was used as a representative example of a gene fragment that can be rapidly amplified and sequenced. Using the Taq DNA polymerase dye terminator sequencing protocol and automated sequencing apparatus from Applied BioSystems, 0.1 to 1.0 pmol of PCR product in a 20-microL reaction volume provided > 97% accurate base detection. Concentrations greater or lower than this range increased the number of ambiguous bases due to alterations in the signal-to-noise ratios. This procedure has been successfully utilized with 140-440-bp PCR products within the optimum concentration range. These results show that low amounts of PCR products are necessary and sufficient for direct sequence analysis.