Optimization of Applied Biosystems 373 sequencer stretch modification

We have optimized the conditions for using the Stretch modification for the Applied Biosystems 373 Automated DNA Sequencers for sequencing double-stranded DNA using 34-cm well-to-read and 48-cm well-to-read configurations. With the manufacturer's recommended settings, uneven spacing within the first 100 bases was observed, which led to miscalls, insertions and deletions in the analyzed data. A significant decrease in accuracy for reads greater than 400 bases was also observed. Various gel concentrations were tested to improve the base spacing for the first 100 bases while maintaining accuracy and usable length of data. A longer average usable length and better resolution of smaller fragments were achieved by increased acrylamide concentration coupled with increased wattage. Using the Applied Biosystems CATALYST 800 Molecular Biology LabStation, Taq dye primer cycle sequencing reactions were optimized for -21 M13 and M13RP1 primers to produce a more even distribution of dye-labeled fragments that increased the overall signal strengths and decreased background signal. These reaction products, run on the Stretch sequencers using the new gel conditions, provided longer reads with increased reliability and accuracy of the data.