External-loop free energy affects dye-labeled terminators premature terminations in DNA cycle-sequencing reactions

Although dideoxy terminators labeled with energy-transfer dyes (BigDyes) provide the most versatile method of auto-Mated DNA sequencing, premature terminations result in a substantially reduced reading length of the DNA sequence. I recently demonstrated that combining the annealing step with the extension step at a single temperature, (60degreesC) reduces, premature terminations of DNA sequences that ordinarily contain premature,terminations when three, temperature steps are used in sequencing. I studied a novel class of DNA sequences of 100-bp length and located upstream from the point that causes premature terminations. I determined the thermodynamics of 49 DNA sequences with premature terminations at three temperature steps by using DNA mfold profiles. Sequencing results for 28 samples were improved with two-step cycle sequencing, whereas two-step cycle-sequencing reactions did not improve the results for 21 sequences. Nearest-neighbor thermodynamic parameters for all 49 sequences I were compared at temperatures 50degreesC and 60degreesC. The parameters predicted that thermodynamic free-base (external-loop) energies (deltadeltaGdegrees) were significantly different for these two study groups of samples. The results indicate that changes in free energy in single-strand base (external-loop) sequences can have a significant effect in reducing premature terminations in DNA sequencing reactions run with energy-transfer-based fluorescent-dye terminators.