Two-step cycle sequencing reduces premature terminations when using primers with high annealing temperatures

Direct cycle sequencing of double-stranded polymerase chain reaction (PCR) products using thermostable polymerase produces fragments that are shorter than expected when the enzyme prematurely detaches as it approaches the 5′-end of the DNA template. These premature terminations result in a substantially reduced reading length of the DNA sequence. Since some DNA templates spontaneously fold and form stable secondary structures at temperatures that are typically used for primer annealing, one factor that may cause premature terminations to occur is the formation of secondary structures in the template during the annealing step of the cycle sequencing reaction. We describe a simple and effective method for reducing premature terminations in DNA sequences. We demonstrate that maintaining the annealing temperature of the cycle sequencing reaction above a critical temperature reduces premature terminations in DNA sequences that regularly contain premature terminations when the temperature of the annealing step is 60°C. In the method described, annealing and extension of the primer along the template take place at the same temperature (72°C). This procedure for reducing premature terminations can be applied when sequencing with primers that are relatively long (at least 27 mer) and have high optimal annealing temperatures.