Single molecule studies, at constant force, of the separation of double-stranded DNA into two separated single strands may provide information relevant to the dynamics of DNA replication. At constant applied force, theory predicts that the unzipped length as a function of time is characterized by jumps during which the strands separate rapidly, followed by long pauses where the number of separated base pairs remains constant. Here, we report previously uncharacterized observations of this striking behavior carried out on a number of identical single molecules simultaneously. When several single phage molecules are subject to the same applied force, the pause positions are reproducible in each. This reproducibility shows that the positions and durations of the pauses in unzipping provide a sequence-dependent molecular fingerprint. For small forces, the DNA remains in a partially unzipped state for at least several hours. For larger forces, the separation is still characterized by jumps and pauses, but the double-stranded DNA will completely unzip in less than 30 min.

We have measured the shear force for short double-stranded DNA sequences pulled by either the 3'3' or 5'5' ends and find that the shear force is independent of the pulling technique. For the 50% GC sequences examined, the force is a linear function of DNA length up to 20 base pairs (bp); however, we show that, as predicted by deGennes, the shear force approaches an asymptotic value in the limit where the number of base pairs approaches infinity, where the shear force for a 32 bp sequence is within 5% of the asymptotic value of 61.4 pN . Fits to deGennes' theory suggest that the shear force is distributed over fewer than 10 bp at the end of the sequence, with the rest of the sequence experiencing negligible shear force. The single base pair rupture force and the ratio of the backbone spring constant to the base pair spring constant determined from fits of the data to deGennes' theory are consistent with ab initio predictions.

We separate double stranded lambda phage DNA by applying a fixed force at a constant temperature ranging from 15°C to 50°C, and measure the minimum force required to separate the two strands, providing the first experimental determination of the phase boundary between single stranded DNA and double stranded DNA in the temperatureforce plane. The measurements also offer information on the free energy of dsDNA at temperatures where dsDNA does not thermally denature in the absence of force. While parts of the phase diagram can be explained using existing models and free energy parameters, others deviate significantly. Possible reasons for the deviations between theory and experiment are considered.

Complete unzipping and rezipping of lambda -phage double-stranded DNA is achieved by applying a constant force. A strong hysteresis is observed at all tested time scales and temperatures. Hysteresis also occurs for partial unzipping, indicating stability for the partially open state over a force range of 2- 5pN . Results are compared to nearest-neighbor model simulations, and reasonable agreement is found.