Measurement of the Phase Diagram of DNA Unzipping in the Temperature-Force Plane

Abstract

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.

The structure of DNA overstretched from the 5′5′ ends differs from the structure of DNA overstretched from the 3′3′ ends

“…SI confirm that these DNA constructs had no nicks. Finally, a bead bound to a DNA molecule that is fully unzipped can remain bound only if there are no nicks in either backbone and several publications have shown that DNA treated using the techniques described in this paper can be completely unzipped (18,30,31). We also note that prior interpretations of overstretching studies have sometimes suggested that nicks exist and affect measurements (17,25,32,33).…”

Nonlinearity in DNA and its Relation to Specific Functions

“…Squares correspond to the experimental results of [14]. Circles represent PBD simulations [19] [14], the theoretical result obtained with a popular thermodynamic model (green line) [14], together with our results (circles) from the described PBD simulations [19]. All results are related to the unzipping of λ-phage DNA (see [14,19] for sequence details).…”

“…A first step towards understanding the relation between thermal denaturation and force-induced separation was taken by Danilowicz et al [14], who recently published the first experimentally determined phase diagram for DNA denaturation as a function of the applied force. The experimental data of this study confirmed intuition in the sense that the force required to unzip the DNA must decrease with increasing temperature.…”

“…Force-temperature phase diagram of λ-phage DNA. Squares correspond to the experimental results of [14]. Circles represent PBD simulations [19] [14], the theoretical result obtained with a popular thermodynamic model (green line) [14], together with our results (circles) from the described PBD simulations [19].…”

“…The development of single-molecule force spectroscopy and dynamical force spectroscopy has made possible the investigation of force-induced separation of double-stranded DNA at various temperatures [14]. Studying the force-induced separation of double-stranded DNA into single-stranded DNA is an important step towards understanding replication and transcription processes.…”