3.2.2 Experimental setup to unzip DNA molecules

Unzipping experiments using optical tweezers consist of two steps: 1) synthesis and preparation of the samples to be pulled and 2) attachment of the molecule between the two beads. The procedures to synthesize DNA, RNA or proteins are very different. Thus, the synthesis of the molecule depends on the type of molecule to be pulled. Section 3.2.3 describes how the DNA molecules to be unzipped are synthesized in the lab.

  1. The preparation of DNA molecules for pulling requires the incubation of the synthesized DNA molecules with the coated AD beads. This process consists in mixing the DNA molecules with beads in a ratio of approximately one bead per molecule. The incubation time is between 15-30 minutes at room temperature. During this time, the digoxigenin labeled handles of the DNA molecules are bonded to the antidigoxigenins that coat the beads. The sample is diluted and introduced in the upper channel of the fluidics chamber (see Fig. C.5 in Appendix C.4). Another sample is prepared by diluting a certain amount of SA beads in buffer. This second preparation is inserted in the lower channel of the fluidics chamber (Fig. C.5). The central channel is then loaded with the corresponding buffer at which the pulling experiment is performed.
  2. The tethering or connection of the molecule between two beads is carried out within the fluidics chamber. First, a SA bead is trapped at the exit of the bearing tube of the lower channel. With the help of the optical trap, the bead is located and immobilized at the tip of the micropipette by air suction. Second, an AD bead (already incubated with DNA) is trapped at the exit of the bearing tube of the upper channel. Finally, the beads are brought close to each other until a tether between the SA bead an the biotin labeled handle of the DNA sample is achieved (see Fig. 3.8).

At this point, the optical trap measures a force when the beads are separated and the system is ready to start the DNA unzipping experiments.

Figure 3.8: Screenshots that show the formation of an attachment between the molecular construct and the beads in the fluidics chamber. (a) Trapping of a SA bead. (b) Immobilization of the SA bead at the tip of the micropipette. (c) Trapping of a AD bead. (d) Tethering of the molecule between the two beads.
\includegraphics[width=\textwidth]{figs/chapter3/settether2.eps}

JM Huguet 2014-02-12