Figure 2. Single-molecule experimental setup.

Single-molecule experimental setup. (a) Duplex λ DNA (48.5 kb) is attached to the surface of the flow cell via the 5′ end of the fork using a biotin-streptavidin interaction, and the 3′ end is attached to a paramagnetic bead using a digoxigenin-anti-digoxigenin interaction. A primed replication fork is formed at the end opposite the bead to allow loading and initiation of the replication proteins. (b) Bead-DNA assemblies are stretched using laminar flow of buffer and imaged using wide-field optical microscopy, permitting simultaneous observation of multiple individual replication reactions. (c) Extension profile of ssDNA (filled circle) and dsDNA (open circle) under low forces. Dashed line shows crystallographic length of fully ds-λ DNA, 16.3 µm²⁹.