Fig. 2.

Specific staining of 3′-3′ attached dsDNA stretches with the intercalating dye YOYO shows that the DNA overstretching transition at 65 pN is a nucleation-limited, first-order phase transition. (A) Fluorescence images of different, extended dsDNA molecules exposed to YOYO show a binary subdivision of the DNA in labeled and unlabeled segments. The vertical dashed lines highlight the locations of the optically trapped beads. Image 1 is taken before the OS transition (L/L₀ < 1) at a force of 50 pN, where the DNA is labeled along its full length. At higher extensions (L/L₀ > 1), where the DNA undergoes the OS transition at a force of 65 pN, discrete unlabeled segments appear at the expense of labeled, double-stranded segments. This subdivision in large labeled and unlabeled segments reveals that the OS transition is nucleation limited. Image 3 shows an unlabeled segment halfway, suggesting that another OS nucleation took place at a nick. Image 6 is taken beyond the OS transition at a force of 80 pN, yet shows that the two strands are still connected by a short YOYO-labeled segment. (B) The fraction of dsDNA plotted as a function of DNA extension. This fraction was obtained from the length of YOYO-labeled segments in fluorescence images such as in (A), assuming them to have the same length as B-form dsDNA at this low degree of labeling (see Fig. S1). The gray dashed line connecting the two gray points indicates the behavior expected for a first-order phase transition from 100% dsDNA at the start of the OS transition (L/L₀ = 1) to no dsDNA at the end (L/L₀ = 1.7).