DNA Unzipping

Protein-DNA interactions dictate gene expression and replication. Although several techniques are able to detect the location of an interaction, few are able to measure its strength, a critical determinant of DNA accessibility. Our lab developed the powerful and versatile DNA unzipping method to measure protein-DNA interactions (Koch et al., BJ, 2002; Koch and Wang, PRL, 2003; Shundrovsky et al., 2006; Hall et al., 2009). Using an optical trap, we mechanically separate a double-stranded DNA (dsDNA) with bound proteins into two single strands. As the unzipping fork reaches a bound protein, the unzipping force increases dramatically, and then reduces suddenly as the interaction is disrupted. Typically, multiple interactions are detected for a given protein, and the unzipping method maps the locations of these interactions to near single-base-pair precision, while also providing a quantitative measure of their strengths.

A typical experimental configuration for unzipping. An optical trap is used to apply a force necessary to unzip through the DNA as the coverslip is moved laterally so that the end of the DNA bound to the coverslip is moved away from the trapped microsphere.