Proof of principle for shotgun DNA mapping by unzipping

摘要

Ability to map polymerases and nucleosomes on chromatin is important for understanding the impact of chromatin remodeling on key cellular processes. Current methods (such as ChIP and ChIP-chip) have produced a wealth of information that demonstrates this importance, but key information is elusive in these ensemble methods. We’re pursuing a new single-molecule chromatin mapping method based on unzipping native chromatin molecules with optical tweezers. The first step we are taking towards this ability is shotgun DNA mapping (SDM). This is the ability to identify the genomic location of a random DNA fragment based on its naked DNA unzipping forces compared with simulated unzipping forces of a published genome. We show that ~32 separate experimental unzipping curves for pBR322 were correctly matched to their simulated unzipping curves hidden in a background of the ~2700 sequences neighboring XhoI sites in the S. cerevisiae (yeast) genome. We describe this method and characterize its robustness as well as discuss future applications.