Abstract
AbstractAbility 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.
Highlights
Abilityto m a p polymerases and nucleoso m e s on chro m atin is important for understanding the impact of chro m atin remo deling on key cellularprocesses
C ertain information can only be obtained via singlem olecule (S M) analysis, such as seeing direct correlations between polymerases and nucleoso m e s on individual fibers or differentiating between so m e proposed m o d els of chro m atin rem od eling
This is due to a significant increase in the unzipping rate above j=1000, because the original purpose of these data sets (Koch 2002) was to probe protein occupancy, w here an increased unzipping rate is desirable and a system atic shiftin unzipping force is not an issue. [Possibly sho w s m o othing from 10 point windo w as well,and say w e chose the 30 point windo w “just cuz”] [Supple m e ntary info / data not sho w n: offset estimated by co m p arison with nonfast data set.]
Summary
Abilityto m a p polymerases and nucleoso m e s on chro m atin is important for understanding the impact of chro m atin remo deling on key cellularprocesses. We de m o n strate that the m o d eling of the pB R 3 2 2 unzipping forces is sufficientlyaccurate so that experimental data are successfully m atched to the pB R 3 2 2 sequence hidden in a background of the [~2700] XhoI frag me nts from the yeast geno m e.
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