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
shotgun DNA mapping (SDM) = Shotgun DNA Mapping; SM = single-molecule; chromatin Immunoprecipitation (ChIP) = Chromatin Immunoprecipitation; Pol II = RNA Polymerase II; SCM = shotgun chromatin mapping
Understanding of these dynamic remodeling processes requires the ability to characterize with high spatial and temporal resolution the changes to chromatin inside living cells. Techniques such as chromatin Immunoprecipitation (ChIP), ChIP-chip, and other existing techniques have provided a wealth of important information, but have drawbacks in terms of sensitivity to small changes in protein occupancy, spatial resolution, and ensemble averaging
We are pursuing a second way of achieving site-specificity which is to unzip random chromatin fragments in a high-throughput fashion, and figuring out from which specific site of the genome it came. We call this shotgun chromatin mapping (SCM) and it based on a method for indentifying the genomic location of naked DNA fragments
Summary
We call this shotgun chromatin mapping (SCM) and it based on a method for indentifying the genomic location of naked DNA fragments (see Fig. 1). We demonstrate that the modeling of the pBR322 unzipping forces is sufficiently accurate so that experimental data are successfully matched to the pBR322 sequence hidden in a background of the ~2700 XhoI fragments from the yeast genome.
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