User:Anthony Salvagno/Notebook/Research/2009/09/30/Biphys Society Abstract 2010
Larry's From 2009
We are developing single-molecule methods for mapping protein-DNA interactions inside living cells by unzipping single chromatin fragments isolated from living cells. One avenue towards this capability involves unzipping random fragments that have been generated by site-specific restriction endonuclease digestion of whole genomic DNA or chromatin, a process we are calling shotgun DNA mapping or shotgun chromatin mapping. A key enabler of shotgun DNA mapping (SDM) will be the ability to assign the individual fragments to their specific sites in the genome, based on the sequence-dependent unzipping force of the underlying naked DNA sequence. We will present proof-of-principle results demonstrating the ability to match experimental data sets for pBR322 unzipping to the correct pBR322 sequence hidden in a library of approximately 3,000 yeast genome sequences arising from the known locations of XhoI recognition sites. We do so via an algorithm that scores the experimental data against simulated unzipping forces from a quasi-equilibrium model (Bockelmann, Essevaz-Roulet, & Heslot, 1997). Our next step is to perform SDM on yeast genomic DNA fragments produced by ligation of XhoI-digested DNA to unzipping constructs. Enhancements of the matching algorithm, data processing, and unzipping simulation will be discussed, along with studies of the robustness of the SDM method as a function of number of sites in genome and other parameters. In addition to the impact on our goal of single-molecule mapping of chromatin from living cells, SDM may have important applications in other areas of genomics, including high-throughput structural DNA mapping and genome-wide mapping of sequence-specific DNA binding proteins.
Points to hit
- What we are doing
- What has been done
- What I hope to achieve
- Future applications
What I want to write about
- Simulations of yeast genome compare and pBR322 compared with unzipping of pBR322
- Digestion of yeast genome
- Ligation of random fragments of yeast genome to unzipping construct
- sequencing of fragments
- OT construction
- unzipping random fragments
- comparing fragments to simulated genome
- identifying correct sequence
- unzipping through RNA Pol II
- testing detection of simple DNA changes (ie cytosine-methylation)
- telomere mapping
- chromatin mapping
What can I get done (realistically) by Feb:
- tweezer calibration (mostly?)
- unzipping fragments
- comparison to genome
- initial telomere prep
- initial chromatin prep
- initial Pol II stuff (NC trip?)
Random Question: Has anyone ever compared in vitro nucleosome locations with in vivo positions? Basically if you put histones in solution with DNA I guess you would get nucleosomes. So has anyone ever compared those random positions (idk if it is a random binding event) with the actual positions that one would find if chromatin is extracted? I think that would be a sweet experiment if no one has done so.
We are developing single-molecule methods for mapping genomic information from living cells by unzipping single DNA fragments isolated from living cells, also known as Shotgun DNA Mapping. Genomic DNA from S. cerevisiae has been digested with restriction endonucleases to produce a library of random fragments. In conjunction, a similar library was created in silica. From this simulations of sequence-dependent unzipping forces were created. Actual genomic DNA fragments were unzipped via Optical Tweezers (1064nm) and the resulting force profiles were compared to the library of force curves in search of a match. The unzipped sequences were then sequenced and matches were compared to the correct sequence. In this poster the current research will be discussed. From this basic level there are many different areas of possible study. The most pressing is the possibility of Shotgun Chromatin Mapping. In this method, unzipping profiles of nucleosomes and RNA Pol II are added to the simulation and locations of various sites will be attained. Another avenue of interest will be the detection of simple structure modifications like cytosine-methylation. Telomere mapping could also be featured as the repetitive nature of this section of the chromosome is strongly observable with OT studies. Last but not least we feel that alternative splicing could be a major impact area since simple changes like deletions and insertions are easily detectable by our system. So come see my poster bitches.
Wow this is bad, but I couldn't think much about this.