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Application of shotgun DNA mapping to yeast genomic DNA
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BPS 2010 Poster Presentation: Shotgun DNA Mapping with Yeast

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This is my poster presentation from the annual Biophysical Society Meeting in San Francisco, CA. I detail the current progress made in Shotgun DNA mapping and include an aside about open notebook science and my scientific life on the internet.

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Transcript of "BPS 2010 Poster Presentation: Shotgun DNA Mapping with Yeast"

  1. 1. Application of shotgun DNA mapping to yeast genomic DNA Anthony Salvagno, Lawrence Herskowitz, Andy Maloney, Kelly Trujillo, Linh Le, Steve Koch University of New Mexico Introduction It is possible to distinguish genomic information based on unzipping DNA with optical tweezers. This is due to the fact that the force signature for unzipping single DNA molecules is sequence- dependent and easily modeled. We can use this information to match an unzipped sequence to a library of sequences obtained through simulations. We call this technique shotgun DNA mapping and have found that we can use it to better understand protein-DNA interactions and the interaction locations. We currently are pursuing applications in chromatin mapping and structural DNA mapping with many future possible ventures. Acquiring Shotgun Clones Unzipping DNA Open Science What are shotgun clones? What do you need to unzip DNA? Shotgun clones are genomic fragments digested from restriction enzymes and inserted into a cloning vector. In order to unzip there are several components you need: (1) optical tweezers and detection system, (2) unzip- There is no target DNA as every clone used in these experiments is completely random. The randomness of the pable DNA and DNA tethers, and (3) software. No component is more important than any other component be- genomic fragments is key to later elements of shotgun DNA mapping. cause without one we couldn’t perform an experiment. Creating Shotgun Clones We started with yeast (S. cerevisiae) and extracted pure genomic DNA. We then digested the genome with both XhoI and EcoRI. After digestion, we ligated the Our Optical Tweezers resulting fragments into pBluescript for We use a 1064nm 4W diode pumped continuous wave laser. blue/white screening. We then used E. We control beam power through the use of an AOM and can coli to clone our plasmids with our shot- manually steer the beam via a one-to-one telescope. We can gun fragments to get shotgun clones. We move the stage through micrometer positioning stages, and for picked several colonies and combined the experiments we move the sample with a 1-d piezoelectric remaining colonies to make a “library” of stage. We use a quadrant photodiode for beam detection. shotgun clones. Some fragments were digested with XhoI only (marked A, C, D, and E) and others were digested with both XhoI and EcoRI (numbered clones). Creating Unzippable DNA Creating unzippable DNA requires a 3 piece ligation. The anchor piece is created from PCR of pRL574 and is 1.1kb in length. It is designed with a BstXI site toward the 3’ end. The adapter piece is a Tethering DNA Open Notebook Science unzippable In order to unzip DNA, we need to be able to pull on it with short oligo (~20bp) designed with 2 sticky ends: one that is compli- our tweezers. To do this we must fix DNA to a glass sur- My notebook is hosted by OpenWetWare.org and is a wiki environment that allows me pBR322 mentary to the anchor and the other which has a SapI/EarI over- to fully customize my notebook. I can embed movies, presentations, spreadsheets, and face. This is achieved through digoxygenin-anti-dig inter- hang. Any DNA you want to unzip must have this SapI/EarI over- hang to be the 3rd piece in the ligation. actions. The dig molecule is located on the 5’end of the no DNA ~400nM dsDNA just about anything. A large portion of my notebook is dedicated to my day-to-day anchor segment of the DNA and we attach anti-dig to glass nonspecifically. We attach 0.51um polystyrene spheres dealings, but I also use it to publish methods, protocols, and data. I publish everything As a proof of principle, we tested ligation parameters on pBR322. regardless of success or failure. What I publish is instantly accessible to the world and is coated with streptavidin to the DNA via a biotinylated First we digested the plasmid with EarI and gel extracted (digestion results in 2 pieces) the desirable fragment. We then added the frag- nucleotide in the adapter oligo. There is a nick about 8 completely searchable by Google. An added advantage is that I can access my note- bases from the biotin and it is this nick that allows us to anchor ment to our 3 piece ligation and achieved success. We digested our separate each strand of DNA. The tethering process itself is book from anywhere in the world, all I need is an internet connection. clones with SapI and performed the same ligation on those. not trivial and relies on the concentration of the DNA, clean ~20nM dsDNA ~4nM dsDNA glass, unclumped microspheres, pure anti-dig, and buffer. Ligating DNA to our unzipping con- Demonstrating how DNA concentration can a ect a typical tether- struct enables us to unzip target DNA. ing experiment. Visually there are more beads in each sample, but the number of apparent stuck beads increase with increasing DNA concentration. Sap14 Sap14 genomic DNA product not clear 2 distinct biotin/streptavidin bands interaction dsDNA anchor dig/anti-dig The image on the left was a rst attempt at ligating shotgun clones digested with SapI onto the interaction unzipping construct. The image on the right is the most recent attempt at the same ligation. The clone in the right image is the same as one of the clones in the left image. cover glass surface Future Plans Acknowledgements References Telomere Mapping Because the telomere region is made up of highly This molecule has 17 nearly identical repetitive DNA, we believe that we can use optical tweezers to detect each repeat ~200bp repeats Bockelmann, U., & et al.(1997). Molecular Stick-Slip Motion Revealed by Open- and analyze various structures of the telomere region. These experiments could probe G-quadruplexes, Telomerase interactions, scafolding proteins, and more. some pictures here Mary Ann Osley ing DNA with Piconewton Forces. Physical Review Letters , 4489-4492. Pranav Rathi Koch, S. J., & et al. (2002). Probing Protein-DNA Interactions by Unzipping a RNA Pol II interactions Transcription is a very complicated process Single DNA Double Helix. Biophysical Journal , 1098-1105. especially in higher order organisms. Unzipping through an assembled RNA Pol II complex could reveal a lot of insight into the nature of the enzyme. We believe Brian Josey Shundrovsky, A ., & et al. (2006). Probing SWI/SNF Remodeling of the Nucleo- some by Unzipping Single DNA Molecules. Nature Structural and Molecular that we can also unzip through Pol II during various stages of transcription for Biology , 549-554. further insight into the process. It will also be useful to have an unzipping profile Karen Adelman Wang, M. D ., & et al. (1997). Stretching DNA with Optical Tweezers. Biophysical when dealing with chromatin mapping in vivo. Chromatin Mapping After Shotgun DNA Cameron Neylon Journal , 1335-1346. Mapping, we hope to be able to map nucleosome loca- Jean-Claude Bradley tions by unzipping through histone proteins bound to dsDNA. Locations would be attainable by retaining the initial unzipping forces, allowing the DNA to rezip, and Diego Ramallo KochLab then unzipping the now naked DNA and using these force curves to match to our database of unzipped fragments. Stefanie Gallegos
  1. ¿Le ha llamado la atención una diapositiva en particular?

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