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Shotgun DNA Mapping<br />Anthony <br />Salvagno<br />

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Welcome to KochLab!<br />Single Molecule DNA Analysis<br />Kinesin Studies<br />F<br />F<br />Image from Block and adapted by Koch<br />Image by Koch<br />

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Kinesin Studies<br />Andy<br />Gliding Motility Assay<br />Surface Passivation<br />Larry<br />Tracking<br />Processivity<br />Brigette<br />Ensemble ATP Hydrolysis<br />Me<br />Bead Motility<br />Making Kinesin<br />60um<br />

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Single Molecule DNA Studies<br />What is DNA?<br />What is Shotgun DNA Mapping?<br />What are Optical Tweezers?<br />What is Molecular Biology?<br />

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DNA: The Code of Life<br />Double stranded polymer<br />Covalently bonded sugar molecules make up the backbone<br />Hydrogen bonded bases join two strands of DNA<br />There are 4 bases<br />Whyfiles.org<br />

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DNA Compaction<br />Lots of DNA in a genome that needs to fit in the nucleus<br />~2m DNA length per cell<br /> ~2nm wide<br />~20um cell diameter<br />~10um nucleus diameter<br />Chromosomes – structure for mitotic cells<br />Chromatin – where everything happens<br />Molecular Biology of the Cell<br />

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Nucleosomes<br />DNA wrapped in histone proteins<br />Proteins:<br />H2A<br />H2B<br />H3<br />H4<br />Form octamer<br />Form stable tetramer<br />Wikipedia<br />

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From DNA to People<br />DNA to RNA to Proteins<br />Known as gene expression<br />Leads to changes in characteristics between organisms<br />Leads to differentiation amongst cell lines<br />Wikipedia<br />Thinkquest.org<br />

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Transcription<br />RNA Polymerase II:<br />Copies single strand of DNA to make RNA <br />Moves with transcription bubble<br />Initiation<br />RNAPII assembly<br />Elongation<br />Active transcription<br />Termination<br />RNAPII disassembly<br />Reassembled Nucleosomes<br />RNA Pol II<br />promoter<br />cryptic<br />promoter<br />Transcription<br />

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Points about Gene Expression<br />Mutations can affect many aspects of gene expression<br />Possible changes because of:<br />DNA sequence modifications<br />Deletions, inversions, insertions, and single base changes (SNPs)<br />Post Translational Modifications<br />

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Why Single Molecule is Powerful<br />Bulk studies provide general insight<br />Information is average from all molecules in sample<br />Different molecules have different properties<br />Studying DNA one molecule at a time can provide unprecedented understanding of a process<br />

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Forces from &lt; 1 pN to 100s pN<br />Length precision ~ 1 nm<br />Thermal energy (kBT) <br />4 pN – nm = 1/40 eV<br />Kinesin 8 nm step, 6 pN stall<br />(molecular motor)<br />RNA Polymerase 0.3 nm step, 25 pN stall<br />DNA Unzipping 15 pN<br />Why Optical Tweezers?<br />

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Examples of Single Molecule Analysis<br />Red Line – protein bound to DNA<br />Black Line – naked DNA<br />Black Dotted Line- predictions of protein locations<br />F<br />F<br />Unzipping can detect proteins bound to DNA<br />Koch et al. 2002<br />

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Examples of Single Molecule Analysis II<br />Unzipping can detect nucleosomes<br />nucleosome<br />

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Shotgun DNA Mapping<br />Want to understand how proteins affect gene expression<br />Need a way to map sequences of DNA to location in genome<br />Library of Simulated Curves<br />Random fragment<br />Experimental Force<br />Endonuclease<br />Genomic DNA<br />Correct Match<br />dsDNA anchor<br />Step 1: Digest genome into fragments<br />Step 2: Unzip fragment and record forces<br />Step 3: Compare experimental forces to a library of simulated curves<br />

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Unzipping Library<br />Used Yeast Genome because less complex than human, but can still have Chromatin<br />Simulated digestion with XhoI<br />Over 1300 fragments<br />Simulated unzipping 2000bp before and after recognition sequence<br />Gives us over 2600 unzipping profiles<br />Unzipping Direction<br />

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Unzipping Simulation<br />Energy depends on:<br />Energy of ssDNA (FJC)<br />Energy of base-pairing (DNA)<br />In order to get force vs unzipping index curve need:<br />EFJC<br />EDNA<br />

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Proof of Principle<br />Simulated unzipping of pBR322 plasmid<br />Simulation info hidden in genomic simulation<br />Old unzipping data (Koch) used for comparison<br />A<br />Correct Match, Score 0.2<br />18<br />Force (pN)<br />12<br />0<br />1500<br />Unzipping fork index (bp)<br />B<br />Mismatch, Score 0.8<br />18<br />Force (pN)<br />12<br />0<br />1500<br />Unzipping fork index (bp)<br />

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Match Data<br />32 unzipped plasmid data compared to library <br />Each time the best match score was the plasmid simulated data<br />

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How do we get real data?<br />

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Optical Tweezers<br />Focused laser light has the ability to trap small particles<br />Simplest trap is composed of just a laser and an objective<br />SM Block<br />

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Optical Trap<br />Bead is tiny dielectric sphere<br />Laser focus creates large E-field gradient<br />Bead attracted to center of focus<br />Want High NA for better trapping<br />

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Data Collection<br />Refraction of laser from bead moves path<br />QPD tracks motion of beam<br />Force in trap approx. as spring<br />F=-kx<br />La Porta Lab<br />

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Our Tweezers<br />

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How do we unzip DNA?<br /><ul><li>Create unzipping construct

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Create Shotgun fragment clones for single molecule analysis

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Attach pieces together and tether to cover slide</li></li></ul><li>The Unzipping Construct<br />Courtesy of Diego<br />

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Restriction Enzymes<br />REs recognize a specific sequence of DNA and cut the DNA at or near the site.<br />

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Piece by Piece Construct Creation<br />Anchor<br />Made from PCR of pRL574<br />Has BstXI overhang with known base sequence<br />Beginning of polymer is labeled with dig molecule for specific binding with anti-dig<br />Adapter<br />Short duplex made 2 single-stranded oligos<br />5’ end has phosphate removed creating a nick<br />5’ end has complementary BstXI overhang<br />3’ end has SapI/EarI overhang<br />SapI<br />GCTCTTCNNNNN<br />CGAGAAGNNNNN<br />GCTCTTCN NNNN<br />CGAGAAGNNNN N<br />BstXI<br />CCANNNNNNTGG<br />GGTNNNNNNACC<br />CCANNNNN NTGG<br />GGTN NNNNNACC<br />Recall:<br />

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Ligating Construct to unzippable DNA<br />Ligate – attach separate DNA strands into one continuous strand<br />Need to ligate in specific way<br />Limited by genomic DNA<br />Low adapter duplex concentration, but gradually increase during the course of the reaction<br />Where does unzippable DNA come from?<br />

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Making Shotgun Clones<br />Why clone?<br />We can have a ton of a specific DNA fragment<br />Some for unzipping<br />Some for sequencing<br />What is shotgunning?<br />Drinking a beer really fast<br />Creating random fragments quickly<br />

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How Cloning Works<br />Plasmids are:<br />Extra chromosomal<br />Capable of replication <br />Useful for cloning<br />Cloning is:<br />Identical copying of fragment of DNA<br />DNA can be inserted into plasmid for replication via Multiple Cloning site <br />Wikipedia.org<br />Fermentas.com<br />

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Cloning<br />LacZ gene turns cell blue<br />Disrupting gene turns cells white<br />Can select specific colonies <br />Each colony contains different genomic fragment<br />fragment<br />Wikipedia<br />No fragment<br />

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Genome Digestion<br />Need to make fragments from pure genomic DNA<br />XhoI digest produces very large fragments<br />XhoI+EcoRI provides much smaller fragment sizes<br />Need smaller fragments for cloning<br />

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DNA Tethering<br />Create flow cell from double stick tape, slide and coverslip<br />Flow anti-dig, surface blocker, tethering DNA, microspheres, and wash sequencially<br />

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What’s Next?<br />

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Calibrate and Unzip<br />Can unzip without calibration<br />Messy data analysis<br />Calibrate with stuck beads and free moving beads<br />Then I can get GOOD unzipping data <br />this can be real soon<br />

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Chromatin Studies<br />Shotgun Chromatin Mapping<br />Can insert random fragments into yeast to get chromatin<br />Want to map nucleosome and protein locations<br />Optical Trap<br />nucleosome<br />Elongating Pol II<br />ssDNA<br />Coverglass<br />Koch<br />

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Transcriptional Studies<br />RNA Pol II unzipping profile<br />Has been achieved for RNA Polymerase I (E. coli)<br />Pol II analysis during initiation, elongation, and termination<br />Stalled Pol II in Elongation from collaborator (K. Adelman)<br />

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A Little About Telomeres<br />During Replication, ends of DNA are lost<br />Telomeric DNA caps ends to prevent disaster<br />Telomerase makes new telomere DNA from short RNA template<br />Wikipedia<br />

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Telomere Studies<br />Telomere mapping<br />Highly repetitive DNA<br />Not easily sequenced<br />Telomerase structure<br />T-loops<br />This DNA Molecule has<br />17 nearly identical<br />~200 bp repeats<br />Koch<br />Griffin et al.<br />

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Can I do it all?<br />Shotgun DNA Mapping<br />Transcription Unzipping<br />Collaborator ready and willing<br />Foundations for Chromatin Mapping<br />Which incorporates transcription<br />Telomere Mapping is gravy<br />Kinesin huge possibility (depending on funding)<br />

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Thank You Everyone!<br />sley<br />Lab<br />Toyoko and Cory too…<br />…And my committee!<br />

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Gel Electrophoresis<br />Electric field applied to charged molecules<br />DNA is negatively charged<br />Gel lattice causes smaller particles to travel faster than larger ones<br />Staining allows visualization of DNA<br />Direction of <br />DNA motion<br />

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Initial Studies<br />Using PHO5 as “calibrator”<br />PHO5 is promoter with 4 well know nucleosome positions<br />We can show mapping works<br />

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Unzipping Sensitivity<br />Unzipping can detect:<br />Insertions<br />Deletions<br />Inversions<br />Seen Right – DNA sequence with deletion (black) compared with original sequence (red)<br />

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Polymerase Chain Reaction<br />Needed to make anchor<br />Start with template DNA and primers<br />Taq polymerase replicates DNA from primer location<br />Undergoes multiple cycles of melting, annealing, and replicating (extension)<br />For anchor one primer has dig molecule attached (digitylated)<br />

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Trapping<br />0<br />

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Calibrating Trap Stiffness with free bead<br />viscosity<br />where<br />radius of particle<br />Power spectrum from<br />Fourier t’form<br />0, mass term insignificant in regime of frequency<br />

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Profile from Stuck Bead(used in calibrating trap)<br />

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Overview of Simulation<br />The simulation is based on a quasi-equilibrium model. This is achieved by calculating the expectation values for Force and unzipping index.<br />EFJC<br />EDNA<br />Bockelmann, U., & et al.(1997). Molecular Stick-Slip Motion Revealed by Opening DNA with Piconewton Forces. Physical Review Letters , 4489-4492<br />Wang, M. D ., & et al. (1997). Stretching DNA with Optical Tweezers. Biophysical Journal , 1335-1346.<br />

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Overview of Simulation<br />EDNAis the energy to break the base pairs.<br />EFJC<br />EDNA<br />Bockelmann, U., & et al.(1997). Molecular Stick-Slip Motion Revealed by Opening DNA with Piconewton Forces. Physical Review Letters , 4489-4492<br />Wang, M. D ., & et al. (1997). Stretching DNA with Optical Tweezers. Biophysical Journal , 1335-1346.<br />

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Overview of Simulation<br />EFJC is the energy of single stranded DNA. As the dsDNA unzips this increases.<br />EFJC<br />EDNA<br />Bockelmann, U., & et al.(1997). Molecular Stick-Slip Motion Revealed by Opening DNA with Piconewton Forces. Physical Review Letters , 4489-4492<br />Wang, M. D ., & et al. (1997). Stretching DNA with Optical Tweezers. Biophysical Journal , 1335-1346.<br />