Shotgun DNA MappingAnthony Salvagno

Welcome to KochLab!Single Molecule DNA AnalysisKinesin StudiesFFImage from Block and adapted by KochImage by Koch

Kinesin StudiesAndyGliding Motility AssaySurface PassivationLarryTrackingProcessivityBrigetteEnsemble ATP HydrolysisFutureBead Motility

Single Molecule DNA StudiesWhat is DNA?What is Shotgun DNA Mapping?What are Optical Tweezers?What is Molecular Biology?

DNA: The Code of LifeDouble stranded polymerCovalently bonded sugar molecules make up the backboneHydrogen bonded bases join two strands of DNAThere are 4 basesWhyfiles.org

DNA CompactionLots of DNA in a genome that needs to fit in the nucleusChromosomes – what we see in a cell through a microscopeChromatin – where everything happensMolecular Biology of the Cell

NucleosomesDNA wrapped in histone proteinsProteins:H2AH2BH3H4Form octamerWikipedia

From DNA to PeopleDNA to RNA to ProteinsKnown as gene expressionLeads to changes in characteristics between organismsLeads to differentiation amongst cell linesWikipediaThinkquest.org

TranscriptionRNA Polymerase II:Copies single strand of DNA to make RNA Moves with transcription bubbleInitiationRNAPII assemblyElongationActive transcriptionTerminationRNAPII disassemblyReassembled NucleosomesRNA Pol IIpromotercrypticpromoterTranscription

Points about Gene ExpressionMutations can affect many aspects of gene expressionPossible changes because of:DNA sequence modificationsPost Translational Modifications

Why Single Molecule is PowerfulBulk studies provide general insightInformation is average from all molecules in sampleDifferent molecules have different propertiesStudying DNA one molecule at a time can provide unprecedented understanding of a process

Examples of Single Molecule AnalysisRed Line – protein bound to DNABlack Line – naked DNABlack Dotted Line- predictions of protein locationsFFUnzipping can detect proteins bound to DNAKoch et al. 2002

Examples of Single Molecule Analysis IIUnzipping can detect nucleosomesnucleosome

Shotgun DNA MappingWant to understand how proteins affect gene expressionNeed a way to map sequences of DNA to location in genomeLibrary of Simulated CurvesRandom fragmentExperimental ForceEndonucleaseGenomic DNACorrect MatchdsDNA anchorStep 1: Digest genome into fragmentsStep 2: Unzip fragment and record forcesStep 3: Compare experimental forces to a library of simulated curves

Unzipping SimulationEnergy depends on:Energy of dsDNA (WLC)Energy of ssDNA (FJC)Energy of base-pairing (DNA)EFJCEDNA

Unzipping LibraryUsed Yeast Genome because less complex than human, but can still have ChromatinSimulated digestion with XhoIOver 1300 fragmentsSimulated unzipping 2000bp before and after recognition sequenceGives us over 2600 unzipping profilesUnzipping Direction

Proof of PrincipleSimulated unzipping of pBR322 plasmidSimulation info hidden in genomic simulationOld unzipping data (Koch) used for comparisonACorrect Match, Score 0.218Force (pN)1201500Unzipping fork index (bp)BMismatch, Score 0.818Force (pN)1201500Unzipping fork index (bp)

Match Data32 unzipped plasmid data compared to library Each time the best match score was the plasmid simulated data

How do we get real data?

Optical TweezersFocused laser light has the ability to trap small particlesSimplest trap is composed of just a laser and an objectiveSM Block

Optical TrapBead is tiny dielectric sphereLaser focus creates large E-field gradientBead attracted to center of focus

Data CollectionRefraction of laser from bead moves pathQPD tracks motion of beamForce in trap approx. as springF=-kxLa Porta Lab

Our Tweezers

How do we unzip DNA?Create unzipping constructCreate Shotgun fragment clones for single molecule analysisAttach pieces together and tether to cover slide

Create unzipping construct

Create Shotgun fragment clones for single molecule analysis

Attach pieces together and tether to cover slide

The Unzipping ConstructCourtesy of Diego

Restriction EnzymesREs recognize a specific sequence of DNA and cut the DNA at or near the site.

Piece by Piece Construct CreationAnchorMade from PCR of pRL574Has BstXI overhang with known base sequenceBeginning of polymer is labeled with dig molecule for specific binding with anti-digAdapterShort duplex made 2 single-stranded oligos5’ end has phosphate removed creating a nick5’ end has complementary BstXI overhang3’ end has SapI/EarI overhangSapIGCTCTTCNNNNNCGAGAAGNNNNNGCTCTTCN NNNNCGAGAAGNNNN NBstXICCANNNNNNTGGGGTNNNNNNACCCCANNNNN NTGGGGTN NNNNNACCRecall:

Ligating Construct to unzippable DNAAdapter oligo contains nick and biotinylated DNA baseNeed to ligate in specific wayLimited by genomic DNALow adapter duplex concentration, but gradually increase during the course of the reactionWhere does unzippable DNA come from?

Making Shotgun ClonesWhy clone?We can have a ton of a specific DNA fragmentSome for unzippingSome for sequencingWhat is shotgunning?Drinking a beer really fastCreating random fragments quickly

YeastWant pure genomic DNANeed to get rid of:Cell wall and membraneProteinsOrganellesNuclear membraneRNARPI.edu

Genome DigestionNeed to make fragments from pure genomic DNAXhoI digest produces very large fragmentsXhoI+EcoRI provides much smaller fragment sizesNeed smaller fragments for cloning

CloningPlasmids are circularized DNA with replication start sequencepBluescript allows for blue/white selectionEach colony contains different fragment of DNA Each cell in colony has the same fragmentWikipediaFermentas.com

DNA TetheringCreate flow cell from double stick tape, slide and coverslipFlow anti-dig, surface blocker, tethering DNA, microspheres, and wash sequencially

What’s Next?

Chromatin StudiesShotgun Chromatin MappingCan insert random fragments into yeast to get chromatinWant to map nucleosome and protein locationsOptical TrapnucleosomeElongating Pol IIssDNACoverglassKoch

Transcriptional StudiesRNA Pol II unzipping profileHas been achieved for RNA Polymerase I (E. coli)Pol II analysis during initiation, elongation, and termination

A Little About TelomeresDuring Replication, ends of DNA are lostTelomeric DNA caps ends to prevent disasterTelomerase makes new telomere DNA from short RNA templateWikipedia

Telomere StudiesTelomere mappingHighly repetitive DNANot easily sequencedTelomerase structureT-loopsThis DNA Molecule has17 nearly identical~200 bp repeatsKochGriffin et al.

Thank You Everyone!Toyoko and Cory too……And my committee!

Gel ElectrophoresisElectric field applied to charged moleculesDNA is negatively chargedGel lattice causes smaller particles to travel faster than larger onesStaining allows visualization of DNADirection of DNA motion

Initial StudiesUsing PHO5 as “calibrator”PHO5 is promoter with 4 well know nucleosome positionsWe can show mapping works

Unzipping SensitivityUnzipping can detect:InsertionsDeletionsInversionsSeen Right – DNA sequence with deletion (black) compared with original sequence (red)

Polymerase Chain ReactionNeeded to make anchorStart with template DNA and primersTaq polymerase replicates DNA from primer locationUndergoes multiple cycles of melting, annealing, and replicating (extension)For anchor one primer has dig molecule attached (digitylated)