Review for Mid-term When: Tuesday, Oct 20, 2015 In class TA Review sessions: go over homeworks Sunday 3-5 pm Monday 4-5:30 pm or 6 pm. Biophysics Office, rm 176 LLP. Formula Sheet– have by Friday at 5pm

Stretching DNA: WLC Fits very well at all stretches

Torsionally stressed single DNA molecule T. Strick et al., J. Stat. Phys., 93, , 1998 Extension vs. supercoiling at constant force Three regimes symmetric under   -  The shortening corresponds to the formation of plectonemes upon writhing. When the force is increased above 0.5 pN, the curve becomes asymmetric: supercoils still form for positive coiling while local denaturation adsorbs the torsional stress for negative . At forces larger than 3 pN no plectonemes are observed: the torsional stress is adsorbed not by writhe but in local structural changes of the molecule. Playing with phone cord: can you explain graphs? Low F: Medium F (>0.5 pN): Large F (>3 pN):

Over- and under-stretching Rotation extension curves for different forces. At higher forces one cannot induce supercoils but denature the DNA molecule. Upon twisting a DNA molecule it takes a number of turns, before the DNA length reduces significantly and plectonemes are formed. The point (N buckling ) where DNA starts to form plectonemes with a constant length reduction per turn is called buckling instability

Reading Material Reading Material: The New Genetics Reading Material: Biochemistry and the Genomic Revolution Reading Material: Introductions to Molecular Biophysics and-genetics/v/dna-deoxyribonucleic-acid and-genetics/v/dna-deoxyribonucleic-acid and-genetics/v/rna-transcription-and-translation and-genetics/v/rna-transcription-and-translation The molecular Biology of the Genes Beginning: DNA  pre-RNA  mRNA  Protein

Lec1: Introduction, Dogma of Mol Bio; Evolution Lec2: Boltzmann, Free Energy, Equilibrium Constant Lec3: The origin of life on earth; atomic structure of the DNA double-helix Lec4: DNA & X-Ray Diffraction Lec5: How to make and use ATP Lec6: ATPase and Chromosome Sequencing Lec7: Sequencing DNA&PCR Lec8: Sequencing DNA and RNA Lec9: Protein Folding Lec 10: Klaus (VMD)—How after test— Lec11 Introduction to DNA binding and twisting Lec12 Mag Tweezer Lec13 Mag Tweezer Lec 14 Mag Tweezers and Review Lecture Material

You must know basic biology DNA  RNA  Proteins (previously shown) You must know that one gene is NOT one polypeptide (in Eukaryotes) because of pre-RNA vs. mRNA. introns, exons, implications for complexity/regulation. Alternative splicing where some exons are cut out and therefore you get multiple proteins from a single gene. >90% of our DNA are introns. Excellent discussion of alternative splicing: Why are we different than C. elegans (and other organisms) even though we have approximately the same number of genes? Different proteins and different side-groups; make sure that you know what type of bonds (covalent, ionic, Hydrogen, van der Waals). Protein folding is important and different types of bonds play a role in this.

Know basic chemistry Know different types of bonds (Covalent, ionic, Hydrogen, van der Waals). Why made up of carbon. What causes things to be stable? – Free Energy height vs. activation energy – Whether you get (free) energy out of food or have to use energy. (b) Hydrolysis: breaking down a polymer Hydrolysis is addition of a water molecule, breaking a bond Enzymes (Catalyst)

Know  G =  E –T  S Why you can replace  E with  G when you have degeneracy (i.e. have entropy) in system.  G and K eq. Boltzmann’s constant: Probability of being in a state with E i is exp(-E i /k B T). If there is more than on state with energy E i (  ; S= k B ln  ), use G i. You must know basic Statistical Mechanics

Bio Techniques: Protein Folding Protein Folding– know how to use the simple model of hydrophobic and hydrophilic bonding pattern to get  G vs. T. Native state IBIB IAIA Unfolded state Intermediate states ENERGY ENTROPY Molten Globule State Know general features of folding landscape.

X-ray Diffraction Why it’s so powerful (get atomic resolution) Specific example of DNA fibers. Change DNA structure: how does x-ray structure change. Change x-ray structure: what does this say about DNA structure Bio-Techniques: X-ray Diffraction

Bio-Techniques: Sequencing DNA Sequencing DNA Idea of shot-gun sequencing. DNA polymerase goes till it stops with dTTP, or dATP, or dGTP, or dCTP. Run it out on gel. You can do with fluorescence. Shot-gun method and then recombine Single molecule sequencing –PacBio (see next slide) Long read lengths; less recombination Have to make it such that you can read a single one—get sensitivity; reducing background (restricting volume of illumination

Bio-Techniques: Pac-Bio Sequencing DNA

All different levels: Comparing genes: prenatal diagnostics; Whole Chromosomes: Trisomy 21; Genes: FISH-Fluorescence In Situ Hybridization Polymerase Chain Reaction (PCR) Identify you uniquely cause you have unique DNA: Forensics (Rape), Military, Sequencing ancient people. Basic (and Clinical) Science: DNA Microarrays (using mRNA or DNA) Bio-Techniques: Comparing DNA sequencing

Bio-Techniques: Comparing DNA/RNA sequencing Comparing different species

DNA bending and twisting Bending—persistence length. Nucleosomes. Twisting—over-and under-winding DNA. Supoercoiling. Detect position of magnetic particle by microscope (very good position in x,y,z) Apply force with magnetic field: determine field strength by Brownian motion Bio-Techniques: Magnetic Tweezers

Class evaluation 1. What was the most interesting thing you learned in class today? 2. What are you confused about? 3. Related to today’s subject, what would you like to know more about? 4. Any helpful comments. Answer, and turn in at the end of class.