Physical properties of DNA and Genome Complexity Problem: Chapter 8: 8 Study guide: p. 109: 11, 1, 3 p. 117: 25-27 Final Exam will be on Thurs May 13, 7:00 - 9:30 pm Denaturation and renaturation of DNA Study of the forces that hold DNA duplexes together Not a chemical reaction but a physical property of DNA Do not confuse with chemical or enzymatic hydrolysis of DNA or RNA It is mostly about H-bonds Simple principle about DNA that is a recurring theme in this course: DNA can be denatured and renatured based on the complementarity of bases. The most stable form of duplex DNA is when the maximum number of H-bonds are formed. Forces that hold the double helix together: 1. Hydrogen bonds - most important. A=T, G=C H-bond, about 1 kCal/mole (4.4 kJ/mole) cf C-C, about 80 kCal/mole (350 kJ/mole) cooperativity gives stability 2. Stacking forces - stacked hydrophobic Pu and Py base pairs allow interactions of electron clouds, van der Waal forces, about 0.5 kCal/mole cooperativity provides stability DG = DH - TDS H = forces that hold the helix together T = temperature (K°) S = entropy of random coils When DG < 0 (or TDS > DH) helix undergoes random coil transition When DG = 0 TDS = DH DNA is half melted Tm = DH/DS The double helix can be reversibly melted Heat or high pH slow fast cool Rigid rod No regular backbone Bases not stacked Viscous Less viscous Absorption spectra of the common nucleotides Measurement for NA concentrations Ch8:9 UV absorption can be used to characterize the physical state of N.A. 260 UV Absorption Spectra of Nucleic Acids 250 200 300 Wavelength, nm Absorption at 260 nm is increased 40% for denatured DNA viscous less viscous Hypochromic: UV260nm absorption is reduced in stacked bases Hyperchromic: UV absorption is increased by 30-40% in unordered bases Tm Sharp change at melting temp. indicates cooperative transition 70 80 90 100 Temperature (°C) 1.4 1.2 1.0 Relative absorbance (260nm) Zipper action Tm Tm Tm = DH/DS, DH depends on the base composition and length, DS is constant Tm can be used to predict base composition of DNA Melting temp (Tm) is dependent on: 1. The G+C content of the DNA molecule (and size) % denaturation = # H-bonds Melting temp (Tm) is dependent on: 2. The pH and ionic strength of solution P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Cations stabilize helix by electrostatic interactions Genome Complexities A.Cot curves (denaturation & renaturation of DNA) B.Genome sequencing (now) Genome relatedness A. DNA hybridization (denaturation & renaturation of DNA) B. Comparative genomics (now) Genomics and Proteomics: the study of genes and proteins at the scale of whole cells and organisms Determination of genome complexity based on rates of reassociation of denatured DNA - Cot curve (C/Co) DNA is sheared and denatured. Co = initial concentration of ss DNA in base pairs C = concentration of ss DNA at a specific time The rate at which 2 complementary strands find each other is dependent on concentration of the complementary sequences and time. Rot analysis - similar concept to measure the transcript levels of genes Repetitive DNA measured by Cot curve Types of repetitive DNA: 1. Satellite DNA, repeats of simple GC or AT-rich seq. 2. Moderately repetitive DNA such as the Alu sequence 3. Repetitive genes such as rRNA and tRNA genes 1-(C/Co) Types of sequences in the human genome Long interspersed elements (6-8kb) short IE (100-300 bp) LINEs 850,000 Retrotransposons 450,000 Simple sequence repeats SINEs 1.5 x 106 Large segmental duplications Genes encoding RNAs Fig 24-8 Given what you know about the complexity of the human genome, what would the Cot curve be like for human genomic DNA? DNA hybridization is a measure of the relatedness between species heat denatured The greater the seq homology, the larger # of hybrids formed, the more related the two species. DNA hybridization is a measure of the relatedness between species Tm #1 70 80 90 100 Temperature (°C) 1.4 1.2 1.0 Relative absorbance (260nm) Tm #2 a b c
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