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Biotech 2011-07-finding-orf-etc

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  • 1. BE 183 Applied Genomic Technologies Lecture 2 Genome Organization and Structure Trey Ideker Departments of Bioengineering and Medicine University of California San Diego
  • 2. Genome Organization• Genome sizes and the C-value paradox• DNA Hybridization: A basic technology• Cot curves and genome complexity• Repeated sequences• Introns and exons• Genome structure 2
  • 3. Genome sizes and the C-value paradox Mol Bio Cell 4ed pp. 33
  • 4. The most basic building block of DNA technology: DNA Hybridization, Denaturation and Annealing
  • 5. DNA Hybridization Scheme
  • 6. Hybridization - Heat denaturation, melting temperature (tm), other factors Temperature, pH, size (# bp’s), G/C to A/T ratio, ionic strength, chem denaturants, detergents, chaotropics Stringency (high and low)
  • 7. Reassociation – the opposite of denaturation 1.0 0.8 0.6 C/C0 0.4 0.2 0.0 7
  • 8. Reassociation kinetics- The Cot curveC = Concentration of ssDNAC0 = Initial ssDNA conc. dCk = reassociation rate const. = −kC 2t1/2 = reassociation half time dtBig C0t1/2 = Slow reassociation C 1This value is proportional to the = number of different types of DNA fragments C0 1 + kC0t 8
  • 9. Comparison of sequence copy number for two organisms with different genome sizes Organism A Organism B Starting DNA 10 pg/ml 10 pg/ml concentration Genome size 0.01 pg 2 pg # genome 1000 5 copies/ml Relative 200 1 concentrationTable 2.1 Primrose and Twyman 9
  • 10. So why the striking difference in species? How do we interpret the curve for cow? 10
  • 11. The Cot curve– many apparently “large” genomes are filled with repetitive sequences (resolution of the C-value paradox)Fig. 4.6The Cell: A Molecular Approach 11
  • 12. Genome Organization• Genome sizes and the C-value paradox• DNA Hybridization: A basic technology• Cot curves and genome complexity• Repeated sequences• Introns and exons• Genome structure 12
  • 13. Satellite DNA CsCl density gradient columnFig. 4.7The Cell: A Molecular Approach 13
  • 14. 14
  • 15. 15
  • 16. Tandem vs. Interspersed repeats• Tandem • Satellites, mini and microsatellites (VNTRs)• Interspersed • Retrotransposons (class I) Autonomous LINE (10% of human genome) • Transposons (class II) Non-autonomous SINE (Alu- 10% of human genome)Resources: Repbase, RepeatMasker 16
  • 17. Genome Structure• Linear/Circular/Segmented• Centromere/Telomere (TTAGGG)• Origin of replication• Heterochromatin/Euchromatin• GC content, GC isochores• CpG islands• Exons/Introns 17
  • 18. Mol Bio Cell 4edG-banding patterns of human chromosomes pp. 199 Giemsa staining- AT rich Naming: e.g., 2p11
  • 19. Split genes: Introns and Exons Intron Exon- Point Mutation ! Sequence of Beta-Globin Gene Exon Hemoglobin Protein Structure Intron Lehninger pp. 196 & 189
  • 20. Distribution of exons in three speciesFigure 2.7 Primrose and Twyman 20
  • 21. Given these features, how might one write a gene finder? 21 21
  • 22. Towards writing a gene finding program:Characteristics of Open Reading Frames (ORFs)Prokaryotes • contiguous ORFs, no introns • very little intergenic sequence • with f(A,C,G,T) = 25%, ORF>300 bp every 36 kb on a single strand • detecting large ORFs is a very good predictor for genes (with good specificity)Eukaryotes • typically 6 exons (150 bp) over ~30 kb • Exceptions • 2.4 Mb (dystrophin gene) • 186 kb with 26 exons (69-3106 bp), 32.4 kb intron (blood coagulation factor VIII gene) • ORFs >225 bp randomly every kb on a single strand • detecting ORFs is NOT a good predictor for eukaryotic genes 22

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