BE 183 Applied Genomic Technologies                   Lecture 2 Genome Organization and Structure                  Trey Id...
Genome Organization•   Genome sizes and the C-value paradox•   DNA Hybridization: A basic technology•   Cot curves and gen...
Genome sizes and the C-value paradox                          Mol Bio Cell 4ed pp. 33
The most basic building block of DNA technology: DNA Hybridization, Denaturation and Annealing
DNA Hybridization Scheme
Hybridization - Heat denaturation, melting temperature (tm), other factors                                  Temperature, p...
Reassociation – the opposite of denaturation                                     1.0                                     0...
Reassociation kinetics- The Cot curveC = Concentration of ssDNAC0 = Initial ssDNA conc.                                   ...
Comparison of sequence copy number for     two organisms with different genome sizes                                Organi...
So why the striking difference in species? How     do we interpret the curve for cow?                                     ...
The Cot curve– many apparently “large” genomes           are filled with repetitive sequences           (resolution of the...
Genome Organization•   Genome sizes and the C-value paradox•   DNA Hybridization: A basic technology•   Cot curves and gen...
Satellite DNA               CsCl density gradient columnFig. 4.7The Cell: A Molecular Approach                13
14
15
Tandem vs. Interspersed repeats• Tandem  • Satellites, mini and microsatellites (VNTRs)• Interspersed  • Retrotransposons ...
Genome Structure•   Linear/Circular/Segmented•   Centromere/Telomere (TTAGGG)•   Origin of replication•   Heterochromatin/...
Mol Bio Cell 4edG-banding patterns of human chromosomes       pp. 199                                                 Giem...
Split genes: Introns and Exons                      Intron                      Exon- Point Mutation !                    ...
Distribution of exons in three speciesFigure 2.7 Primrose and Twyman                   20
Given these features, how might one write a                gene finder?                                         21        ...
Towards writing a gene finding program:Characteristics of Open Reading Frames (ORFs)Prokaryotes    •   contiguous ORFs, no...
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Biotech 2011-07-finding-orf-etc

  1. 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. 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. 3. Genome sizes and the C-value paradox Mol Bio Cell 4ed pp. 33
  4. 4. The most basic building block of DNA technology: DNA Hybridization, Denaturation and Annealing
  5. 5. DNA Hybridization Scheme
  6. 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. 7. Reassociation – the opposite of denaturation 1.0 0.8 0.6 C/C0 0.4 0.2 0.0 7
  8. 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. 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. 10. So why the striking difference in species? How do we interpret the curve for cow? 10
  11. 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. 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. 13. Satellite DNA CsCl density gradient columnFig. 4.7The Cell: A Molecular Approach 13
  14. 14. 14
  15. 15. 15
  16. 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. 17. Genome Structure• Linear/Circular/Segmented• Centromere/Telomere (TTAGGG)• Origin of replication• Heterochromatin/Euchromatin• GC content, GC isochores• CpG islands• Exons/Introns 17
  18. 18. Mol Bio Cell 4edG-banding patterns of human chromosomes pp. 199 Giemsa staining- AT rich Naming: e.g., 2p11
  19. 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. 20. Distribution of exons in three speciesFigure 2.7 Primrose and Twyman 20
  21. 21. Given these features, how might one write a gene finder? 21 21
  22. 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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