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Chapt “7:” Recombinant DNA and Genomics

Chapt “7:” Recombinant DNA and Genomics. Goals: What is recombinant DNA technology? What is genomics? ……. a nutshell, sort of. Structure of nucleotides. Base, nucleoside, nucleotide, D/RNA (polymer). Bases- diversity of life thru permutations of FOUR bases. RNA: U, C, G, A ---NTP

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Chapt “7:” Recombinant DNA and Genomics

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  1. Chapt “7:” Recombinant DNA and Genomics Goals: • What is recombinant DNA technology? • What is genomics? ……. a nutshell, sort of

  2. Structure of nucleotides • Base, nucleoside, nucleotide, D/RNA (polymer)

  3. Bases- diversity of life thru permutations of FOUR bases • RNA: U, C, G, A ---NTP • DNA: T, C, G, A ---dNTP

  4. DNA: alpha helix, gouble strand complementary and antiparallel

  5. De/Re- naturation

  6. DNA-protein

  7. DNA cloning vectors Natural biology-chemistry Bacterial processes Palindromes NNNACGTNN-> NNNA CGTNN NNNTGCANN NNNTGC ANN CGNNN NNNGC Enzymes Biotechnology

  8. Practice

  9. Restriction/modification

  10. Restriction endonucleases

  11. Fine tuning recombinant tech

  12. Other ways to clone

  13. Genes, ESTs

  14. Gel electrophoresis: paper, starch, polyacrylamide, agarose

  15. Agarose, acrylamide

  16. DNA sequencing

  17. Application of replication biochemistry: DNA sequencing

  18. DNA sequencing chemistry: Fluorescence-based methodology

  19. Biotechnology drives basic biology drives biotechnology….ABI series: 370, 373 and 377 • Non-radioactive. • Semi-automated and automate-able. • Large scale and high throughput operations- industrial strength! • Allows more questions. • Presents bioinformatics challenges or “opportunities.”

  20. Screen ABI373A: multiplex 4->1

  21. Capillary electrophoresis

  22. Base calling and sequence assembly issues

  23. Basecalling issues

  24. ABI 377 data

  25. Real world data: Sequencing progress Ad 4

  26. Ad 1 assembly: Consensus

  27. MAP alignment of variola and vaccinia consensus sequences

  28. BLAST etc • Human neurofibromatosis NF-1 • Yeast Ira “GAP-type protein • both regulate Ras • Inappropriate cell division, tumor formation

  29. Applications of large-scale DNA technology

  30. Applications to biological systems • Immunology. • T-cell biology. • Molecular diagnostics. • Thyroid hormone resistance. • Pharmaceutical and Biotech. • Gene discovery and target validation. • HIV drug development. • Gene expression: Fluorescent differential display. • Comparative virology. • Adenoviruses. • Human adenoviruses. • Poxviruses.

  31. 1975 1980 1990 2000 Biology: Science in Transition Genetic Engineering DNA Sequencing Automated DNA Sequencing Human Genome Project H. Influenzae C. elegans • Biology is changing rapidly from a “data-poor” to a data-rich and information-rich science. • “New” Fields: • Computational Biology • Bioinformatics • Systems biology Human Genome PCR DNA Chips Growth of Genome Databases

  32. Comparative genomics- Why? • Faster cheaper better technology and methodology. • Even more so for multiple similar additional genomes. • Scientific and funding interests- • Example: E. coli K12 vs O157:H7. • ca. 1Mb more DNA. • 1400 new genes. • 5 additional strains in progress. • Example: ca. 20 different Bacillus anthracis strains. • Example: Staphylococcus aureus- 5 strains. • Example: Chlamydia pneumoniae- 5 strains.

  33. Conserved genes of poxviruses- need more tools Bacterial genomes: Need to build better “mouse trap.”

  34. GCG SeqWeb Compare: Adenovirus genomes

  35. FLAG: Fast Local Alignment for Gigabases

  36. Genomics data mining: “BreakThrough” strains • Ad “BreakThrough 4/5” strain. • Isolated from a vaccinated recruit. • Serotyped as Ad4 by microneutralization. • Genome determination and analysis suggest it is Ad5. • BLAST; genome alignments; hexon and fiber trees. • Re-serotyped as Ad5. • BT4/5 hexon sequence is identical to Ad5.

  37. Poxvirus genomes: GeneOrder analysis I

  38. Bioinformatics Mycoplasma genitalium ORFs

  39. devGeneOrder3.0: Application to small bacterial genomes, ca. 2002 Mycoplasmasizedatepredicted coding regions M. genitalium 580kb 01/8/01 470 M. pneumoniae 816kb 04/2/01 688 M. pulmonis 964kb 10/2/01 782* • *Functions assigned to 486; 92 match hypothetical proteins; 204** without significant matches. • Gene annotation. • **204 potential targets for pharma and diagnostics. • How many unique targets within Mycoplasma group?

  40. devGeneOrder3.0 application to small bacterial genomes

  41. Whole genome snapshots

  42. Microarray expt: yeast mRNAs • Cell growth ~ C source

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