96 97 98 99 00 95 20 40 60 80 100 Genom e projects year Genetics Genomics
  279 completed and  543  m icrobial  g enomes  i n- p rogress          Bos   taurus  (cow)         Canis   familiaris  (d...
1000 microbial genomes 4,000 genes each, 25 % unique i.e. 1.000.000  unique  genes Bible  -   12,143  different words (out...
<ul><li>Functional genomics  </li></ul><ul><li>Genome libraries and their screening </li></ul><ul><li>DNA microarrays (exp...
Genome libraries and their screening
Minimal set of clones 19 klon ů ( E. coli  DH10B pBeloBAC11)
Functional screening of genomic library With immunized serum
 
DNA microarrays –  regulatory gene identifications
DNA microarrays –  regulatory gene identifications
 
Cloning of individual PCR products into plasmid pUNI-D-TOPO Fusion of pUNI and pHOST plasmids
Detection of antibodies using ELISA Statistically significant reactions of immunized serum with bacterial proteins Statist...
<ul><li>Komparative genomics </li></ul><ul><li>Genome sequencing </li></ul><ul><li>DNA microaarrays (with PCR products) </...
Comparison of two bacterial genomes using DNA microaarrays containing PCR products 3113 individual hybridizations
DNA fingerprinting P4 Genomic DNA Interval   A Interval B Primer control Restriction digest P1 P2 P3 XL PCR
in  T. pallidum  subsp.  pertenue  Samoa D TPI48 interval  1 – standard (1 kb) 2, 7, 12 –  T. pallidum  subsp.  pallidum  ...
 
DNA Genome comparisons  (NimbleGen) 29-mers (with  22   bp  overlaps) 2 x 162 574  hybridizations  per array Phase II. Seq...
in situ   oligonucleotide synthesis NimbleGen Systems Inc. <ul><li>Digital Micromirror Device (DMD) </li></ul><ul><li>Up t...
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L13 functional and_comparative_genomics

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L13 functional and_comparative_genomics

  1. 1. 96 97 98 99 00 95 20 40 60 80 100 Genom e projects year Genetics Genomics
  2. 2.   279 completed and 543  m icrobial g enomes i n- p rogress      Bos taurus (cow)      Canis familiaris (dog)      Felis catus (cat)      Homo sapiens (human)      Mus musculus (mouse)      Ovis aries (sheep)      Pan troglodytes (chimpanzee)      Rattus norvegicus (rat)      Sus scrofa (pig) Plasmodium falciparum      Danio rerio ( zebrafish )      Gallus gallus (chicken) Anopheles gambiae (mosquito) Apis mellifera (honey bee) Drosophila melanogaster (fruit fly Strongylocentrotus purpuratus (purple sea urchin) Arabidopsis thaliana ( thale cress) Avena sativa (oat) Glycine max (soybean) Hordeum vulgare (barley) Lycopersicon esculentum (tomato) Oryza sativa (rice) Triticum aestivum (wheat) Zea mays (corn)
  3. 3. 1000 microbial genomes 4,000 genes each, 25 % unique i.e. 1.000.000 unique genes Bible - 12,143 different words (out of 783,137) Complete works of Shakespeare - 24,000 differing words There are fewer than 100,000 words in the French language 895,000 words in the English Language 1 gram of soil: 5,000 – 38,000 of different bacterial species
  4. 4. <ul><li>Functional genomics </li></ul><ul><li>Genome libraries and their screening </li></ul><ul><li>DNA microarrays (expression profiling) </li></ul><ul><li>Heterologous expression </li></ul>
  5. 5. Genome libraries and their screening
  6. 6. Minimal set of clones 19 klon ů ( E. coli DH10B pBeloBAC11)
  7. 7. Functional screening of genomic library With immunized serum
  8. 9. DNA microarrays – regulatory gene identifications
  9. 10. DNA microarrays – regulatory gene identifications
  10. 12. Cloning of individual PCR products into plasmid pUNI-D-TOPO Fusion of pUNI and pHOST plasmids
  11. 13. Detection of antibodies using ELISA Statistically significant reactions of immunized serum with bacterial proteins Statisticky signifikantní chemiluminiscenční signál 56 dní po infekci Před infekcí 56 dní po infekci
  12. 14. <ul><li>Komparative genomics </li></ul><ul><li>Genome sequencing </li></ul><ul><li>DNA microaarrays (with PCR products) </li></ul><ul><li>Genome fingerpri n ts </li></ul><ul><li>DNA oligonucleotide microaarrays </li></ul>
  13. 15. Comparison of two bacterial genomes using DNA microaarrays containing PCR products 3113 individual hybridizations
  14. 16. DNA fingerprinting P4 Genomic DNA Interval A Interval B Primer control Restriction digest P1 P2 P3 XL PCR
  15. 17. in T. pallidum subsp. pertenue Samoa D TPI48 interval 1 – standard (1 kb) 2, 7, 12 – T. pallidum subsp. pallidum Nichols 3, 8, 13 – T. paraluiscuniculi 4, 9, 14 – T. pallidum subsp. pertenue Samoan D 5, 10, 15 – T. pallidum subsp. pallidum SS14/4934 6, 11, 16 – T. pallidum subsp. pallidum SS14/4933 <ul><li>deletion 2, 5 kb u T. paraluiscuniculi </li></ul><ul><li>different restriction pattern for Hin d III </li></ul>11460 bp 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 10 kb 3 kb 1 kb Eco R I Hin d III Bam H I
  16. 19. DNA Genome comparisons (NimbleGen) 29-mers (with 22 bp overlaps) 2 x 162 574 hybridizations per array Phase II. Sequencing chip DNA sequencing Each nucleotide is detected by at least 8 oligos DNA
  17. 20. in situ oligonucleotide synthesis NimbleGen Systems Inc. <ul><li>Digital Micromirror Device (DMD) </li></ul><ul><li>Up to 3 86.000 features per chip </li></ul>

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