Hoofdstuk 20 2008 deel 3

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Hoofdstuk 20 2008 deel 3

  1. 1. 12a DNA sequencing: the Sanger method <ul><li>Nobel prize to David Sanger </li></ul><ul><li>Use of dideoxy-nucleotides or ddNTPs </li></ul><ul><li>ddNTP can be incorporated but not elongated </li></ul>
  2. 2. <ul><li>Primer: </li></ul><ul><ul><li>Short (15-20) string of dNTPs </li></ul></ul><ul><ul><li>Hybridizes to complementary strand </li></ul></ul><ul><ul><li>Primes DNA synthesis (Ch 16!) </li></ul></ul>12b DNA sequencing: the Sanger method 3’ 5’ 3’ 5’ Taq 5’ 3’
  3. 3. 12c DNA sequencing: the Sanger method
  4. 4. 12c DNA sequencing: the Sanger method
  5. 5. 12c DNA sequencing: the Sanger method
  6. 6. 12c DNA sequencing: the Sanger method Alberts 4 th ed 8.37
  7. 7. 12c DNA sequencing: the Sanger method Alberts 4 th ed 8.37
  8. 8. 15 Micro-array analysis Measures gene expression levels of many genes <ul><li>Procedure </li></ul><ul><li>Isolate mRNA </li></ul><ul><ul><li>Cells, tissues, tumors,.. </li></ul></ul><ul><li>Make cDNA </li></ul><ul><ul><li>Using fluorescent nucleotides </li></ul></ul><ul><li>Hybridize cDNA with array-chip </li></ul><ul><li>Chip contains short sequence of many genes </li></ul><ul><ul><li>Primers </li></ul></ul><ul><ul><li>PCR-products </li></ul></ul><ul><ul><li>… </li></ul></ul>
  9. 9. 15 Micro-array analysis Measures gene expression levels of many genes <ul><li>Procedure </li></ul><ul><li>Isolate mRNA </li></ul><ul><ul><li>Cells, tissues, tumors,.. </li></ul></ul><ul><li>Make cDNA </li></ul><ul><ul><li>Using fluorescent nucleotides </li></ul></ul><ul><li>Hybridize cDNA with array-chip </li></ul><ul><li>Chip contains short sequence of many genes </li></ul><ul><ul><li>Primers </li></ul></ul><ul><ul><li>PCR-products </li></ul></ul><ul><ul><li>… </li></ul></ul>
  10. 10. 15 Micro-array analysis Measures gene expression levels of many genes <ul><li>Procedure </li></ul><ul><li>Isolate mRNA </li></ul><ul><ul><li>Cells, tissues, tumors,.. </li></ul></ul><ul><li>Make cDNA </li></ul><ul><ul><li>Using fluorescent nucleotides </li></ul></ul><ul><li>Hybridize cDNA with array-chip </li></ul><ul><li>Chip contains short sequence of many genes </li></ul><ul><ul><li>Primers </li></ul></ul><ul><ul><li>PCR-products </li></ul></ul><ul><ul><li>… </li></ul></ul>
  11. 11. 15 Micro-array analysis Measures gene expression levels of many genes <ul><li>Procedure </li></ul><ul><li>Isolate mRNA </li></ul><ul><ul><li>Cells, tissues, tumors,.. </li></ul></ul><ul><li>Make cDNA </li></ul><ul><ul><li>Using fluorescent nucleotides </li></ul></ul><ul><li>Hybridize cDNA with array-chip </li></ul><ul><li>Chip contains short sequence of many genes </li></ul><ul><ul><li>Primers </li></ul></ul><ul><ul><li>PCR-products </li></ul></ul><ul><ul><li>… </li></ul></ul>
  12. 12. 15 Micro-array analysis Measures gene expression levels of many genes <ul><li>Procedure </li></ul><ul><li>Isolate mRNA </li></ul><ul><ul><li>Cells, tissues, tumors,.. </li></ul></ul><ul><li>Make cDNA </li></ul><ul><ul><li>Using fluorescent nucleotides </li></ul></ul><ul><li>Hybridize cDNA with array-chip </li></ul><ul><li>Chip contains short sequence of many genes </li></ul><ul><ul><li>Primers </li></ul></ul><ul><ul><li>PCR-products </li></ul></ul><ul><ul><li>… </li></ul></ul>
  13. 13. Only briefly mentioned techniques <ul><li>But very important </li></ul><ul><li>Not tested for these in exam! </li></ul>Knock-out In vitro mutagenesis RNA interference Transgenic animals Homologous recombination SNPs STR RFLPs
  14. 14. 16-18 Can a differentiated cell lead to a new organism? The Dolly-affair <ul><li>Procedure </li></ul><ul><li>Isolate donor cell </li></ul><ul><li>Isolate nucleus </li></ul><ul><li>Isolate oocyte </li></ul><ul><li>Remove DNA from egg </li></ul><ul><li>Inject nucleus into egg </li></ul><ul><li>Grow </li></ul><ul><li>Implant </li></ul><ul><li>Wait </li></ul>
  15. 15. 16-18 Can a differentiated cell lead to a new organism? The Dolly-affair <ul><li>Procedure </li></ul><ul><li>Isolate donor cell </li></ul><ul><li>Isolate nucleus </li></ul><ul><li>Isolate oocyte </li></ul><ul><li>Remove DNA from egg </li></ul><ul><li>Inject nucleus into egg </li></ul><ul><li>Grow </li></ul><ul><li>Implant </li></ul><ul><li>Wait </li></ul>
  16. 16. 16-18 Can a differentiated cell lead to a new organism? The Dolly-affair <ul><li>Procedure </li></ul><ul><li>Isolate donor cell </li></ul><ul><li>Isolate nucleus </li></ul><ul><li>Isolate oocyte </li></ul><ul><li>Remove DNA from egg </li></ul><ul><li>Inject nucleus into egg </li></ul><ul><li>Grow </li></ul><ul><li>Implant </li></ul><ul><li>Wait </li></ul>
  17. 17. 16-18 Can a differentiated cell lead to a new organism? The Dolly-affair <ul><li>Procedure </li></ul><ul><li>Isolate donor cell </li></ul><ul><li>Isolate nucleus </li></ul><ul><li>Isolate oocyte </li></ul><ul><li>Remove DNA from egg </li></ul><ul><li>Inject nucleus into egg </li></ul><ul><li>Grow </li></ul><ul><li>Implant </li></ul><ul><li>Wait </li></ul>
  18. 18. 16-18 Can a differentiated cell lead to a new organism? The Dolly-affair <ul><li>Procedure </li></ul><ul><li>Isolate donor cell </li></ul><ul><li>Isolate nucleus </li></ul><ul><li>Isolate oocyte </li></ul><ul><li>Remove DNA from egg </li></ul><ul><li>Inject nucleus into egg </li></ul><ul><li>Grow </li></ul><ul><li>Implant </li></ul><ul><li>Wait </li></ul>
  19. 19. 20 Stem cells <ul><li>Very hot issue </li></ul><ul><li>Medical applications </li></ul>
  20. 20. 20 Stem cells <ul><li>Very hot issue </li></ul><ul><li>Medical applications </li></ul>
  21. 21. 20 Stem cells <ul><li>Very hot issue </li></ul><ul><li>Medical applications </li></ul>
  22. 22. 20 Stem cells <ul><li>Very hot issue </li></ul><ul><li>Medical applications </li></ul>
  23. 23. 22 Gene therapy <ul><li>DNA technology to correct genetic disorders </li></ul><ul><li>Especially suitable for blood disorders </li></ul><ul><ul><li>Proven correction in 2002! </li></ul></ul><ul><li>Procedure </li></ul><ul><li>Clone gene </li></ul><ul><li>Insert in virus </li></ul><ul><li>Isolate cells </li></ul><ul><li>Grow cells </li></ul><ul><li>Infect cells </li></ul><ul><li>Select right cells </li></ul><ul><li>Inject corrected cells </li></ul>
  24. 24. 22 Gene therapy <ul><li>DNA technology to correct genetic disorders </li></ul><ul><li>Especially suitable for blood disorders </li></ul><ul><ul><li>Proven correction in 2002! </li></ul></ul><ul><li>Procedure </li></ul><ul><li>Clone gene </li></ul><ul><li>Insert in virus </li></ul><ul><li>Isolate cells </li></ul><ul><li>Grow cells </li></ul><ul><li>Infect cells </li></ul><ul><li>Select right cells </li></ul><ul><li>Inject corrected cells </li></ul>
  25. 25. Diagnosis of disease <ul><li>Made possible by DNA technology: </li></ul><ul><ul><li>PCR </li></ul></ul><ul><ul><ul><li>Detect virus RNA or DNA in serum: HIV! </li></ul></ul></ul><ul><ul><li>Detecting abnormal forms of genes </li></ul></ul><ul><ul><ul><li>SNPs </li></ul></ul></ul><ul><ul><ul><ul><li>Single nucleotide polymorphism </li></ul></ul></ul></ul><ul><ul><ul><li>STR analysis </li></ul></ul></ul><ul><ul><ul><li>RFLP: Huntington </li></ul></ul></ul><ul><ul><ul><li>DNA sequencing </li></ul></ul></ul>
  26. 26. 25 Transgenic organisms <ul><li>Genetic engineering of bacteria </li></ul><ul><ul><li>Cleaning water </li></ul></ul><ul><li>Sheep, goats </li></ul><ul><ul><li>Protein in milk </li></ul></ul><ul><li>Plants </li></ul><ul><ul><li>Relatively easy </li></ul></ul><ul><ul><ul><li>one cell will develop into plant </li></ul></ul></ul>
  27. 27. Golden rice <ul><li>Genes for synthesis of ß-carotene inserted into rice DNA </li></ul><ul><ul><li>Vitamin A deficiency </li></ul></ul>
  28. 28. Summary of the techniques <ul><li>DNA analysis </li></ul><ul><ul><li>Electrophoresis </li></ul></ul><ul><ul><li>Sequencing </li></ul></ul><ul><ul><li>Southern blotting </li></ul></ul><ul><ul><li>RFLP </li></ul></ul><ul><li>Practical applications </li></ul><ul><ul><li>Diagnosis of disease </li></ul></ul><ul><ul><li>Gene therapy </li></ul></ul><ul><ul><li>Fingerprints-RFLP </li></ul></ul><ul><ul><li>Genetic engineering </li></ul></ul><ul><li>DNA cloning </li></ul><ul><ul><li>Restriction enzymes </li></ul></ul><ul><ul><li>Recombinant DNA </li></ul></ul><ul><ul><li>Cloning a gene into a plasmid </li></ul></ul><ul><ul><li>DNA libraries </li></ul></ul><ul><ul><li>PCR </li></ul></ul>

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