Hoofdstuk 20 2008 deel 1

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Hoofdstuk 20 2008 deel 1

  1. 1. Chapter 20 DNA technology
  2. 2. Outline of this chapter on techniques <ul><li>DNA analysis </li></ul><ul><ul><li>Restriction enzymes </li></ul></ul><ul><ul><ul><li>Recombinant DNA </li></ul></ul></ul><ul><ul><li>Libraries </li></ul></ul><ul><ul><li>Making cDNA </li></ul></ul><ul><ul><li>Hybridization </li></ul></ul><ul><ul><li>PCR, RT-PCR </li></ul></ul><ul><ul><ul><li>Reverse transcriptase-polymerase chain reaction </li></ul></ul></ul><ul><ul><li>Electrophoresis </li></ul></ul><ul><ul><li>Southern blotting </li></ul></ul><ul><ul><li>DNA sequencing </li></ul></ul><ul><ul><li>Micro-arrays </li></ul></ul><ul><ul><li>Cloning </li></ul></ul><ul><ul><ul><li>Plants </li></ul></ul></ul><ul><ul><ul><li>Bacteria </li></ul></ul></ul><ul><ul><ul><li>Animals </li></ul></ul></ul><ul><ul><ul><li>Use of stem cells </li></ul></ul></ul><ul><li>Practical applications </li></ul><ul><ul><li>SNPs </li></ul></ul><ul><ul><ul><li>Single nucleotide polymorphism </li></ul></ul></ul><ul><ul><li>RFLP, diagnosis of disease </li></ul></ul><ul><ul><li>Gene therapy </li></ul></ul><ul><ul><li>Transgenic plants </li></ul></ul><ul><li>DNA cloning </li></ul><ul><ul><li>Origin of gene </li></ul></ul><ul><ul><li>Use of a plasmid </li></ul></ul><ul><ul><li>Cloning gene into plasmid </li></ul></ul><ul><ul><li>Selection of recombinant bacteria </li></ul></ul><ul><ul><li>Multiplication of bacteria </li></ul></ul>Computermodule supports study of this chapter
  3. 3. “ Wat moet ik leren van dit hoofdstuk?” <ul><li>Te bestuderen: </li></ul><ul><ul><li>Afhankelijk van de nog beschikbare tijd, maar in ieder geval: </li></ul></ul><ul><ul><li>Kloneren van een gen in een plasmide </li></ul></ul><ul><ul><ul><li>Ook practicum! </li></ul></ul></ul><ul><ul><li>PCR: polymerase chain reaction </li></ul></ul><ul><ul><li>DNA sequencing </li></ul></ul>
  4. 4. 2 Overview of the use of a bacterial plasmid in cloning genes
  5. 5. 2 Overview of the use of a bacterial plasmid in cloning genes
  6. 6. 2 Overview of the use of a bacterial plasmid in cloning genes Transformation
  7. 7. 2 Overview of the use of a bacterial plasmid in cloning genes Transformation
  8. 8. 2 Overview of the use of a bacterial plasmid in cloning genes Transformation
  9. 9. 2 Overview of the use of a bacterial plasmid in cloning genes Transformation
  10. 10. 2 Applications of cloned genes
  11. 11. 3 Using a restriction enzyme to make recombinant DNA
  12. 12. 3 Using a restriction enzyme to make recombinant DNA <ul><li>Palindromic sequence: </li></ul><ul><ul><ul><li>GAA|TTC CTT|AAG </li></ul></ul></ul><ul><li>Sticky ends </li></ul><ul><ul><li>5’ overhang </li></ul></ul><ul><ul><ul><li>… .G ….CTTAA </li></ul></ul></ul><ul><ul><li>3’ overhang </li></ul></ul><ul><ul><ul><li>… .GAATT ….C </li></ul></ul></ul><ul><li>Blunt ends </li></ul><ul><ul><ul><li>… GAA …CTT </li></ul></ul></ul><ul><li>Restriction </li></ul><ul><ul><li>bacteria </li></ul></ul><ul><ul><li>modification by methylation </li></ul></ul><ul><li>Ligation gives two combinations!! </li></ul>
  13. 13. 3 Using a restriction enzyme to make recombinant DNA <ul><li>Palindromic sequence: </li></ul><ul><ul><ul><li>GAA|TTC CTT|AAG </li></ul></ul></ul><ul><li>Sticky ends </li></ul><ul><ul><li>5’ overhang </li></ul></ul><ul><ul><ul><li>… .G ….CTTAA </li></ul></ul></ul><ul><ul><li>3’ overhang </li></ul></ul><ul><ul><ul><li>… .GAATT ….C </li></ul></ul></ul><ul><li>Blunt ends </li></ul><ul><ul><ul><li>… GAA …CTT </li></ul></ul></ul><ul><li>Restriction </li></ul><ul><ul><li>bacteria </li></ul></ul><ul><ul><li>modification by methylation </li></ul></ul><ul><li>Ligation gives two combinations!! </li></ul>
  14. 14. 3 Using a restriction enzyme to make recombinant DNA <ul><li>Palindromic sequence: </li></ul><ul><ul><ul><li>GAA|TTC CTT|AAG </li></ul></ul></ul><ul><li>Sticky ends </li></ul><ul><ul><li>5’ overhang </li></ul></ul><ul><ul><ul><li>… .G ….CTTAA </li></ul></ul></ul><ul><ul><li>3’ overhang </li></ul></ul><ul><ul><ul><li>… .GAATT ….C </li></ul></ul></ul><ul><li>Blunt ends </li></ul><ul><ul><ul><li>… GAA …CTT </li></ul></ul></ul><ul><li>Restriction </li></ul><ul><ul><li>bacteria </li></ul></ul><ul><ul><li>modification by methylation </li></ul></ul><ul><li>Ligation gives two combinations!! </li></ul>
  15. 15. 3 Using a restriction enzyme to make recombinant DNA <ul><li>Palindromic sequence: </li></ul><ul><ul><ul><li>GAA|TTC CTT|AAG </li></ul></ul></ul><ul><li>Sticky ends </li></ul><ul><ul><li>5’ overhang </li></ul></ul><ul><ul><ul><li>… .G ….CTTAA </li></ul></ul></ul><ul><ul><li>3’ overhang </li></ul></ul><ul><ul><ul><li>… .GAATT ….C </li></ul></ul></ul><ul><li>Blunt ends </li></ul><ul><ul><ul><li>… GAA …CTT </li></ul></ul></ul><ul><li>Restriction </li></ul><ul><ul><li>bacteria </li></ul></ul><ul><ul><li>modification by methylation </li></ul></ul><ul><li>Ligation gives two combinations!! </li></ul>
  16. 16. 3 Using a restriction enzyme to make recombinant DNA <ul><li>Palindromic sequence: </li></ul><ul><ul><ul><li>GAA|TTC CTT|AAG </li></ul></ul></ul><ul><li>Sticky ends </li></ul><ul><ul><li>5’ overhang </li></ul></ul><ul><ul><ul><li>… .G ….CTTAA </li></ul></ul></ul><ul><ul><li>3’ overhang </li></ul></ul><ul><ul><ul><li>… .GAATT ….C </li></ul></ul></ul><ul><li>Blunt ends </li></ul><ul><ul><ul><li>… GAA …CTT </li></ul></ul></ul><ul><li>Restriction </li></ul><ul><ul><li>bacteria </li></ul></ul><ul><ul><li>modification by methylation </li></ul></ul><ul><li>Ligation gives two combinations!! </li></ul>
  17. 17. 4 & 5 Cloning a bird gene in a plasmid (COO + practicals!) <ul><li>Cloning vector </li></ul><ul><ul><li>A plasmid </li></ul></ul><ul><ul><li>Contains </li></ul></ul><ul><ul><ul><li>amp-gene: resistance against ampicillin </li></ul></ul></ul><ul><ul><ul><ul><li>a penicillin </li></ul></ul></ul></ul><ul><ul><li>lacZ gene (ß-galactosidase) </li></ul></ul><ul><ul><ul><li>Converts X-gal into a blue product </li></ul></ul></ul><ul><li>Screen for amp R , lacZ - </li></ul><ul><li>One colony is a clone from one bacterium </li></ul>
  18. 18. 4 & 5 Cloning a bird gene in a plasmid (COO + practicals!) <ul><li>Cloning vector </li></ul><ul><ul><li>A plasmid </li></ul></ul><ul><ul><li>Contains </li></ul></ul><ul><ul><ul><li>amp-gene: resistance against ampicillin </li></ul></ul></ul><ul><ul><ul><ul><li>a penicillin </li></ul></ul></ul></ul><ul><ul><li>lacZ gene (ß-galactosidase) </li></ul></ul><ul><ul><ul><li>Converts X-gal into a blue product </li></ul></ul></ul><ul><li>Screen for amp R , lacZ - </li></ul><ul><li>One colony is a clone from one bacterium </li></ul>Filmpje: cloning A library of hummingbird genes

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