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Advanced Gene Technology

Advanced Gene Technology. DNA,RNA, Recombinant DNA Technology. Nucleic Acids – DNA and RNA. DNA + RNA. DNA + RNA. DNA + RNA. RNA. DNA. DNA structure -> sequence. Polymerase reaction: 5’-> 3’. The central dogma. Page 93. Gene expression. Page 93. What is a gene?.

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Advanced Gene Technology

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  1. Advanced Gene Technology

  2. DNA,RNA, Recombinant DNA Technology

  3. Nucleic Acids – DNA and RNA

  4. DNA + RNA DNA + RNA DNA + RNA RNA DNA

  5. DNA structure -> sequence

  6. Polymerase reaction: 5’-> 3’

  7. The central dogma Page 93

  8. Gene expression. Page 93

  9. What is a gene?

  10. Eukaryotes – Intron-Exon concept Page 95

  11. DNA mRNA Ribosome Polymerase Protein

  12. Recombinant DNA Technology

  13. Recombinant DNA Technology Clones -> Cells or organisms with identical DNA

  14. Restrictionendonucleases 5’-> 3’ 3’ <- 5’

  15. Gel Electrophoresis

  16. Gel Electrophoresis

  17. Gel Electrophoresis

  18. X-Ray structure of a complex of ethidium bromid with DNA. Page 1125

  19. Construction of a restriction map. Page 104

  20. Restriction map for the 5243-bp circular DNA of SV40. Page 104

  21. Construction of a recombinant DNA molecule through the use of synthetic oligonucleotide adaptors Page 109

  22. Ligation conditions • Temperature: 4º-10ºC -> takes long time 16ºC -> good temperature, but maybe inconvenient RT (room temp) -> much faster, compromise • Concentration of DNA : high -> intermolecular (between different molecules) ligation fevered low -> intramolecular (within one molecule) ligation fevered • Optimal vector-insert ratio: from 1:3 to 3:1 (molar ratio -> vector: insert) depending on size e.g.: vector: 5kb + insert: 500 bp -> molar ratio of 1:1 -> 500ng vector + 50 ng insert vector: 6kb + insert: 50kb -> 1:1 -> 500ng vector + 5ug insert -> WV/SV:WI/SI

  23. Plasmid Cloning Vectors

  24. Plasmid Cloning Vectors

  25. Insertional inactivation Gene in cloning site: • LacZ -> pUC18 (lacZ complements the host defect in lacZ) -> pUC18 into host organism -> active lacZ (β-galactosidase) from plasmid-> cleavage of X-gal (blue colonies) -> gene cloned into polylinker -> lacZ gene disrupted -> no cleavage of X-gal (white colonies)

  26. Insertional inactivation Gene in cloning site: • Resistance marker -> pBR322 (cloning sites within antibiotica resistence marker) -> plasmid into host -> resistance against 2 antibiotica -> gene cloned within one resistance marker -> gene for antibiotica resistance marker disrupted -> sensitive against one antibioticum

  27. Transformation and Selection

  28. Horizontal gene transfer • - Transformation -> uptake of naked DNA (chemical transformation, electroporation) • - Conjugation -> DNA transfer by cell – cell contact • Transduction -> DNA transfer by bacteriopage infection • Other methods of Gene transfer -> used with fungi, animal and plant cells: • Microinjection • protoplasts

  29. Electron micrograph of bacteriophage λ. Bacteriophages Page 107 Electron micrograph of the filamentous bacteriophage M13.

  30. Bacteriophage T2 injecting its DNA into an E. coli Page 84

  31. Life Cycle of Bacteriophage

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