1. Advanced Gene Technology

2. DNA,RNA, Recombinant DNA Technology

3. Nucleic Acids – DNA and RNA

5. DNA + RNA DNA + RNA DNA + RNA RNA DNA

6. DNA structure -> sequence

8. Polymerase reaction: 5’-> 3’

9. The central dogma Page 93

11. Gene expression. Page 93

13. What is a gene?

17. Eukaryotes – Intron-Exon concept Page 95

21. DNA mRNA Ribosome Polymerase Protein

22. Recombinant DNA Technology

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

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

28. Gel Electrophoresis

29. Gel Electrophoresis

30. Gel Electrophoresis

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

32. Construction of a restriction map. Page 104

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

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

38. 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

40. Plasmid Cloning Vectors

41. Plasmid Cloning Vectors

42. 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)

43. 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

45. Transformation and Selection

46. 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

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

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

49. Life Cycle of Bacteriophage