1. Gene cloning  Steps  Preparation of insert  Modification  Ligation  Transformation

2. Endonucleases Cloning involves cutting and joining DNA

3. Vectors and DNA are cut by endonucleases

5. Vector cleavage:  Cleavage of vector cannot be random  Each must be cleaved at same position. DNA cleavage:  DNA extraction results in fragments too large to clone (80 kb)  Gene only 2 – 3 kb

7. How is DNA cut?  Many different restriction endonucleases  Each have a specific sequence where the cut is made. (Type II restriction endonucleases)

8. Most recognition sequences are palindromic

9. Blunt End vs. Sticky End

10. Endonucleases with different recognition sequences can produce the same sticky ends

11. Restriction Sites are not evenly spaced. Endonucleases

12. Performing a restriction digestion  DNA from extract is added to tube.  Endonuclease requires certain environment to work correctly.  pH (usually 7.4ish)  Ionic conc. (NaCl)  Mg 2+ concentration  Temperature Variation from these conditions can cause inefficient cleavage or non-specific cleavage Endonucleases

13. Performing a restriction digestion  Conditions of solution usually set by adding concentrated buffer that came with endonuclease.  Endonuclease then added.  Mixture is incubated at a specific temperature (usually 37C an hour) Endonucleases

14. Stopping the restriction digestion After the DNA has been cleaved, the restiction enzyme must be deactivated.  Prevents non-specific cleavage or cleavage of DNA added at a later step Enzyme destroyed by temperature increase, phenol extraction, or EDTA Endonucleases

16. Ligation

17. DNA Ligase  All living cells produce DNA ligase  (For genetic engineering, DNA ligase from E. coli used)  DNA ligase repairs phosphodiester bonds between adjacent nucleotides.  Natural breaks  DNA replication  Recombination (2 repairs made)

18. DNA Ligase  Ligation can occur between blunt ends or sticky ends.  Blunt end ligations not efficient. Ligase has to wait for chance encounter.  Blunt end ligations are usually done at high concentrations.

19. DNA Ligase  Sticky end ligation much more efficient.  Hydrogen bonding forms relatively stable state.

20. Adding Sticky Ends (Linkers) Linkers: short, double-stranded, blunt-ended, DNA fragments  Contain a sticky end restriction site  Linkers are easily added to end of blunt end molecule because they can be added in such large quantities

21. Cleavage results in sticky end molecule

22. Adding Sticky Ends (Adaptors)  One drawback to linkers is the DNA molecule may contain the restriction site.  Adaptors are small oligonucleotides with one blunt end and one sticky end.

23. Potential problem with linkers

24. Adding Sticky Ends (Adaptors)  One drawback to linkers is the DNA molecule may contain the restriction site.  Adaptors are small oligonucleotides with one blunt end and one sticky end.  Potential problem with adaptors: they may ligate to each other; form blunt ends

25. Potential problem with adaptors

27. Adding Sticky Ends (Adaptors)  This adaptor problem is solved by chemically modifying the 5’ end.  Removal of 5’ phospahate prevents ligation of adaptors to each other

28. 5’-P allows for ligation of 2 sticky ends

29. Solution to adaptor problem

30. Adding Sticky Ends (Adaptors)  This adaptor problem is solved by chemically modifying the 5’ end.  Removal of 5’ phospahate prevents ligation of adaptors to each other  After adaptor addition, phosphate added with polynucleotide kinase

31. Solution to adaptor problem

32. Adding Sticky Ends (homopolymer tail)  Terminal deoxynucleotidyl transferase adds single stranded tail  In the presence of one nucleotide, a homopolymer tail forms.  Different tails are added to the vector (Cs) and the DNA (Gs)

33. Addition of homopolymer tail

34. Adding Sticky Ends (homopolymer tail)  Tails probably won’t be the same length.  Can be repaired immediately, or length may be stable enough to allow entry into bacteria, where the gaps can be repaired.

35. Addition of homopolymer tail

37. DNA into Cells  Object of cloning is to create many copies of a specific piece of DNA.  Cloning can result in 1000x increase (colonies) or a million x increase (liquid culture)

39. DNA into Cells  Object of cloning is to create many copies of a specific piece of DNA.  Cloning can result in 1000x increase (colonies) or a million x increase (liquid culture) The trick is to separate the desired DNA fragment from other fragments.

40. Following ligation, the mixture will contain:  Desired recombinant molecule  Unligated vectors  Unligated DNA fragments  Vector molecules w/o insert (self-ligated)  Recombinant molecules with wrong inserts

41. Products after ligation

42. Unligated DNA fragments usually aren’t taken up by bacteria.  Must seperate desired recombinants from undesired recombinants and self-ligated vectors Even if they are, degraded inside cell

44. Transformation: uptake of DNA  Most bacteria, including E. coli, only take up a limited amount of DNA.  In genetic engineering, bacteria are treated to increase uptake.  Following treatment, cells are said to be competent.

45. Preparing Competent Cells  DNA entry into cell involves first binding, then entering.  CaCl 2 treatment increases binding of DNA to outside of cell.  Movement of DNA into cell is stimulated by a brief raise in temperature.

47. Selecting for recombinants  Even in competent cells, only 0.01% have been transformed.  Must first separate transformed from not transformed.  Done by selectable marker in the plasmid.

48. If plasmid contains antibiotic resistant gene, then only bacteria with plasmid will grow in presence of that antibiotic.

49. Selecting for recombinants  If bacteria were immediately “plated” after entry into cell, there would not be enough resistant protein for survival.  Bacteria are incubated before they are plated in order to make sure enough of the resistant protein is made.

51. Selecting for recombinants  Even using antibiotic resistance, non- recombinant transformed cells (containing self-ligated plasmid) still will grow  Usually, recombinants are identified by inserting DNA into plasmid gene (inactivating it)

52. Inactivated gene must be identified phenotypically

53. Selecting for recombinants Can Inactivate:  Antibiotic resistant gene  Lac Z (color change)

54. Disrupt antibiotic resistant gene Example:  Plasmid contains amp resistant gene and tet resistant gene  DNA is inserted into tet resistant gene

55. Colonies grown on amp medium; only transformed cells will grow (both self-ligated plasmids and recombinants) Bacteria without plasmid will not grow

56. Portion of colonies transferred to tet medium. Only self-ligated transformed cells will grow (these are the unwanted colonies)

57. Can determine position of recombinant colonies on original amp plate.

58. Disrupt Lac Z’ gene  Lac Z gene codes for β-galactosidase (breaks down lactose)  Some bacteria strains have a modified Lac Z; they are missing Lac Z’  These bacteria can only breakdown lactose if they have the Lac Z’ in a plasmid.

60. Disrupt Lac Z’ gene Example:  Plasmid contains amp resistant gene and Lac Z’ gene  DNA is inserted into Lac Z’ gene

61. Disrupt Lac Z’ gene  Therefore, non-recombinant transformed cells will be able to breakdown lactose.  Recombinant transformed cells will not be able to breakdown lactose. Instead of lactose, X-gal is used: X-gal β-galactosidase IPTG Blue Product

62. Disrupt Lac Z’ gene  Therefore, non-recombinant transformed cells will turn blue.  Recombinant transformed cells will remain white.

63. Agar also contains ampicillin to prevent non- transformed growth.

64. Phage DNA in Cells (in vitro packaging)  Transfection (transformation for phages) not very efficient.  Would be useful if recombinant molecules were packaged in protein head and tail.  Requires making a large amount of the capsule proteins.

65. In vitro packaging: making capsules Defective phages can’t replicate; they only make proteins

66. In vitro packaging: making capsules Different phages with different capsule defects; Neither form capsules to complete infection

67. In vitro packaging Proteins and phage DNA mixed and capsules are formed.

68. Phage Infection  After addition of phage particles, infected cells spread on lawn of bacteria.  Infected cells will lyse, and phages will move on to infect and lyse neighboring cells  Lysed cells will create a clear zone called a plaque

70. Identify Recombinant Phages  Insertional inactivation of lacZ gene  Insertional inactivation of cI gene Self ligated vectors can be identified as blue (lacZ) or turbid (cI) plaques.