Genetic engineering involves transferring genes between organisms using recombinant DNA techniques. This allows genes to be isolated, cloned, and moved within and between different species. Cloning a gene involves using restriction enzymes to cut DNA at specific sequences, and DNA ligase to join DNA fragments together. Cloned genes have many research uses such as determining gene sequences, altering phenotypes, and obtaining protein products of genes.

1. RECOMBINANT DNA TECHNOLOGY

12. How is DNA cloned? Cell-based DNA cloning Cell-free DNA cloning (PCR)

13. Clone in dividing cells

14. Clone in P C R

15. Restriction Endonucleases --The Molecular Scissors Host enzymes that prevent the invasion of foreign DNAs such as viral DNA, by cutting them up. These enzymes cut within the foreign DNAs, rather than chewing them away from the ends. These enzymes recognize a specific DNA sequence (4-12bp) which is twofold symmetry and cut both DNA strands Some enzymes make staggered cuts GAATTC CTTAAG Restriction Endonucleases Some make even cuts CCCGGG GGGCCC

17. Sticky end Sticky end

19. DNA ligase covalently links two DNA strands Restriction enzyme Restriction enzyme Ligase Ligase 5’ 3’ 5’ 3’

24. Plasmid: a cloning vector or vehicle

30. Selectable Markers:

33. cDNA Library preparation

40. Plasmids

45. BACs: Bacterial Artificial Chromosomes

46. YACs:Yeast Artificial Chromosomes

50. Bacterial transformation Introduction of DNA into bacteria Spontaneous uptake – low probability E. coli – cells treated with CaCl 2 Less than 1 of 10 3 cells acquire a plasmid Selection of transformed cells: resistance to antibiotics using chromogenic substances Antibiotics: molecules produced by microorganism that kill other microorganism peniciline, tetracycline, ciplroflaxine – inhibits gyrase in the complex with DNA – inhibits DNA replication Chromogenic substances:

59. Transgenic Mammals

61. Recombinant DNA Vaccines? Strategy for a subunit vaccine for herpes simplex

62. Gene Therapy Treatment of SCID (severe combined immunodeficiency). SCID affects the maturation of immune cells that develop in bone marrow. SCID sufferers lack the enzyme ADA (adenosine deaminase).