1. Genetic Changes  Humans have changed the genetics of other species for thousands of years by selective breeding  Causing Artificial Selection  Natural processes also at work Mutation Mutation

2. Discovery of Restriction Enzymes  Hamilton Smith was studying how bacteria defend themselves from bacteriophage attack  Discovered bacteria have an enzyme that chops up viral DNA

3. Specificity of Cuts  Restriction enzymes cut DNA at a specific sequence  Number of cuts made in DNA will depend on number of times the “target” sequence occurs

4. Restriction enzymes Discovery of Restriction Enzymes

5. Making Recombinant DNA 5’ 3’ G C T T A A A A T T C G G C T T A AG 3’ 5’ one DNA fragmentanother DNA fragment 3’ 5’

6. Making Recombinant DNA nick 5’ 3’ 5’ GA A T T C C T T A AG nick GA A T T C C T T A AG DNA ligase action

7. Fig. 16-2, p.244 Stepped Art G CTTAA AATTCG 3’ 5’ 3’ 5’ CTTAA AATTC G cut fragments G DNA ligase action nick G CTTAA AATTCG 3’ 5’ 3’ 5’ another DNA fragment AATTC 3’ 5’3’ 5’ G one DNA fragment 3’ 5’ G CTTAA 3’ 5’ enzyme recognition site G CTTAA AATTCG 3’ 5’

8. Using Plasmids  Plasmid = small circle of bacteria DNA  Foreign DNA can be inserted into plasmids  Making them recombinant plasmids  Plasmids can be used as a vector  vectors can deliver DNA into another cell

9. Fig. 16-3a, p.244 Plasmids

10. Fig. 16-3b, p.244 Plasmids

11. host cells containing recombinant plasmids Same Enzyme Cuts plasmid DNA Human & Bacteria DNA are mixed Restriction Enzyme Cuts Human DNA DNA w/ sticky ends Plasmids w/Human DNA Plasmid DNA w/ sticky ends

12. a A restriction enzyme cuts a specific base sequence everywhere it occurs in DNA. c The same enzyme cuts the same sequnece in plasmid DNA. d The plasmid DNA also has sticky ends e The DNA fragments and the plasmid DNA are mixed with DNA ligase. f The result? A collection of recombinant plasmids that incorporate foreign DNA fragments. g Host cells that can divide rapidly take up the recombinant plasmids. b The DNA fragments have sticky ends. Fig. 16-4, p.245 Using Plasmids

13. Formation of recombinant DNA Making Recombinant DNA

14. Base-pairing of DNA fragments Base-Pairing

15. Familial Hypercholesterolemia  Gene encodes protein that serves as cell’s LDL (cholesterol) receptor  Normal genes keep blood level of LDLs low  Mutated alleles lead to abnormally high cholesterol levels & heart disease

16. Example of Gene Therapy  Woman with familial hypercholesterolemia  Part of her liver was removed  Virus vector used to insert normal gene for LDL receptor into her cultured liver cells  Modified liver cells placed back in patient

17. Amplifying DNA = Make many copies of a piece of DNA  Fragments can be inserted into fast-growing microorganisms like bacteria or or  Polymerase chain reaction (PCR) can be used to make many copies of the DNA

18. Gel Electrophoresis  DNA is placed at one end of a gel  A current is applied to the gel  DNA molecules are negatively charged and move toward positive end of gel  Smaller molecules move faster than larger ones

19. Gel Electrophoresis  Used to separate DNA fragements by size  http://www.sumanasinc.com/webcontent/a nimations/content/gelelectrophoresis.html

20. Nucleotides for Sequencing  Standard nucleotides (A, T, C, G)  Modified versions of these nucleotides Labeled so they fluoresce Labeled so they fluoresce Structurally different so that they stop DNA replication when they are added to a strand Structurally different so that they stop DNA replication when they are added to a strand

21. Recording the DNA Sequence T C C A T G G A C C T C C A T G G A C T C C A T G G A T C C A T G G T C C A T G T C C A T T C C A T C C T C T electrophoresis gel one of the many fragments of DNA migrating through the gel one of the DNA fragments passing through a laser beam after moving through the gel T C C A T G G A C C A DNA is placed on gel Fragments move off gel in size order; pass through a laser beam Color each fragment fluoresces is recorded on printout

22. p.248 Recording the Sequence

23. electrophoresis gel one of many fragments of DNA migrating through the gel one of the DNA fragments passing through a laser beam after moving through the gel Fig. 16-8a, p.248 Recording the Sequence

24. Fig. 16-8b, p.248 Recording the Sequence

25. Automated DNA sequencing Recording the Sequence

26. DNA Fingerprints  Each person has unique DNA fragments  Inherited from parents  Even full siblings are different

27. Tandem Repeats  Mutations where short sequences of DNA repeat over and over  There are many sites in genome where tandem repeats occur

28.  Different people will have a different number of repeats in their Tandem Repeat  Tandem Repeats that occur in areas of DNA that do not code for any genes

29. RFLPs  Restriction fragment length polymorphisms  DNA is cut with restriction enzymes before and after an area with tandem repeats  Since each different person has a different number of repeats, the DNA fragments can be many different lengths  Different lengths are detected by gel electrophoresis

30. Analyzing DNA Fingerprints  DNA is stained or made visible by use of a radioactive probe  Pattern of bands is used to: Identify or rule out criminal suspects Identify or rule out criminal suspects Identify bodies Identify bodies Determine paternity Determine paternity

31. DNA fingerprinting DNA Fingerprints

32. Fig. 16-9a, p.249 Gel Electrophoresis

33. Fig. 16-9b, p.249 Gel Electrophoresis

34. Genome Sequencing  1995 – DNA Sequence of bacterium Haemophilus influenzae determined  Automated DNA sequencing now main method  Draft sequence of entire human genome determined in this way

35. Fig. 16-10a, p.250 Genome Sequencing

36. Fig. 16-10b, p.250 Genome Sequencing

37. Genetic Engineering  Genes are isolated, modified, and inserted into an organism  Made possible by recombinant technology Cut DNA up and recombine pieces Cut DNA up and recombine pieces Amplify modified pieces Amplify modified pieces

38. Engineered Proteins  Bacteria can be used to grow medically valuable proteins Insulin (for diabetics) Insulin (for diabetics) blood-clotting factors blood-clotting factors Vaccines Vaccines

39. Cleaning Up the Environment  Bacteria naturally break down organic wastes  Some can be engineered to break down pollutants like oil spills or  To take in toxic materials

40. Can Genetically Engineered Bacteria “Escape”?  Genetically engineered bacteria are designed so that they cannot survive outside lab  Genes are included that will be turned on in outside environment, triggering death

41. p.252