1. Lecture 2 Mandatory Articles/Finding structure on the web. Restriction enzymes: a) History b) uses/properties PCR: a) basics b) considerations Agarose Gel electrophoresis Tips for this week’s lab

2. Mandatory Articles Sept. 29 Article 1 Oct. 6 Article 2 Oct. 13 Thanksgiving Oct. 20 Article 3 Oct. 27 Article 4 Nov. 3 Article 5 Nov. 10 Article 6 Nov. 17 Article 7

3. Finding structures on the Web http://www.ncbi.nlm.nih.gov/ Choose structures Search for the one you are interested in. Download file and viewer and look at your structure.

12. Socratic Method Questions are the fundamental guiding element that lead to answers, or boundaries to the unanswerable. Method of critical thinking/investigation. Development of student intellectual autonomy. I use a loose Socratic style in lecture.

13. Restriction enzymes Enzymes and recombinant DNA technology. How were restriction enzymes found originally? What are the properties of restriction enzymes? What are their uses?

14. Early 1970s: Genomic Studies: How to cut DNA into manageable fragments? Chemical/mechanical means non-specific, non-reproducible Breakthrough needed Restriction Endonucleases: Molecular Scissors for Cutting DNA

15. But where did restriction enzymes come from? They were discovered as a method of protecting bacteria from bacteriophage infection. Called the restriction modification system

22. Restriction Enzymes Recognize Palindromic Sequences

23. Restriction enzymes Considerations Why do we have MgCl 2 ? Why do we have NaCl? Why do we have a buffer?

25. Restriction enzymes can be used to create a “MAP” of DNA ? MAP ? Cleavage of DNA with restriction enzymes provides landmarks and sequence information.

26. Restriction enzymes can be used to create a “MAP” of DNA Digestion of DNA with restriction enzymes cloning into vectors Construction of restriction maps of individual clones Restriction enzymes played a pivotal role in cloning of the Human Genome

27. FORMATION OF RECOMBINANT DNA

28. Polymerase Chain Reaction Considerations Why do we have MgCl 2 ? Why do we have a buffer? Why do we have dNTPs? Why do we have primers? Why three temperatures per cycle?

29. POLYMERASE CHAIN REACTION STEPS INVOLVED Taq Polymerase Forward Primer Reverse Primer

30. Unlike most enzymes, Taq DNA polymerase can withstand high temperatures necessary for DNA strand separation and can be left in the reaction.

31. PCR: Representative Temperature Profile

32. AMPLIFICATION OF DNA POLYMERASE CHAIN REACTION

33. POLYMERASE CHAIN REACTION (Con’t)

34. EXPONENTIAL AMPLIFICATION OF PRODUCT

35. 1. Primers should be 18-25 bases in length; 2. Base composition should be 50-60% (G+C); 3. Primers should end (3') in CG or GC: this creates “tight” ends and increases efficiency of priming 4. T m = 4(G + C) + 2(A + T) o C: T a should be 2-5 o C below T m ( 55-60 o C) 5. 3'-ends of primers should not be complementary: primer-dimer 6. Primer self-complementary (ability to form hairpins) should be avoided. 7. Three or more Cs or Gs at the 3'-ends of may promote mispriming at G or C- rich sequences (because of stability of annealing), and should be avoided. DESIGNING PRIMERS Adapted from Innis and Gelfand, 1991

36. Gel electrophoresis Considerations On what basis are molecules separated? How does a gel work? Which way do molecules go and why? What colour are electrodes and anodes?

37. AGAROSE GEL ELECTROPHORESIS Agarose: A polysaccharide extracted from seaweed. Used to separate DNA fragments based on size

38. Structure of Ethidium Bromide (Fluorescent Dye) Detecting DNA using Ethidium Bromide When excited by UV light, EtBr emits fluorescent light at 590 nm EtBr is a very dangerous mutagen. Intercalates between bases of DNA

39. How are different sizes of DNA strands separated on agarose gel? Mixture of DNA molecules

40. DNA separation is based on fragment size Decreasing Size (+) (-) PCR1 PCR2

41. Size markers and their use to determine size

42. Mobility of a DNA fragment is proportional to the log of its size in bases.

43. Plot of relative mobility of DNA vs log of size on semi-log graph paper

44. Varying concentrations of agarose How does one make a 0.7%, 1.0% & 1.5% agarose gel? Why are there varying concentrations of agarose gels? DNA Ladders

45. Varying concentrations of agarose Why are there varying concentrations of agarose gels? Higher concentrations provide better resolution for smaller DNA fragments Lower concentrations provide better resolution for larger DNA fragments DNA Ladders

46. Tips Major reason for this experiment not working is not having all the reaction components in the correct tube. Tick list Droplets on tube wall Master Mix

47. Tick list

48. Droplet method

49. Check pipet tip for solution. Eject solution on the side of tube. Check for a droplet. To get the solution in the droplet to the bottom of the tube tap the tube on the bench.

50. Master Mix On the board.

52. 100bp Ladder.1.5 1 2 NTC. 1.5 1 2 DNABufferMgCl 2 Primer 1dNTPsTaq Concentration (X)

53. 100bp Cycle # ladder 5 10 15 20 25 30