1. 1 Purifying DNA & RNA Source Amounts & Purity Damage or Loss

2. 2 Bind DNA Cells Pure DNA Extract Remove junk

3. 3 DNA, RNA Solution Denatured Protein Phenol Cell Extract ShakeSpin 1. Remove high MW junk

4. 4 (to 70%) SPIN (fast) Add and salt 2. Remove low MW junk and concentrate

5. 5 BIND + SALT (aided by alcohol) ELUTE - NO SALT (just add water) Or Bind DNA

6. 6 EXTRACT IN High Salt SILICA PARTICLES WATER DNA High Salt Wash

7. 7 Purifying one type of DNA away from other DNA molecules -Plasmids from bacterial chromosomal DNA (Form) - phage DNA (Location) -Restriction fragments, PCR products (Size)

8. 8 SDS, alkali

9. 9

10. 10 alkali neutralize

11. 11 RNA PURIFICATION Lyse & denature proteins FASTAcidic phenol Or bind glass/silica Small RNAs?

12. 12

13. 13

14. 14

15. 15 A? B?C? M

16. 16 Oligo-dT beads for polyA+ mRNA

17. 17 High Salt

18. 18

19. 19

20. 20 Chemical Synthesis of oligonucleotides Uses? Block and unblock sequentially so that only one nucleotide adds at a time

21. 21 Phosphoramidite 5’ 3’ Protected amino groups * *

22. 22 Couple Growing chain Blocked 5’-OH

23. 23 Unblock 1st “nucleotide”

24. 24 Add and activate next “nucleotide”

25. 25 Couple

26. 26 Product 99%

27. 27 1% Cap

28. 28

29. 29 1st nuc on bead. (blocked at 5’-OH) unblock couple cap Remove from bead Purify 3’- 5’ Add next nuc. (blocked 5’) De-protect Unblock 5’-OH

30. 30 PNA RNA harder DNA variants like

31. 31

32. 32

33. 33 If DNA is too large for conventional electrophoresis….

34. 34 Pulsed-field electrophoresis

35. 35

36. 36

37. 37 Polyacrylamide Gels can resolve small DNAs differing in length by one nucleotide

38. 38 Dideoxy sequencing converts sequence Information (A, C, G, T) into size differences e.g. if a DNA has T residues at positions 2, 5, 13, 16… this can be converted into a set of DNAs of length n+ 2, 5, 13, 16.. (which can be measured by denaturing polyacrylamide gel electrophoresis)

39. 39

40. 40 ddCTP

41. 41 ddATP dCTP dGTP dTTP dATP LABEL

42. 42 2,5,13,16 9,10,15,19 1,4,7,8,12,14,20 3,6,11,17,18 ddA ddC ddG ddT

43. 43

44. 44 2,5,13,16 9,10,15,19 1,4,7,8,12,14,20 3,6,11,17,18 ddA ddC ddG ddT n +

45. 45

46. 46 2,5,13,16 9,10,15,19 1,4,7,8,12,14,20 3,6,11,17,18 ddA ddC ddG ddT

47. 47 ddA ddC ddG ddT

48. 48

49. 49

50. 50 What do you need to sequence DNA? Where do the reagents come from? Must the DNA be pure? How much is needed How much good sequence can you obtain?

51. 51 Nucleic acid hybridization Key to life and almost every procedure in molecular biology

52. 52

53. 53

54. 54

55. 55

56. 56 Hybridize to DNA on blot

57. 57 Recognized by specific Ab Recognized by (strept)avidin

58. 58 RNA Probe

59. 59

60. 60 Northern RNA blot

61. 61 COLONY HYBRIDIZATION

62. 62 DNA (chromosome) in situ FISH

63. 63

64. 64

65. 65

66. 66 RNA in situ with non-radioactive probe