1. Chemistry 2100 Chapter 26

2. The Central Dogma The central dogma of molecular biology: –Information contained in DNA molecules is expressed in the structure of proteins. –Gene expression is the turning on or activation of a gene. 2

3. transcription

6. Transcription The architecture of yeast RNA polymerase II. Transcription of DNA (helical structure) into RNA (red) is shown. The template strand of DNA (blue) and the coding strand (green) and also shown. 6

7. Transcription Organization and transcription of a split eukaryote gene. 7

12. Why detect Transcription Factor targets? Transcription factors are medically relevant – ~10% of human genes – Crucial roles in development and cell life cycle – Misregulation and mutation cause disease – Critically, most cancers involve TF overactivity Darnell, Nature Reviews Cancer 2, 740 (2002)

13. Traditional methods for Transcription Factor detection Expression Microarrays Gel Shift Assays The challenge: Most of these methods are indirect, slow (hours), or can’t differentiate active and inactive protein. Western Blots

14. Bio-mimicry is a powerful motivation Velcro: inspired by burrs Conformation Switching Probes Marvin J S et al. PNAS 1997;94:4366-4371

15. Optical Conformation Switching TF Switch Sensors

16. Rationally Tuning TF Sensors % switches open KS = 10 KS = 1 KS = 0.1 KS = 0.01 KS = 0.001 KS = [ ] KS [target] KD (1+ KS) + KS [target] % switches open = KD = [ ] Target [M]

17. TF Beacon Actual Performance

18. Quantitative Detection in 4 easy steps

20. initiation

23. elongation

30. A – U – G – U – U – U – G – U – C – A – G – U..... mRNA tRNA peptide Met –––– Phe –––– Val –––– Ser.... (5') U – A – C – A – A – A – C – A – G – U – C – A

31. AU CU

32. Pyrimidine Dimers from UV http://highered.mcgraw-hill.com/olc/dl/120082/micro18.swf

33. The Genetic Code 33

44. DNA T – A – C – A – A – A – C – A – G – T – C – A..... A – T – G – T – T – T – G – T – C – A – G – T..... (3') (5') peptide Met –––– Phe –––– Val –––– Ser.... A – U – G – U – U – U – G – U – C – A – G – U..... mRNA (5')

45. DNA T – A – C – A – A – A – C – A – G – T – C – A..... A – T – G – T – T – T – G – T – C – A – G – T..... (3') (5') peptide Met –––– Phe –––– Val –––– Ser.... A – U – G – U – U – U – G – U – C – A – G – U..... mRNA (5')

46. DNA T – A – C – A – A – A – C – A – G – T – C – A..... A – T – G – T – T – T – G – T – C – A – G – T..... (3') (5') peptide Met –––– Phe –––– Val –––– Ser.... A – U – G – U – U – U – G – U – C – A – G – U..... mRNA (5')

47. DNA T – A – C – A – A – A – C – A – G – T – C – A..... A – T – G – T – T – T – G – T – C – A – G – T..... (3') (5') peptide Met –––– Phe –––– Val –––– Ser.... A – U – G – U – U – U – G – U – C – A – G – U..... mRNA (5')

48. peptide Met –––– Phe –––– Val –––– Ser.... A – U – G – U – U – U – G – U – C – A – G – U..... mRNA (5') peptide Met –––– Phe –––– Asp –––– Ser.... A – U – G – U – U – U – G – A – C – A – G – U..... mRNA (5') Mutations

49. point substitution mutation peptide Met –––– Phe –––– Val –––– Ser.... mRNA (5') peptide Met –––– Phe –––– Asp –––– Ser.... A – U – G – U – U – U – G – A – C – A – G – U..... mRNA (5') A – U – G – U – U – U – G – U – C – A – G – U – A....

50. point substitution mutation peptide Met –––– Phe –––– Val –––– Ser.... mRNA (5') peptide Met –––– Phe –––– Asp –––– Ser.... A – U – G – U – U – U – G – A – C – A – G – U – A.... mRNA (5') A – U – G – U – U – U – G – U – C – A – G – U – A....

51. point substitution mutation peptide Met –––– Phe –––– Val –––– Ser.... A – U – G – U – U – U – G – U – C – A – G – U – A.... mRNA (5') peptide Met –––– Phe ––– Asp ––– Ser.... A – U – G – U – U – U – G – A – C – A – G – U – A.... mRNA (5')

52. peptide Met –––– Phe –––– Val –––– Ser.... mRNA (5') frameshift mutation peptide Met –––– Phe –––– Val –––– Val.... A – U – G – U – U – U – G – U – C – A – G – U – A.... mRNA (5') A – U – G – U – U – U – G – U – C – A – G – U – A....

53. peptide Met –––– Phe –––– Val –––– Ser.... mRNA (5') frameshift mutation peptide Met –––– Phe –––– Val –––– Val.... A – U – G – U – U – U – G – U – C – A – G – U – A.... mRNA (5') A – U – G – U – U – U – G – U – C – A – G – U – A....

54. peptide Met –––– Phe –––– Val –––– Ser.... mRNA (5') frameshift mutation peptide Met –––– Phe –––– Val –––– Val.... A – U – G – U – U – U – G – U – C – A – G – U – A.... mRNA (5') A – U – G – U – U – U – G – U – C – A – G – U – A....

55. PCR Polymerase Chain Reaction

57. DNA Fingerprinting

58. DNA Sequencing

60. Shotgun Sequencing

61. Electrochemical Sequencing http://www.youtube.com/watch?v=yVf2295JqUg

64. Recombinant DNA

66. Gene Therapy