1. Primer/probe design Crucial for successful DNA & RNA analysis! Main source of specificity for PCR

2. Primer/probe design Also important for microarrays, sequencing, Southerns Concerns Specificity Complementarity: Hairpins homoduplexes heteroduplexes may not melt May be extended by DNA polymerase

3. Primer/probe design Also important for microarrays, sequencing, Southerns Concerns Specificity Complementarity: Melting T Should match!

4. Primer/probe design Also important for microarrays, sequencing, Southerns Concerns Specificity Complementarity: Melting T Should match! Every site calculates them differently!

5. Primer/probe design Also important for microarrays, sequencing, Southerns Concerns Specificity Complementarity: Melting T Targeting specific locations amplifying specific sequences

6. Primer/probe design Also important for microarrays, sequencing, Southerns Concerns Specificity Complementarity: Melting T Targeting specific locations amplifying specific sequences creating mutations: need mismatch towards 5’ end so 3’ end binds well

7. Primer/probe design Also important for microarrays, sequencing, Southerns Concerns Specificity Complementarity: Melting T Targeting specific locations amplifying specific sequences creating mutations: need mismatch towards 5’ end so 3’ end binds well Add restriction sites at 5’ end: may need to reamplify an amplicon

8. Primer/probe design Also important for microarrays, sequencing, Southerns Concerns Specificity Complementarity: Melting T Targeting specific locations amplifying specific sequences creating mutations: need mismatch towards 5’ end so 3’ end binds well Add restriction sites at 5’ end: may need to reamplify an amplicon Use Vent or another polymerase with proof-reading, taq’s error frequency is too high.

9. Primer/probe design Also important for microarrays, sequencing, Southerns Concerns Specificity Complementarity: Melting T Targeting specific locations Amplifying sequences from related organisms If use protein alignments need to make degenerate primers; eg CCN means proline, so need to make primers with all 4 possibilities

10. Primer/probe design Also important for microarrays, sequencing, Southerns Concerns Specificity Complementarity: Melting T Targeting specific locations Amplifying sequences from related organisms If use protein alignments need to make degenerate primers; eg CCN means proline, so need to make primers with all 4 possibilities CodeHOP is a way around this: have a perfect match for 10-12 bases at 3’ end, then pick most likely candidates for the rest.

11. Primer/probe design Also important for microarrays, sequencing, Southerns Concerns Specificity Complementarity: Melting T Targeting specific locations Amplifying sequences from related organisms If use protein alignments need to make degenerate primers; eg CCN means proline, so need to make primers with all 4 possibilities CodeHOP is a way around this: have a perfect match for 10-12 bases at 3’ end, then pick most likely candidates for the rest. Based on codon usage

12. Primer/probe design Stem-loop primers for short RNAs where only have enough info for one primer

13. Optimizing PCR Choosing enzyme Template (RNA or DNA?)

14. Optimizing PCR Choosing enzyme Template (RNA or DNA?) Fidelity

15. Optimizing PCR Choosing enzyme Template (RNA or DNA?) Fidelity Temperature stability

16. Optimizing PCR Choosing enzyme Template (RNA or DNA?) Fidelity Temperature stability Processivity

17. Optimizing PCR Choosing enzyme Template (RNA or DNA?) Fidelity Temperature stability Processivity Km dNTP DNA

18. Optimizing PCR Choosing enzyme Template (RNA or DNA?) Fidelity Temperature stability Processivity Km dNTP DNA Vmax

19. Optimizing PCR Choosing enzyme Template (RNA or DNA?) Fidelity Temperature stability Processivity Km dNTP DNA Vmax Tolerance of imperfect conditions Dirty DNA dNTP analogs or modified dNTP [Mg] (or other divalent cation)

20. Optimizing PCR Choosing enzyme Template (RNA or DNA?) Fidelity Temperature stability Processivity Km dNTP DNA Vmax Tolerance of imperfect conditions Dirty DNA dNTP analogs or modified dNTP [Mg] (or other divalent cation) Fragment ends

21. Optimizing PCR Choosing enzyme Template (RNA or DNA?) Fidelity Temperature stability Processivity Km dNTP DNA Vmax Tolerance of imperfect conditions Dirty DNA dNTP analogs or modified dNTP [Mg] (or other divalent cation) Fragment ends Cost

22. Optimizing PCR Choosing enzyme Template (RNA or DNA?): Reverse transcriptases from retroviruses make DNA copies of RNA Tth DNA Polymerase from Thermus thermophilus reverse transcribes RNA in the presence of Mn 2+

23. Optimizing PCR Choosing enzyme Template (RNA or DNA?): Reverse transcriptases from retroviruses make DNA copies of RNA Tth DNA Polymerase from Thermus thermophilus reverse transcribes RNA in the presence of Mn 2+ Then dilute rxn & add Mg 2+ to do PCR

24. Optimizing PCR Choosing enzyme Template (RNA or DNA?) Tth DNA Polymerase from Thermus thermophilus reverse transcribes RNA in the presence of Mn 2+ Then dilute rxn & add Mg 2+ to do PCR Tfl DNA Polymerase from Thermus flavus has no RT activity: can mix with RNA & RT w/o activity then go directly to PCR after RT is done

25. Choosing enzyme Template Fidelity Taq from Thermus aquaticus has no proof-reading

26. Choosing enzyme Template Fidelity Taq from Thermus aquaticus has no proof-reading goes faster, but error freq of 1 in 3000 Vent from Thermococcus litoralis has error frequency of 1 in 30,000

27. Choosing enzyme Template Fidelity Taq from Thermus aquaticus has no proof-reading goes faster, but error freq of 1 in 3000 Vent from Thermococcus litoralis has error frequency of 1 in 30,000 Pfu from Pyrococcus furiosus has error frequency of 1 in 400,000

28. Choosing enzyme Template Fidelity Taq from Thermus aquaticus has no proof-reading goes faster, but error freq of 1 in 3000 Vent from Thermococcus litoralis has error frequency of 1 in 30,000 Pfu from Pyrococcus furiosus has error frequency of 1 in 400,000 Genetically engineered proof-reading Phusion from NEB has error frequency of 1 in 2,000,000

29. Choosing enzyme Template Fidelity Temperature stability E.coli DNA polymerase I denatures at 75˚ C T 1/2 of Taq @ 95˚ C is 0.9 hours, < 0.1 hour @ 100˚ C

30. Choosing enzyme Template Fidelity Temperature stability E.coli DNA polymerase I denatures at 75˚ C T 1/2 of Taq @ 95˚ C is 0.9 hours, < 0.1 hour @ 100˚ C T 1/2 of Phusion @ 96˚ C is >6 hours, 2 hours @ 98˚ C

31. Choosing enzyme Template Fidelity Temperature stability E.coli DNA polymerase I denatures at 75˚ C T 1/2 of Taq @ 95˚ C is 0.9 hours, < 0.1 hour @ 100˚ C T 1/2 of Phusion @ 96˚ C is >6 hours, 2 hours @ 98˚ C T 1/2 of Vent @ 95˚ C is 6.7 hours, 1.8 hours @ 100˚ C

32. Choosing enzyme Template Fidelity Temperature stability E.coli DNA polymerase I denatures at 75˚ C T 1/2 of Taq @ 95˚ C is 0.9 hours, < 0.1 hour @ 100˚ C T 1/2 of Phusion @ 96˚ C is >6 hours, 2 hours @ 98˚ C T 1/2 of Vent @ 95˚ C is 6.7 hours, 1.8 hours @ 100˚ C T 1/2 of Deep Vent from Pyrococcus species GB-D (grows @ 104˚ C)is 23 hours @ 95˚ C, 8 hours @ 100˚ C

33. Choosing enzyme Template Fidelity Temperature stability Processivity (how far does it go before falling off) Q5 is 10x more processive than Pfu, 2x more than Taq

34. Choosing enzyme Template Fidelity Temperature stability Processivity (how far does it go before falling off) Q5 is 10x more processive than Pfu, 2x more than Taq lets you make longer amplicons in shorter time

35. Choosing enzyme Template Fidelity Temperature stability Processivity (how far does it go before falling off) Q5 is 10x more processive than Pfu, 2x more than Taq lets you make longer amplicons in shorter time Taq = 8 kb max cf 40 kb for Q5

36. Choosing enzyme Template Fidelity Temperature stability Processivity (how far does it go before falling off) Km dNTP: 13 µM for Taq, 60 µM for Vent

37. Choosing enzyme Template Fidelity Temperature stability Processivity (how far does it go before falling off) Km dNTP: 13 µM for Taq, 60 µM for Vent DNA: 2 nM for Taq, 0.01 nM for Deep Vent

38. Choosing enzyme Template Fidelity Temperature stability Processivity (how far does it go before falling off) Km Vmax: >1,000 nt/s when attached Binding is limiting, processivity determines actual rate 1000 bp/min is good for PCR

39. Choosing enzyme Template Fidelity Temperature stability Processivity (how far does it go before falling off) Km Vmax Tolerance of imperfect conditions Dirty DNA: in general, non-proofreading polymerases tolerate dirtier DNA than proof-readers except Phusion

40. Choosing enzyme Template Fidelity Temperature stability Processivity (how far does it go before falling off) Km Vmax Tolerance of imperfect conditions Dirty DNA: in general, non-proofreading polymerases tolerate dirtier DNA than proof-readers except Phusion dNTP analogs or modified dNTP non-proofreading polymerases do better, but varies according to the modification

41. Choosing enzyme Template Fidelity Temperature stability Processivity (how far does it go before falling off) Km Vmax Tolerance of imperfect conditions Dirty DNA: in general, non-proofreading polymerases tolerate dirtier DNA than proof-readers except Phusion dNTP analogs or modified dNTP non-proofreading polymerases do better, but varies according to the modification [Mg]: Vent is more sensitive to [Mg] and needs 2x more than Taq

42. Choosing enzyme Template Fidelity Temperature stability Processivity (how far does it go before falling off) Km Vmax Tolerance of imperfect conditions Fragment ends: proof-readers (eg Vent) give blunt ends

43. Choosing enzyme Template Fidelity Temperature stability Processivity (how far does it go before falling off) Km Vmax Tolerance of imperfect conditions Fragment ends: proof-readers (eg Vent) give blunt ends Non-proof-readers (eg Taq) give a mix of blunt & 3’A

44. Choosing enzyme Template Fidelity Temperature stability Processivity (how far does it go before falling off) Km Vmax Tolerance of imperfect conditions Fragment ends: proof-readers (eg Vent) give blunt ends Non-proof-readers (eg Taq) give a mix of blunt & 3’A Can use 3’A for t:A cloning GAATTCAtcgca CTTAAGtagcgt

45. Choosing enzyme Template Fidelity Temperature stability Processivity (how far does it go before falling off) Km Vmax Tolerance of imperfect conditions Fragment ends: proof-readers (eg Vent) give blunt ends Cost @ NEB: http://www.neb.com/nebecomm/default.asp Taq = $59.00 for 400 units Vent = $62.00 for 200 units Deep Vent = $90.00 for 200 units Q5 = $ 103.00 for 100 units

46. Optimizing PCR [enzyme] [Template] [Mg 2+ ] Annealing Temperature Denaturation temperature

47. Optimizing PCR [enzyme] 0.4-2 units/100 µl for proofreaders : start with 1

48. Optimizing PCR [enzyme] 0.4-2 units/100 µl for proofreaders : start with 1 1-5 units/100 µl for non-proofreaders : start with 3

49. Optimizing PCR [enzyme] [Template] 1-10 ng/100 µl reaction for plasmids 10 - 1000 ng/100 µl reaction for genomic DNA Excess DNA can give extra bands, also brings more contaminants

50. Optimizing PCR [enzyme] [Template] 1-10 ng/100 µl reaction for plasmids 10 - 1000 ng/100 µl reaction for genomic DNA Excess DNA can give extra bands, also brings more contaminants [dNTP] 50-500 µM for Taq: start with 200, lower increases fidelity, higher increases yield 200-400 µM for proof-readers: if too low start eating

51. Optimizing PCR [enzyme] [Template] [Mg 2+ ] 0.5 - 4 mM for Taq: start with 1.5; lower if extra bands, raise if low yield

52. Optimizing PCR [enzyme] [Template] [Mg 2+ ] 0.5 - 4 mM for Taq: start with 1.5; lower if extra bands, raise if low yield 1- 8 mM for proofreaders: start with 2, lower if extra bands, raise if low yield

53. Optimizing PCR [enzyme] [Template] [Mg 2+ ] Denaturation Temperature Go as high as you can w/o killing enzyme before end 94˚C for Taq 96-98˚C for Vent 98˚C for Deep Vent & Q5

54. Optimizing PCR [enzyme] [Template] [Mg 2+ ] Denaturation Temperature Annealing Temperature Start 5 ˚C below lowest primer Tm

55. Optimizing PCR [enzyme] [Template] [Mg 2+ ] Denaturation Temperature Annealing Temperature Start 5 ˚C below lowest primer Tm Adjust up and down as needed

56. Optimizing PCR [enzyme] [Template] [Mg 2+ ] Denaturation Temperature Annealing Temperature Start 5 ˚C below lowest primer Tm Adjust up and down as needed # cycles: raise if no bands, lower if OK yield but extra bands

57. Optimizing PCR Most common problems = wrong [DNA], dirty DNA, [Mg 2+ ] annealing temperature & # cycles

58. Optimizing PCR Most common problems = wrong [DNA], dirty DNA, [Mg 2+ ] annealing temperature & # cycles Can try “PCR enhancers” to overcome dirty DNA Use Ammonium SO 4 in buffer cf KCl Use molecules that alter Tm eg DMSO & formamide Use molecules that stabilise Taq eg Betaine & BSA

59. Optimizing PCR Most common problems = wrong [DNA], dirty DNA, [Mg 2+ ] annealing temperature & # cycles If extra bands persist, use Taq bound to antibody Inactive until denature antibody 7’ at 94˚ C

60. Optimizing PCR Most common problems = wrong [DNA], dirty DNA, [Mg 2+ ] annealing temperature & # cycles If extra bands persist, use Taq bound to antibody Inactive until denature antibody 7’ at 94˚ C Alternatively, try touch-down: start annealing @ too high & lower 1˚ C each cycle ( binds correct target first)

61. Regulating transcription Telling RNA pol to copy a DNA sequence

62. Regulating transcription Telling RNA pol to copy a DNA sequence Transcription factors bind promoters & control initiation of transcription

63. Regulating transcription Telling RNA pol to copy a DNA sequence Transcription factors bind promoters & control initiation of transcription 1/signal gene senses

64. Regulating transcription Telling RNA pol to copy a DNA sequence Transcription factors bind promoters & control initiation of transcription 1/signal gene senses 1 binding site/signal gene senses

65. Transcription factors Bind surface -> base-pairs form unique patterns in major & minor grooves

66. Transcription factors Bind surface -> base-pairs form unique patterns in major & minor grooves Scan DNA for correct pattern

67. Transcription factors Bind surface -> base-pairs form unique patterns in major & minor grooves Scan DNA for correct pattern need 15 - 20 H-bonds = 5-8 base-pairs

68. Transcription Prokaryotes have one RNA polymerase makes all RNA core polymerase = complex of 5 subunits (      ’  )

69. Transcription Prokaryotes have one RNA polymerase makes all RNA core polymerase = complex of 5 subunits (      ’  )  not absolutely needed, but cells lacking  are very sick

70. Initiating transcription in Prokaryotes 1) Core RNA polymerase is promiscuous

71. Initiating transcription in Prokaryotes 1)Core RNA polymerase is promiscuous 2)sigma factors provide specificity

72. Initiating transcription in Prokaryotes 1)Core RNA polymerase is promiscuous 2)sigma factors provide specificity Bind promoters

73. Initiating transcription in Prokaryotes 1)Core RNA polymerase is promiscuous 2)sigma factors provide specificity Bind promoters Different sigmas bind different promoters