1. Molecular Biology 101 for Laboratory Professionals: Part Two Erik Munson Clinical Microbiology Wheaton Franciscan Laboratory Wauwatosa, Wisconsin The presenter states no conflict of interest and has no financial relationship to disclose relevant to the content of this presentation. 1

2. OUTLINE I.Cell biology vignette II.Molecular diagnostic application III.Life-creating, life-changing events A.DNA structure B.DNA replication C.DNA replication D.Transcription 2

3. Review 3

4. COMPONENTS DNA Phosphate Pentose (deoxyribose) Base (thymine) O O-O- PO-O- O-O- OHH O HOCH 2 N O CH 3 N H O 2’ 4

5. COMPOSITE OHH O CH 2 OH O O O-O- P OO-O- O P OO-O- N N N N NH H Adenine N O N N H H H Cytosine 5

6. N N N N NH H N N N N O NH H H N O CH 3 NH O N O N N H H Adenine Guanine Thymine Cytosine H H keto group amino group HYDROGEN BONDING 6

7. UNWIND DOUBLE HELIX 7

8. POLYMERASE SPECIFICITY Catalyze ester bond ONLY between first 5’ phosphate of new nucleotide and 3’ hydroxyl of previous nucleotide Polymerase ONLY allows addition of phosphate to pre-existing hydroxyl 5’ to 3’ direction 8

9. OHH O CH 2 O O-O- P OO-O- N N N N NH H Adenine Cytosine O P OO-O- N O N N H H H OHH O CH 2 O-O- 5’ 3’ DNA polymerase () x 3 9

10. Molecular Diagnostics Classifications 10

11. Probe anneals to target of interest TARGET DETECT Probe NUCLEIC ACID HYBRIDIZATION 11

12. PROBE TECHNOLOGY More effective on colonial growth than on primary clinical specimens 12

13. Amplify target of interest prior to detection TARGET Primer DETECTAMPLIFY NUCLEIC ACID AMPLIFICATION TESTING (NAAT) 13

14. ANALYTICAL SENSITIVITY Manual of Clinical Microbiology, 6th edition; 1995 14

15. Denature (95 ° C, 5 min) Anneal/hybridize (30°C, 2 min) Repeat 19 times; add Klenow each time Klenow extension (30°C, 2 min) PROTOCOL (Mullis et al.) 15

16. Taq polymerase isolated from extreme thermophile Thermus aquaticus Thermostability eliminates necessity to replenish enzyme with each new cycle REVOLUTIONARY FINDING Science 239: 487-491; 1988 16

17. Denature (95 ° C) Anneal/hybridize (62 ° C) ~40 cycles Extension (72 ° C) “OPTIMIZED” PCR PROTOCOL 17

18. Elapsed time: 3 hours 18

19. Elapsed time: 4 hours 19

20. Elapsed time: 4.5 hours 20

21. Elapsed time: 6.5 hours 21

22. Elapsed time: 7 hours 22

23. Alternatively… Elapsed time: 5.5 hours 23

24. OLDSCHOOLOLDSCHOOL 24

25. 25

26. Diagnostic Application: Real-time PCR Elapsed time: ~1 hour 26

27. Surface area to volume ratio HOW DOES THIS HAPPEN?!?! 27

28. Surface area to volume ratio (increased) HOW DOES THIS HAPPEN?!?! 28

29. Real-time detection Surface area to volume ratio No target control HOW DOES THIS HAPPEN?!?! 29

30. BASELINE Real-time detection Surface area to volume ratio HOW DOES THIS HAPPEN?!?! 30

31. BASELINE Real-time detection Surface area to volume ratio HOW DOES THIS HAPPEN?!?! 31

32. BASELINE CTCT Cycle threshold (C T ) Real-time detection Surface area to volume ratio HOW DOES THIS HAPPEN?!?! 32

33. 10 6 copies DNA 10 3 copies DNA REAL-TIME CHEMISTRY Anal. Biochem. 245: 154-160; 1997 SYBR green Preferentially binds double-stranded DNA Fluorescence increases upon binding 33

34. REAL-TIME CHEMISTRY SYBR green Preferentially binds double-stranded DNA Fluorescence increases upon binding Specific and non-specific DNA products SOLUTION: melting curve analysis 34

35. Two strands of nucleic acid will melt apart and fluorescence will decrease Slowly increase temperature (post-reaction) Function of: length of amplified target nature of sequence percentage G C - - - MELTING CURVE ANALYSIS 35

36. A. Hepatitis B sAg gene C. Human  -globin gene B. Combination of A and C Anal. Biochem. 245: 154-160; 1997 MELTING CURVE ANALYSIS 36

37. Characteristic melting peak (T m ) Primer dimers, non-specific products with different T m ; give broader peaks A. Hepatitis B sAg gene C. Human  -globin gene B. Combination of A and C Anal. Biochem. 245: 154-160; 1997 MELTING CURVE ANALYSIS 37

38. RQ probe REAL-TIME CHEMISTRY FRET probes Fluorescent Resonance Energy Transfer Means of increasing specificity Fluorescent (reporter) and quencher dyes SYBR green 38

39. 5’ EXONUCLEASE PCR (TaqMan) Taq polymerase also exhibits 5’ exonuclease activity 39

40. Taq polymerase also exhibits 5’ exonuclease activity Extra, non-extending labeled hybridization probe (internal to target) Primer extension displaces, cleaves probe Specificity due to location of signal probe fluorescence indicates PCR product 5’ EXONUCLEASE PCR (TaqMan) 40

41. TARGET 3’5’ TARGET 5’3’ 5’ EXONUCLEASE PCR (TaqMan) 41

42. TARGET 3’5’ TARGET 5’3’ Denaturation 5’ EXONUCLEASE PCR (TaqMan) 42

43. PRIMER TARGET 3’5’ 3’ PRIMER TARGET 5’3’ 5’ Primer annealing 5’ EXONUCLEASE PCR (TaqMan) 43

44. PRIMER TARGET 3’5’ 3’ PROBE RQ 5’ 3’ PRIMER TARGET 5’3’ 5’ Probe annealing 5’ EXONUCLEASE PCR (TaqMan) 44

45. PRIMER TARGET 3’5’ 3’ PROBE RQ 5’ 3’ PRIMER TARGET 5’3’ 5’ Taq pol Primer extension (polymerization) 5’ EXONUCLEASE PCR (TaqMan) 45

46. PRIMER TARGET 3’5’ 3’ PROBE RQ 5’ 3’ PRIMER TARGET 5’3’ 5’ Taq pol Primer extension (polymerization) 5’ EXONUCLEASE PCR (TaqMan) 46

47. PRIMER TARGET 3’5’ 3’ OBE Q 3’ PRIMER TARGET 5’3’ 5’ Taq pol Probe cleavage via 5’ exonuclease activity R FLUORESCENCE EMISSION 5’ EXONUCLEASE PCR (TaqMan) 47

48. PRIMER TARGET 3’5’ 3’ Q PRIMER TARGET 5’3’ 5’ Taq pol Primer extension (polymerization) completed R FLUORESCENCE EMISSION 5’ EXONUCLEASE PCR (TaqMan) 48

49. FRET probes SYBR green Molecular beacons REAL-TIME CHEMISTRY Nat. Biotechnol. 16: 49-53; 1998 49

50. Hairpin oligonucleotide probe with internally- quenched fluorophore (close proximity) Resulting hybrid more stable than stem; conformational change forces stem apart Fluorophore and quencher move away; fluorescence restored MOLECULAR BEACONS Nat. Biotechnol. 16: 49-53; 1998 50

51. More sensitive More specific Can be quantitative Less prone to contamination More compatible with automation Assessment of inhibition (internal control) ADVANTAGES OF REAL-TIME PCR 51

52. FLUORESCENCEFLUORESCENCE Analyte of interest Internal control TIME positive BASELINE Two-color stain 52

53. FLUORESCENCEFLUORESCENCE Analyte of interest Analyte of interest Internal control Internal control TIME positivenegative BASELINE 53

54. FLUORESCENCEFLUORESCENCE Analyte of interest Analyte of interest Internal control Internal control Analyte of interest Internal control TIME positivenegativeunresolved BASELINE 54

55. 55

56. Transcription 56

57. BASIC TENETS--I Three types of RNA rRNA (ribosome structure; 5S rRNA, 16S, 23S) tRNA (brings amino acid to ribosome) mRNA (carries DNA message to ribosome) DNA is transcribed into RNA RNA is single-stranded molecule Ribose (-OH at carbon #2) Uracil substituted for thymine OH O HOCH 2 2’ 57

58. RNA polymerase binds to promoter Core enzyme Sigma factor ( ) Not continuous Energy conservation Initiator, terminator regions BASIC TENETS--II σ 58

59. 59

60. POLYMERIZATION No proofreading--not a big deal Many mRNA are short-lived Many copies are made … if RNA not functional, new one will be made RNA nucleotides added to free 3’-OH Template(non-coding)DNA3’ GTACAC5’ Complement(coding)DNA5’ CATGTG3’ TranscriptRNA5’ CAUGUG3’ Transcription 60

61. TERMINATION 3’ 5’ INVERTED REPEAT 61

62. TERMINATION Rho-dependent termination Rho-independent termination 62

63. FINAL PRODUCT (TRANSCRIPT) Start codon Stop codon Possible regulatory function Recognition of mRNA at ribosome 63

64. Diagnostic Application: Transcription-mediated amplification 64

65. Reverse transcriptase-mediated formation of ds cDNA sequences containing binding sites for T7 DNA-dependent RNA polymerase Transcription RNA transcripts re-enter cycle BASIC TENETS OF TMA 65

66. TARGET RNA STEPS INVOLVED IN TMA J. Clin. Virol. 25: S23-S29; 2002 66

67. TARGET RNA PRIMER #1POL STEPS INVOLVED IN TMA J. Clin. Virol. 25: S23-S29; 2002 67

68. TARGET RNA PRIMER #1POL Reverse Transcriptase STEPS INVOLVED IN TMA J. Clin. Virol. 25: S23-S29; 2002 68

69. TARGET RNA POLTARGET cDNA STEPS INVOLVED IN TMA J. Clin. Virol. 25: S23-S29; 2002 69

70. POLTARGET cDNA STEPS INVOLVED IN TMA J. Clin. Virol. 25: S23-S29; 2002 70

71. POLTARGET cDNA PRIMER #2 STEPS INVOLVED IN TMA J. Clin. Virol. 25: S23-S29; 2002 71

72. POLTARGET cDNA PRIMER #2 Polymerase STEPS INVOLVED IN TMA J. Clin. Virol. 25: S23-S29; 2002 72

73. POLTARGET ds cDNA TARGETPOL STEPS INVOLVED IN TMA J. Clin. Virol. 25: S23-S29; 2002 73

74. POLTARGET ds cDNA TARGETPOL RNA polymerase STEPS INVOLVED IN TMA J. Clin. Virol. 25: S23-S29; 2002 74

75. POLTARGET ds cDNA TARGETPOL RNA polymerase STEPS INVOLVED IN TMA J. Clin. Virol. 25: S23-S29; 2002 75

76. POLTARGET POL RNA polymerase TARGET Transcription RNA STEPS INVOLVED IN TMA J. Clin. Virol. 25: S23-S29; 2002 76

77. TARGET RNA polymerase TARGET STEPS INVOLVED IN TMA J. Clin. Virol. 25: S23-S29; 2002 77

78. Multiple, complex reactions simultaneously at one temperature (isothermal) Amplify RNA targets without RNA isolation or DNase pretreatment Rapid kinetics POTENTIAL ADVANTAGES OF TMA “Low” risk of contamination 78

79. Detection of hepatitis C in previously- negative clinical specimens Multicopy rRNA target In vitro lower limit of detection experiments SOME LIGHT READING Am. J. Gastroenterol. 96: 2968-2972; 2001 Hepatology 32: 818-823; 2000 J. Clin. Microbiol. 44: 400-405; 2006 J. Med. Microbiol. 54: 357-360; 2005 J. Clin. Microbiol. 35: 1369-1372; 1997 79

80. THE END Stuff we’ve done Nucleic acid hybridization Nucleic acid amplification Liquid phase (hybridization protection) Solid phase (Southern hybridization) In situ hybridization (& FISH) Polymerase Chain Reaction (& real-time) Transcription-mediated amplification 80

81. THE END See you at the Dells Stuff we’ve done Nucleic acid hybridization Nucleic acid amplification Liquid phase (hybridization protection) Solid phase (Southern hybridization) In situ hybridization (& FISH) Polymerase Chain Reaction (& real-time) Transcription-mediated amplification 81