REPLICATION AND POLYMERASE CHAIN REACTION (PCR)

Transcription

1 REPLICATION AND POLYMERASE CHAIN REACTION (PCR) Debbie S. Retnoningrum School of Pharmacy, ITB References: 1. Glick, BR and JJ Pasternak, 2003, pages 27 28; Groves MJ, 2006, pages Brown TA, 2006, pages 6 7; Replication & PCR 1

2 The Central Dogma of Molecular Biology Replication & PCR 2

3 REPLICATION Replication is DNA duplication Synthesis of two DNA molecules from 1 DNA molecule Base pairing concept Direction of synthesis: 5 3 in the new strand Catalyzed by DNA polymerase Replication & PCR 3

4 Components of DNA replication Started at origin of replication (ori) Single strand binding protein (SSB) Helicase (opens the double helix) Primase (RNA polymerase) DNA polymerase Replication & PCR 4

5 DNA REPLICATION: Extension of new DNA strand dntps: datp, dttp, dgtp, dctp Role of DNA polymerase Replication & PCR 5

6 DNA POLYMERASE Prokaryote Pol I Pol II Pol III Eukariot Pol-α Pol-β Pol-δ Pol-γ Pol-ε Function(s) Gap filling & DNA repair Gap filling & DNA repair Replication Replication of lagging strand Gap filling & DNA repair Replication of leading strand Replication of mitochondria Gap filling & DNA repair Replication & PCR 6

7 Polymerase Chain Reaction (PCR) Karry Mullis, inventor of PCR Nobel prize winner Replication & PCR 7

8 PCR CYCLES CYCLE 1 CYCLE 2 CYCLE 3 T 94 o C 72 o C 55 o C TIME (SECOND) Replication & PCR 8

9 PCR CYCLES AND DNA AMPLIFICATION Replication & PCR 9

10 Polymerase Chain Reaction (PCR) Replication & PCR 10

11 PCR COMPONENTS DNA TEMPLATES SHORT OLIGONUCLEOTIDES (primers) Thermostabile DNA polymerase :Taq Polymerase (Thermus aquaticus)& buffer dntps (datp, dttp, dctp dan dgtp) Replication & PCR 11

12 Sequenced genomes Replication & PCR 12

13 Examples of genome information in genbank Replication & PCR 13

14 Amplification of target DNA from genome Replication & PCR 14

15 Amplification of target DNA from genome Replication & PCR 15

16 DETECTION OF PCR PRODUCT Replication & PCR 16

17 HOME BREW PCR COBAS AMPLICOR Replication & PCR 17

18 PCR FOR PATHOGEN DETECTION P1 GENE TARGET P2 PCR PRODUCT: 490 BASE PAIRS GENE TARGET SHOULD BE PATHOGEN SPECIFIC Replication & PCR 18

19 PCR FOR PATHOGEN DETECTION DETECTION OF Mycobacterium tuberculosis DNA TARGET: IS6110 HIGH COPY NUMBER SPESIMEN: SPUTUM DETECTION OFChlamydia trachomatis DNA TARGET: GEN MAJOR OUTER MEMBRANE PROTEIN = MOMP Spesimen: vaginal swap (4), blood (5), eye swab (6) Replication & PCR 19

20 MODIFIED PCR PCR HIBRIDIZATION (PCR PRODUCT WAS HYBRIDIZED WITH PROBE) MULTIPLEX PCR Nested PCR PCR Sequencing Real time PCR Replication & PCR 20

21 PRINCIPLE OF HYBRIDIZATION Hybridization Addition of probe Denaturation Replication & PCR 21

22 PCR HYBRIDIZATION USING COLORIMETRIC DETECTION lysis PCR Colorimertic detection Σ Target molecule Σsignal denaturation substrate hybridization Replication & PCR 22

23 COLORIMETRIC DETECTION TMB H2O2 Avidin Biotin Horse radish peroxidase PCR product Probe Replication & PCR 23

24 Multiplex PCR Primer pairs for target DNA > 1 Example: Escherichia coli: TARGET GENES LT1, ST1, SLT1, SLT2, VT1, VT2, VTE 7 PRIMER PAIRS 7 PCR PRODUCTS Replication & PCR 24

25 RESULT OF MULTIPLEX PCR S A B C D LT1 ST1 SLT1 SLT2 VT1 VT2 VTE Replication & PCR 25

26 Example of Multiplex PCR Replication & PCR 26

27 Nested PCR FLAGELIN GENE OF Salmonella typhi ST1 458 bp ST3 First PCR ST4 ST2 Second PCR 343 bp Replication & PCR 27

28 First Round S. typhi Nested PCR 1. DNA Marker (1, 0.75, 0.5, 0.3, 0.15, kb) 2. Negative control S. typhi 3. Positive control S. typhi (0.458 kb) 4. S. thypi non infected patient (negative) 5. S. thypi infected patient (positive) 6. S. thypi infected patient (positive) Second Round S. typhi Nested PCR 1. DNA Marker (1, 0.75, 0.5, 0.3, 0.15, kb) 2. Negative control S. typhi 3. Positive control S. typhi (0.343 kb) 4. S. thypi non infected patient (negative) 5. S. thypi infected patient (positive) 6. S. thypi infected patient (positive) Replication & PCR 28

29 PCR SEQUENCING Replication & PCR 29

30 PCR REAL TIME CONVENTIONAL PCR : AMPLIFICATION AND DETECTION STEPS OCCUR IN CONSECUTIVE REAL TIME PCR: AMPLIFICATION AND DETECTION STEPS OCCUR SIMULTANEOUSLY Replication & PCR 30

31 DETECTION IN CONVENTIONAL PCR Replication & PCR 31

32 Detection range of real time PCR Replication & PCR 32

33 HYBRIDIZATION PROBE Replication & PCR 33

34 HYBRIDIZATION PROBE Replication & PCR 34

35 Example of realtime PCR result Replication & PCR 35