Kayhan Azadmanesh MD, Ph.D. Virology Department

Transcription

1 Principles of molecular tests Kayhan Azadmanesh MD, Ph.D. Virology Department Pasteur Institute t of Iran 1

2 DNA molecule (1954) 2

3 Southern Blot (1975) 3

4 We needed to know the sequence of the genes and probes! F. Sanger (1977) 4

5 Now we can amplify our target : Polymerase chain reaction Kary Mullis

6 Polymerase Chain Reaction Primers and DNA Strand denaturation and primer annealing PCR Dreischrittreaktion Primer extension Denaturation 95 C Cycling Exponential amplification of PCR products Extension 72 C Annealing C 6

7 Earlier PCR machines! 7

8 Denaturation Target Sequence Target Sequence ٨

9 Annealing ٩

10 Extension ١٠

11 The Result of First PCR Cycle ١١

12 Target Amplification No. of Cycles No. Amplicon Copies of Target 1 cycle = 2 Amplicon 2 cycle = 4 Amplicon 3 cycle = 8 Amplicon 4 cycle = 16 Amplicon 5 cycle = 32 Amplicon 6 cycle = 64 Amplicon 7 cycle = 128 Amplicon ,048, ,073,741,824 ١٢

13 Polymerase Chain Reaction Plateau phase t PCR Produc Linear phase Exponential phase End point analysis on agarose gels Cycles 13

14 Visualize the result 14

15 15

16 Mass spectrometric Identification of PCR products 16

17 Real Time PCR 17

18 SYBR Green (1) At the beginning i of amplification, i the reaction mixture contains the denaturedd DNA, the primers, and the dye. The unbound dye molecules weakly fluoresce, producing a minimal background fluorescence signal which is subtracted during computer analysis. (2) After annealing of the primers, a few dye molecules can bind to the double strand. DNA binding results in a dramatic increase of the SYBR Green I molecules to emit light upon excitation. (3) During elongation, more and more dye molecules bind to the newly synthesized DNA. If the reaction is monitored continuously, an increase in fluorescence is viewed in real-time. Upon denaturation of the DNA for the next heating cycle, the dye molecules are released and the fluorescence signal falls. 18

19 5 Nuclease Assay using TaqMan probes FAM, VIC TAMRA dyes Forward Primer R TaqMan Probe PCR specificity (primer) Q Hybridization specificity (probe) Reverse 5 Primer

20 5 Nuclease Assay using TaqMan probes R Q Fluorescence Resonance Energy Transfer (FRET) from high energy to low energy dye No reporter signal with intact 20probe

21 5 Nuclease Assay using TaqMan probes R R 5 p 3 5 Q Displacement of probe by 5 nuclease activity of polymerase 21

22 5 Nuclease Assay using TaqMan probes R Q Cleavage of probe by 5 nuclease activity of Taq qpolymerase FRET disabled, generation of reporter signal 22

23 Probes for real time PCR 23

24 Molecular Beacon 24

25 Thermal cycling and data aquisition 25

26 Dyes and their colours 26

27 Concept of Threshold and Ct Value Rn Thresh old Ct value Baseline 27 Cycles

28 28 threshold = 3

29 From Fluorescence to Results Step 2 Comparison of Ct Values The well contains 400 target copies Ct s s Ct values Log copy number

30 threshold 30

31 Standard Curve Ct value es y = x R 2 = Efficiency = 10 (-1/slope) Log copy number Example: Slope = E = 10 (-1/-3.3) 1 = 10 (0.30) 1 = If slope = efficiency i becomes 1 = or 99.5% 31

32 Full Efficiency y=x(1+e) n If PCR efficiency i is 1, the equation becomes y = x 2 n y = Number of PCR products x = Initial target copy number E = Efficiency n = Number of PCR cycles 32

33 CYCLE AMOUNT OF DNA AMOUNT OF DNA AMOUNT OF DNA AMOUNT OF DNA 100% EFFICIENCY 90% EFFICIENCY 80% EFFICIENCY 70% EFFICIENCY , ,048 1, ,096 2,213 1, ,192 4,205 2, ,384 7,990 3,748 1, ,768 15,181 6,747 2, , , , , ,072 54,804 21,859 8, , ,127 39,346 14, , ,842 70,824 23, ,048, , ,482 40, ,097, , ,468 69, ,194,304 1,356, , , ,388,608 2,578, , , ,777,216 4,898,763 1,338, , ,554,432 9,307,650 2,408, , ,108,864 17,684,534 4,335, , ,217,728 33,600,615 7,804,726 1,667, ,435,456 63,841,168 14,048,506 2,835, ,870, ,298,220 25,287,311 4,819, ,073,741, ,466,618 45,517,160 8,193,466 AMO OUNT OF DNA AMOUNT OF DNA 1,200,000,000 1,000,000, ,000, ,000, ,000, ,000, ,000,000,000 1,000,000, ,000,000 10,000,000 1,000, ,000 10,000 1, % EFF 90% EFF 80% EFF 70% EFF PCR CYCLE NUMBER 100% EFF 90% EFF 80% EFF 70% EFF PCR CYCLE NUMBER

34 Genotyping HPV Line Probe Assay (LiPA) kaline Phosphatase Strepavidin Biotin 3 Genotype-specific p DNA probes on nitrocellulose strip 34 Biotin-labeled L1 region of amplified target t

35 Genotyping by Reverse Hybridization HPV types identified by INNO-LiPA HPV Genotyping Extra: *Hi High risk: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, 82 * Probable high risk: 26, 53, 66 * Low risk: 6, 11, 40, 43, 44, 54, 70 * Additional types: 69, 71, 74 35

36 A typical Sequencing result 36

37 HPV genotypes 37

38 Principles of bdna 38

39 Overview 41-Plex H-Typing RT-PCR Assay Developed to Detect Inf-A in Humans & Birds 37 Labeled AI primer sets Reverse transcriptase PCR assay 3 H1 6 H2 5 H3 5 H5 9 H7 6 H9 2 M(7) 1 NS(8) 6 H gene types from Inf A Plus 3 Pan A signatures nsity Red Inten 39 Orange Intensity

40 Identification of M. tuberculosis Wild Type drug resistance No signal Multidrug-resistant TB His 526>Tyr (rpob) No signal Ser 315>Thr (katg) IS 6110 IS 6110 probe probe Routine therapy Second line drugs should be used 40

41 41