LightCycler 480 Real-Time PCR System. Alvan Wang

Transcription

1 LightCycler 480 Real-Time PCR System Alvan Wang

2 1. LC 480 Basic training 2. Experiment set up 3. LC480 Software 2

3 LC 480 Basic training : 1. Real time PCR principle introduction 2. Detection formats 3. Absolute/Relative Quantification 4. Genotyping 3

4 Introduction Real-time PCR is the combination of conventional PCR and detection within one reaction vessel. Amplification of DNA with polymerase chain reaction (PCR) is monitored in real time (fluorescence measurement) Why we have to monitor the amplification of DNA in Real time? 4

5 Monitoring of PCR Reactions If a PCR would be started with 1 molecule of DNA 36 The amplification signal will be detected after about 36 cycles

6 Monitoring of PCR Reactions If a PCR would be started with 10 molecule of DNA The amplification signal will be detected after about 32.8 cycles

7 Monitoring of PCR Reactions If a PCR would be started with 100 molecule of DNA The amplification signal will be detected after about 29.1 cycles

8 Monitoring of PCR Reactions If a PCR would be started with 1000 molecule of DNA The amplification signal will be detected after about 26 cycles

9 Monitoring of PCR Reactions If a PCR would be started with molecule of DNA The amplification signal will be detected after about 22.6 cycles

10 Monitoring of PCR Reactions If a PCR would be started with molecule of DNA The amplification signal will be detected after about 19.1 cycles

11 Monitoring of PCR Reactions If a PCR would be started with molecule of DNA The amplification signal will be detected after about 15.8 cycles

12 Monitoring of PCR Reactions If a PCR would be started with molecule of DNA The amplification signal will be detected after about 12.5 cycles

13 Monitoring of PCR Reactions If a PCR would be started with molecule of DNA The amplification signal will be detected after about 9.4 cycles

14 If we can monitor of PCR reaction in real time If a PCR would be started with molecule of DNA The amplification signal will be detected after about 5.9 cycles

15 There is a correlation between Cycle no. and concentration 10XE9 10XE8 10XE7 10XE6 10XE5 10XE4 10XE3 10XE2 10XE1 10xE The higher concentration of target nucleic acid in the starting material, the sooner a significant increase in fluorescent signal will be observed, yielding a lower Cycle no.

16 LC 480 Basic training : 1. Real time PCR principle introduction 2. Detection formats 3. Absolute/Relative Quantification 4. Genotyping 16

17 LightCycler Detection Formats Non-specific binding dye Specific labeled probes SYBR Green I Hybridization Probe Hydrolysis Probe (TaqMan) Elongation Phase Annealing Phase Elongation Phase 17

18 LightCycler Assay Formats 95 40~45 cycle SYBR Green I Format

19 Analysis Module Tm Calling 19

20 LightCycler Assay Formats Fluorescence Resonance Energy Transfer (FRET) 20

21 LightCycler Assay Formats Hydrolysis Probe-TaqMan Probe 1. 多色 Multiplex 2. 不需 melting curve 21

22 LightCycler Assay Formats Hybridization Probes No primer-dimer or side-products disturbance! High specificity Don t need to run the Melting curve High efficiency, 22

23 LC 480 Basic training : 1. Real time PCR principle introduction 2. Detection formats 3. Absolute/Relative Quantification 4. Genotyping 23

24 Cp(Cross Point) Ct(Threshold Cycle) Cq(Quantification Cycle) Calculation

25 PCR Quantification Theoretical and Practical Aspects N N = N 0 x 2 n Theory N = N 0 x (E const ) n log-phase-pcr n N 0 N N Real N = N 0 x (E var ) n end-point-pcr n N 0 N: number of amplified molecules n: number of amplification cycles N 0: initial number of molecules E: amplification efficiency 25

26 Fit Point method (user-influenced) Fit Point method (user-influenced) 26

27 Crossing Point Calculation 2 nd Derivative Maximum Method st Derivative Max 2 nd Derivative Max Fluorescence Signal Crossing point Fully automated method Without user influence Based only on the shape of the amplification curve 27

28 Concentrations and Crossing Points Calculation of crossing points (Cp) Optional with Fit Point method (user-influenced) Standard method (automatic) 2nd Derivative Maximum Method 二次微分最大值 28

29 There is a correlation between Cp and concentration The higher concentration of target nucleic acid in the starting material, the sooner a significant increase in fluorescent signal will be observed, yielding a lower Cycle no. 10XE9 10XE8 10XE7 10XE6 10XE5 10XE4 10XE3 10XE2 10XE1 10xE

30 LC 480 Basic training : 1. Real time PCR principle introduction 2. Detection formats 3. Absolute/Relative Quantification 4. Genotyping 30

31 Fluorescence Crossing Point Fluorescence Absolute Quantification with External Standards: Principle Target Cycles Unknown Sample Result absolute value (e.g. copy number) Standards Cycles Log Concentration Standard Curve 31

32 Relative Quantification Scheme Wanted: Target gene expression level T = 100 T = 10 T = 1 = 2 = 2 = 0.2 R = 50 R = 5 R = 5 Required: Target gene concentration Reference gene concentration Calibrator sample (optional) = calibrator 32

33 Relative Quantification - Without Efficiency correction Type gene Cp Sample Target 22 Reference = = 2 1 = 2-1 = 2 Ct = 2 - Ct = 1/2 Type gene Cp Calibrator Target 25 (control) Reference 20 = = 2 5 = 2-5 = 2 Ct = 2 - Ct = 1/32 33

34 Relative Quantification - Without Efficiency correction Type gene Cp Sample Target 22 Reference 21 Type gene Cp Calibrator Target 25 (control) Reference 20 = = 2 1 = 2-1 = 1/2 = 2 Ct = 2 - Ct = 2-1-(-5) = 2 4 = 16 = = 2 5 = 2-5 = 2 - Ct = 2 Ct = 2 - Ct = 1/32 34

35 LC 480 Basic training : 1. Real time PCR principle introduction 2. Detection formats 3. Absolute/Relative Quantification 4. Genotyping 35

36 Melting Curve Genotyping Principle How to detect? perfect match Anchor Probe Mutation Probe mismatch Anchor Probe Mutation Probe

37 Melting Curve Genotyping Principle Low Medium High Temperature

38 Melting Curve Based Genotyping Genotyping of Single Point Mutation Melting Curves Fluorescence Mismatch Perfect Match Melting Peaks -df/dt Mutation WT C Temperature 38

39 Experiment Set Up: 1. Primer/Probe design 2. Universal probe library 3. Preparation of Standard curve/pcr efficiency 39

40 Primer&Probe design: Target gene sequence 40

41 Primer&Probe design: Sequence Alignment and find the conserve region BioEditor 41

42 Primer&Probe design: Website for primer design 42

43 Primer&Probe design: 43

44 Primer&Probe design: Hydrolysis probe 44

45 PCR 配方 and condition Hydrolysis probe(taqman) Forward primer (10uM) Reverse primer (10uM) Probe(10uM) LightCycler 480 Probe Master (2x) Water (PCR-grade) Sample (1~100ng) 1ul (0.3~1uM, recommended 0.5uM) 1ul (0.3~1uM, recommended 0.5uM) 0.4ul (0.05~0.2uM) 10ul 2.6ul 5ul 45

46 Experiment Set Up: 1. Primer/Probe design 2. Universal probe library 3. Preparation of Standard curve/pcr efficiency 46

47 Specificity Real-Time PCR Specificity or Flexibility? Today you have to decide whether using a specific probe-based assay or a flexible SYBR Green I assay! Probe based assays? Universal ProbeLibrary What about having both characteristics together in one assay? SYBR Green I* Flexibility * and other intercalating dye based assays

48 Universal ProbeLibrary Only 165 probes for over 5 millions assays Organism Species Name Assays Available Coverage Rate Human Homo sapiens > % Mouse Mus musculus > % Rat Rattus norvegicus > % Primates Pan troglodytes > % Drosophila Drosophila melanogaster > % Arabidopsis Arabidopsis thaliana > % C. Elegans Caenorhaboditis elegans > % Maize Zea mays > % Rice Oryza sativa > % Zebrafish Anopheles gambiae > % Yeast Saccharomces cerevisiae > % Total >

49 Universal ProbeLibrary (UPL) What is it? Universal ProbeLibrary (UPL) a unique combination of pre-validated hydrolysis probes and free online assay design that allows you to design custom gene expression assays to quantify virtually any transcript in any genome. 165 Pre-validated Hydrolysis Probes + Free Online Assay Design Center = UPL

50 Assay design with ProbeFinder ProbeFinder free web tool at:

51 Assay design with ProbeFinder Step1: Choose an organism

52 Assay design with ProbeFinder Step2: Enter your sequnce/ gene of interest Option1: Enter your sequence ID Option2: Paste sequences intron spanning assay

53 Assay design with ProbeFinder Step3: Output information of optimal assay The optimal probe No. Primer information (sequence, Tm, %GC) Amplicon information

54 Assay design with ProbeFinder Step3: Output information of optimal assay Forward primer # 37 probe Reverse primer intron intron

55 Assay design with ProbeFinder Step3: Output information of optimal assay

56

57 Experiment Set Up: 1. Primer/Probe design 2. Universal probe library 3. Preparation of Standard curve/pcr efficiency 57

58 Absolute Quantification with External Standards: Example 58

59 Example for Relative Quantification Type gene Cp Calibrator Target 25 Reference 20 Sample Target 22 Reference 21 I. Without efficiency correction Relative amount = 2 -ΔΔCT = 2 [(22-21)-(25-20)] = 2 [1-5] =2 4 = 16 II. With efficiency correction E T = 1.6 E R = 1.8 Relative amount = E T CpT (C) - CpT (S) X E R CpR (S) - CpR (C) = 1.6 (25-22) X 1.8 (21-20) = X =

60 LightCycler480 Software: 1. New experiment from template 2. Subset & sample set up 3. Data analysis & report 60

61 System Start-Up 61

62 System Start-Up 62

63 Open the software and Login to the data base For PC login ID: operator PW: LC480 admin LightCycler480 63

64 LightCycler 480 Software Version 1.5 Front screen: New experiment from template 64

65 Start Run 65

66 Apply template to create a new experiment 溫度 偵測模式時間升降溫速度 : 升溫 4.4 /s 降溫 2.2 /s 66

67 Start your experiment by your template 67

68 Subset Step 2 Step 1 Step 3 68

69 Sample Editor AQ example : Step 4 : Select all well and click 69

70 Sample Editor RQ example : Step 4 : Select all well and click 70

71 Analysis 分析模式簡要說明 : 分析模式 Abs Quant / 2nd Derivative Max Abs Quant / Fit Points Advanced Relative Quantification Basic Relative Quantification Endpoint Genotyping Melt Curve Genotyping Tm Calling 說明絕對定量 (2 nd Max) 絕對定量 (Fit Point) 進階相對定量 (2 nd Max or Fit Point) 基礎相對定量 (Fit Point only) 基因分型 ( 適用於 TaqMan probe) 基因分型 ( 適用於 Hyprobe) Tm 分析 71

72 Analysis 72

73 AQ Analysis 73

74 AQ Analysis 74

75 RQ Analysis 75

76 RQ Analysis 76

77 RQ Analysis 77

78 Report 78

79 Q&A

80 Doing now what patients need next