LightCycler 480 Real-Time PCR System. Alvan Wang

Transcription

1 LightCycler 480 Real-Time PCR System Alvan Wang

2 1. LC480 Basic training 2. Genotyping by LC LC480 SW introduction 2

3 LC 480 Basic training : 1. Assay Formats and Cp Calculation 2. Absolute/Relative Quantification 3

4 LightCycler Detection Formats Non-specific binding dye Specific labeled probes SYBR Green I Hybridization Probe Hydrolysis Probe (TaqMan) Elongation Phase Annealing Phase Elongation Phase 4

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

6 Analysis Module Tm Calling 6

7 LightCycler Assay Formats Fluorescence Resonance Energy Transfer (FRET) 7

8 LightCycler Assay Formats Hydrolysis Probe-TaqMan Probe 1. 多色 Multiplex 2. 不需 melting curve 8

9 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

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

11 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 11

12 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 12

13 Fit Point method (user-influenced) Fit Point method (user-influenced) 13

14 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 14

15 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

16 LC 480 Basic training : 1. Assay Formats and Cp Calculation 2. Absolute/Relative Quantification 16

17 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 17

18 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 18

19 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 19

20 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 20

21 Absolute Quantification with External Standards: Example 21

22 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 =

23 Q&A

24 Single Nucleotide Polymorphism(SNP) 單一核苷酸多型性 :DNA 序列單一位置發生變異 1. Synonymous SNP: 轉譯出的胺基酸不變 2. Nonsynonymous SNP: 轉譯出的胺基酸有變 24

25 Single Nucleotide Polymorphism(SNP) 使個人體質 易患上某種疾病 或 改變對某些藥物的反應 1. 非小細胞肺癌的標靶藥物, 對 K-RAS 發生 T790M 突變的患者療效不佳 2. 大腸直腸癌的標靶藥物, 對 K-RAS 發生 codon 12,13 突變的患者療效不佳 3. 已知 BRAF(V600E) 突變是大腸直腸癌重要的不良預後標記 1. RFLP 2. Sequence How to detect? 3. MALDI-TOF 4. Real time PCR 25

26 Genotying by LC480: 1. End Point Genotyping Assay 2. Melting Curve Genotyping Assay 3. High Resolution Melting 26

27 Genotyping by LC 480 Real time PCR Endpoint genotyping

28 Endpoint Genotyping (allelic discrimination) Principle How to detect? 28

29 Endpoint Genotyping (allelic discrimination) Principle 29

30 Endpoint Genotyping (allelic discrimination) Principle Fluorescence FAM VIC C GG sample GT sample TT sample TT sample Cycles GT sample A Fluorescence VIC FAM GG sample Cycles

31 Genotyping by LC 480 Real time PCR Melting genotyping

32 Endpoint Genotyping Data Influence of DNA Concentration Fluorescence FAM Cycles 1 and 20 ng DNA 1ng 20ng Fluorescence VIC Cycles 32

33 Endpoint Genotyping Data Influence of reaction cycle 20 cycle 40 cycle 30 cycle 35 cycle 33

34 Endpoint Genotyping Data Influence of reaction cycle 20 cycle 40 cycle 30 cycle 35 cycle 34

35 Endpoint Genotyping Data Not for Multiple SNP or High-Throughput SNP Analysis 35

36 SNP detection with Endpoint Genotyping TaqMan probe system (Endpoint Genotyping) 1. Probe 分辨率 How to detect?? 2. Repeat sequence 3. 鄰近的 SNP 4. DNA conc. 5. Reaction cycle 36

37 Melting Curve Genotyping Principle perfect match Anchor Probe Mutation Probe mismatch Anchor Probe Mutation Probe

38 Melting Curve Genotyping Principle Low Medium High Temperature

39 Melting Curve Based Genotyping Genotyping of Single Point Mutation Melting Curves Fluorescence Mismatch Perfect Match Melting Peaks -df/dt Mutation WT C Temperature 39

40 Endpoint Genotyping Data Influence of DNA concentration and reaction cycle 1 and 20 ng DNA 20ng 1ng 40 cycle 35 cycle 40

41 Endpoint Genotyping Data Influence of DNA concentration and reaction cycle 41

42 Endpoint Genotyping Data Influence of DNA concentration and reaction cycle -- A -- B -- A:B =1:9 -- A:B =3:7 -- A:B =5:5 -- A:B =7:3 -- A:B =9:1 42

43 LightCycler Genotyping: Example SNP with Three Different Alleles

44 Genotyping by LC End point genotyping 2. Melting genotyping by probe 3. High Resolution Melting 44

45 Analysis Module Tm Calling 45

46 Amplicon Melting Principle of Gene Scanning by Melting 1 /read 46

47 Amplicon Melting Principle of Gene Scanning by (HRM) High Resolution Melting 1 /read 0.1 /read 47

48 High Resolution Melting Application Mutation detection SNP detection Methylation Genotyping etc 48

49 Prerequisites and Innovations What Is Needed to Perform HRM? Novel intercalating dye. Precise Instrument. Analysis software. 49

50 Novel intercalating dye SYBR Green I 非飽合螢光物質 較弱的模糊訊號 失真的結果 LightCycler 480 ResoLight Dye 飽合性螢光物質 強且穩定的訊號 50

51 Novel Signal-Improved HRM Dye Provided in High-Resolution Melting Master SYBR Green I vs LightCycler 480 ResoLight Dye 51

52 Prerequisites and Innovations What Is Needed to Perform HRM? Novel intercalating dye. Precise Instrument. Analysis software. 52

53 Preparing a Gene Scanning Experiment PCR Programs: High Resolution Melting Example: Melting 0.2 /read HRM 0.04 /read 53

54 Q&A

55 LightCycler480 Software: 1. New experiment from template 2. Subset & sample set up 3. Data analysis & report 55

56 System Start-Up 56

57 System Start-Up 57

58 Open the software and Login to the data base For PC login ID: operator PW: LC480 admin LightCycler480 58

59 LightCycler 480 Software Version 1.5 Front screen: New experiment from template 59

60 Start Run 溫度 偵測模式時間升降溫速度 : 升溫 4.4 /s 降溫 2.2 /s 60

61 Start Run 61

62 Start Run 62

63 Start your experiment by your template 63

64 LightCycler480 Software: 1. New experiment from template 2. Subset & sample set up 3. Data analysis & report 64

65 Subset Step 2 Step 1 Step 3 65

66 Sample Editor AQ example RQ example 66

67 Sample Editor HRM example : 67

68 LightCycler480 Software: 1. New experiment from template 2. Subset & sample set up 3. Data analysis & report 68

69 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 69

70 Analysis 70

71 AQ Analysis 71

72 AQ Analysis 72

73 RQ Analysis 73

74 RQ Analysis 74

75 RQ Analysis 75

76 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 76

77 HRM Analysis 77

78 HRM Analysis 78

79 HRM Analysis 79

80 HRM Analysis 80

81 HRM Analysis 81

82 Report 82

83 Q&A

84 Doing now what patients need next