Catherine Technical Product Specialist

Transcription

1 量 PCR 理 Catherine Technical Product Specialist

2 Traditional Quantitative System StepOne Real-Time PCR System Northern Blots Analysis Competitive PCR RT-PCR followed by South blots Disadvantages Labor intensive, many manual steps Semi-quantitative Low Resolution Limited dynamic Low throughput 2 Date AB CONFIDENTIAL

3 量 PCR 量 (GMO) 量 量 : HBV, HCV,HPV Quantifier Detection mirna -- SNP 聯 3 Date AB CONFIDENTIAL

4 Real-time PCR is sequence detection in a closed-tube Y=X(1+e) n Log [DNA] Plateau lens cap PCR vessel thermal block Geometric Y=X2 n Cycle # sample 4 Date AB CONFIDENTIAL

5 Amplification plot features 0.90 Normalised reporter fluorescence (R n ) Threshold Baseline region Sample No Template control Lower expression level C T PCR cycle number Geometric Phase 5 Date AB CONFIDENTIAL Y= N0 2 n, CT 度 數

6 High StepOne ThroughPut Real-Time PCR System Nucleotide Quantitation System -2 PCR 96 PCR Amplification Curves 量 Pipette Samples StepOne / 7300 / 7500 / 7500fast / 7900 fluorescent signal Read Results real-time PCR amplification 10,000 20,000 PCR cycles Standard curve Relative quantitation 6 Date AB CONFIDENTIAL

7 ABI Real Time PCR StepOne Real-Time PCR System Total Solutions for you! ABI TaqMan Chemistry & Real-Time PCR 理論 ABI 量 & Ct 念 ABI 量 PCR efficiency 100% Genomic Assays for SNP & Gene Expression HUMAN and MOUSE 來 High Throughput Multi Channels Speed ABI 7700 ABI 7900HT 量 PCR ABI Date AB CONFIDENTIAL 2000 理量 ABI ABI 7500 StepOne

8 易 8 Date AB CONFIDENTIAL

9 Basic Operation Soft power button on the LCD touchscreen 9 Date AB CONFIDENTIAL

10 The StepOne Real-Time PCR System Introduction 48 well 0.1 ml tube Single LED excitation 3 color System (FAM / SYBR Green, VIC /JOE, ROX dyes) Fast and Standard ramping in the same block (1-100%) Standalone or co-located Remote monitoring, LAN 10 Date AB CONFIDENTIAL

11 StepOne Real-Time PCR System: The Basics One block, Two speeds Fast cycling: 40 cycles in under 40 minutes Standard cycling: 40 cycles in under 2 hours 48-wells 10-30µl reaction volume 4 11 Date AB CONFIDENTIAL

12 StepOne Real-Time PCR System: The Basics Supported consumables: 48-well plates with optical adhesive cover 48-well plates with caps Fast tubes Fast 8-tube strips 12 Date AB CONFIDENTIAL

13 Networkability: Remote Monitoring, Notification or LAN 13 Date AB CONFIDENTIAL

14 Summary 路 便 : 48 well plate or Single tube - 精 PCR : Peltier technology 兩 Sample ramp rate --- 不 更 Block - Standard mode 40 cycles 2hr - Fast mode 40 cycles 40min 14 Date AB CONFIDENTIAL

15 Chemistry Comparison TaqMan Probe SYBR Green 1 Dye Specificity Flexibility Optimization 15 Date AB CONFIDENTIAL Primer binding Probe hybridization PCR conditions Multiple Targets per tube SNP Detection +/- Application Universal guidelines Primer binding - PCR conditions Universal guidelines Check Primer dimer formation

16 Fluorogenic 5' Nuclease Assay R 5 3 Q Forward Primer R FRET Q 5 5 *FRET= Fluorescence Resonance Energy Transfer Reverse Primer Date AB CONFIDENTIAL

17 Fluorogenic 5' Nuclease Assay Displacement during Polymerization 5 3 Forward Primer R Q 5 5 R Reverse Primer 3 5 Cleavage Q Date AB CONFIDENTIAL

18 PCR Reaction Monitored in Real-Time Generating Amplification Curves Fluorescent signal is proportional to the amount of PCR product amplifying in the tube. 18 Date AB CONFIDENTIAL

19 next-generation chemistry: TaqMan MGB Probes R AGGCCTTGAGAGATAT (non-fluorescent quencher) NFQ Q Q MGB (minor groove binder) R AGGCCTTGAGAGATAT Q GCTACACAGTCCGGAACTCTCTATAGCATCACAC All probes will be short (13-20-mers) 19 Date AB CONFIDENTIAL High resolution, Low Background

20 TaqMan StepOne Real-Time chemistry PCR System Noiseless data due to the second level of specificity provided by the probe Multiplex compatible 行兩 量 each probe can be differently coloured and thereby mixed with others Signal proportional to product 度 signal related to amount of amplified product (not mass/length) Optimized assays available Assays on Demond (AoD), Assays by Design (AbD), PDAR 20 Date AB CONFIDENTIAL

21 Signal Generation with SYBR Green 1 dye SYBR Green 1 dye binds to the minor groove of ds DNA Detects specific and unspecific products 21 Date AB CONFIDENTIAL

22 SYBR Green I Dye Double Stranded DNA: Dye will fluoresce when bound to minor grove Denaturation: No Signal Chemistry reports the accumulation of dsdna in PCR Need to verify target specific amplification by other means 22 Date AB CONFIDENTIAL

23 Specificity check SYBR Green 1 dye How? When? Gel or Real-Time PCR System Post PCR Analysis add Melting Curve stage 178bp T m =77ºC Gel electrophoresis to determine PCR product size Melt Analysis to determine PCR product Tm (performed on the Real-Time PCR instrument). 23 Date AB CONFIDENTIAL

24 SYBR StepOne Green Real-Time PCR I System Chemistry 省 Probe 理 Primer Dimer PCR 離 multiplex PCR Compatible with dissociation curve analysis 利 dissociation curve assay PCR 24 Date AB CONFIDENTIAL

25 _master mix StepOne Real-Time PCR System All Chemlink Hot start (5~ 15 min) Carry over protection, UNG Real Master Mix, all in one 4 0 C, ROX ROX normalize 25 Date AB CONFIDENTIAL

26 All in One Features 量 Absolute Quantification - Standard curve 量 Realtive Quantification Ct study 量 Realtive Quantification - Relative standard curve Melting Curve Analysis SNP Genotyping Assay Presence/Absence 26 Date AB CONFIDENTIAL

27 量 ( Absolute Quantitation) - 量 - StepOne Real-Time PCR System The well contains 400 target copies Y= mx+b Ct s Ct values 27 Date AB CONFIDENTIAL Log copy number

28 量 Relative Quantification Analysis Methods Standard Curve Gene A Gene B Heart Liver Lung Brain Date AB CONFIDENTIAL Comparative C t 2 - ( Ct) Relative Quantity of Expression t = 0 t = 1 h t = 6 h t = 24 h

29 Relative Quantitation theory 量 the C T a measure of the ratio of one target to another 29 Date AB CONFIDENTIAL

30 C T is a constant value more total RNA less total RNA relative fluorescence B A threshold B A threshold C T =5 cycle number C T =5 When the PCR efficiency of both systems is the same the C T remains constant 30 Date AB CONFIDENTIAL

31 Converting C T into a ratio A (target gene) / B (endogenous gene) = 2 C T e.g. if the C T between mrna species A & B = 5 cycles (i.e. it took 5 more cycles to see amplification of A) then there is: 2 5 = 1 / 2 5 = 1 / 32 as much A as B 31 Date AB CONFIDENTIAL

32 Ct Definition Ct = Ct (Unknown) - Ct (Control) Xrel = (1+E) - Ct If Efficiency = ~100 %, Relative Quantity = 2 - Ct 32 Date AB CONFIDENTIAL

33 Relative Quantification Calibrator The sample used as the basis for comparative results t =0 t=12 t=24 t=48 time Convert purified total RNA to cdna [GOI] [GOI] [GOI] [GOI] [ENDO] [ENDO] [ENDO] [ENDO] Control for sample variability 33 Date AB CONFIDENTIAL

34 C t Calculations (Comparative C t ) IL-2 18S DC t DDC t 2 -DDCt T=0 (calibrator) T=12hr T=24hr T=48hr Relative Quantity of Expression t = 0 t = 1 h t = 6 h t = 24 h 0 34 Date AB CONFIDENTIAL

35 The Application of SNP Genotyping on Real Time PCR System

36 Genotyping competing probes discriminating alleles and single nucleotide polymorphisms Allele A Allele B FAM Q VIC Q VIC Q FAM Q Mismatch Mismatch FAM = homozygous allele A VIC = homozygous allele B FAM & VIC = heterozygous (A & B) 36 Date AB CONFIDENTIAL

37 Delta Tm ( T m ) or Mismatch Probe Stabilization MGB T m =65 T m =49 ( Tm = 17 C) 70 C 65 C 60 C 55 C 50 C T m = T m of perfectly matched probe - T m of mismatched Discrimination of alleles is achieved by using an annealing/ extension temperature within the T m window 37 Date AB CONFIDENTIAL

38 allelic discrimination (SNP) data StepOne Real-Time PCR System Homozygous Allele 2 (FAM colour signal) Heterozygotes (FAM + VIC signal) NTC s (no template controls) Homozygous Allele 1 (VIC colour signal) 38 Date AB CONFIDENTIAL

39 StepOne Software Introduction

40 All in One Features 量 Absolute Quantification - Standard curve 量 Realtive Quantification Ct study 量 Realtive Quantification - Relative standard curve Melting Curve Analysis SNP Genotyping Assay Presence/Absence 40 Date AB CONFIDENTIAL

41 Software Wizard 41 Date AB CONFIDENTIAL

42 Automatic StepOne Real-Time PCR plate Systemlayout in the Design wizard Real Time PCR 42 Date AB CONFIDENTIAL

43 Run Module 易 43 Date AB CONFIDENTIAL

44 Reaction Protocol Setup 44 Date AB CONFIDENTIAL

45 Additional New Features in the Software Absolute & Relative Standard Curve Relative Quantification can read Absolute Quantification Data 不 45 Date AB CONFIDENTIAL

46 Genotyping Assay ( Real-Time data collected in the same file as pre- and post-read) Amplification plot 易 troubleshooting 46 Date AB CONFIDENTIAL

47 Result: Export to Excel, PowerPoint, or save as JPEG 47 Date AB CONFIDENTIAL

48 48 Date AB CONFIDENTIAL

49 StepOne 不 精靈 易 - reaction condition setup 不 data 都 不 failed export to Excel, Power Point JPEG Remote Monitoring, Notification data 49 Date AB CONFIDENTIAL

50 Can the StepOne System be used as a conventional thermal cycler? You can create a thermal cycler protocol on the touchscreen and use the StepOne System as a thermal cycler. 50 Date AB CONFIDENTIAL

51 Performance specifications customers have come to expect with AB instruments - High Precision : 2- fold discrimination with 99.7% confidence - High Sensitivity : 10 copies per 30 ul - Broad Dynamic Range : 9 logs - Dual Reporter and Single Reporter Uniformity 51 Date AB CONFIDENTIAL

52 RNAse P Instrument Verification Plate 5K population SD= K population SD= Date AB CONFIDENTIAL

53 9-Log Dynamic Range Dilution series of gdna using 18S TaqMan probes and primers 53 Date AB CONFIDENTIAL

54 Dual Reporter in Fast Mode 18S SD=0.088 TGFβ SD=0.080 Multiplexed 18S VIC-labeled and TGFβ FAM-labeled TaqMan probes and primers 54 Date AB CONFIDENTIAL

55 Summary!! 便 : 48 well plate or Single tube - 精 PCR : Peltier technology - No Gradient!! 度 兩 Sample ramp rate --- 不 更 Block - Standard mode 40 cycles 2hr - Fast mode 40 cycles 40min data - (download data monitor experiment process) 了 Real-Time PCR PCR 55 Date AB CONFIDENTIAL

56 Applied StepOne Real-Time Biosystems PCR SystemIntegrated Solution on Genomic Assays Assay Development TaqMan Gene Expression Assays TaqMan SNP Genotyping Assays Custom TaqMan GX & SNP Assays mirna assays Custom Primers/Probe Design Software Universal PCR Condition on Applied Biosystems Real Time PCR System Automatic Data Analysis Auto-clustering in SNP Genotyping Gene Expression: automatic Constructing standard curve Automatic relative quantitation Data analysis 56 Date AB CONFIDENTIAL

57 Real-Time PCR

58 Two-Step StepOne Real-Time PCR System RT (High Capacity cdna Archive Kit P/N ) PCR: cdna template or gdna Probe Reagent TaqMan Probe Systems 20 x probe/ primer mix (TaqMan Gene Expression Assay) (Custom TaqMan Gene Expression Assay) Or Primer Express software design (300 nm primer nm probe) P/N (200 Rx) + 2x TaqMan Universal PCR Master Mix SYBR Green I Systems Primer Express software design 行 (optimized primer conc.) P/N (200 Rx) + 2x SYBR Green I PCR Master Mix 58 Date AB CONFIDENTIAL

59 One-step StepOne Real-Time RT-PCR: System RNA template Probe Reagent TaqMan Probe Systems 20 x probe/ primer mix P/N (200 Rx) TaqMan One-Step RT-PCR Master Mix Reagents (TaqMan Gene Expression Assay) Kit (Custom TaqMan Gene Expression Assay) Or Or Primer Express software design (300 nm primer nm probe) N (200 Rx) TaqMan EZ RT PCR core Reagents SYBR Green I Systems Primer Express software design 行 (optimized primer conc.) P/N SYBR Green RT-PCR Reagent (P/N N ) 59 Date AB CONFIDENTIAL

60 Performance of Power SYBR Green Master Mix 60 Date AB CONFIDENTIAL

61 All include ROX as the internal passive reference 61 Date AB CONFIDENTIAL

62 StepOne Real Real-Time PCR System Time PCR Reverse Transcription : High Capacity cdna archive Kit Real Time PCR: TaqMan Chemistry 2x TaqMan Master Mix 1x 12.5 ul 20x Probe/primer Assay Mix 1.25 ul Water NA cdna ng 5-10 ul Component 10x Reverse Transscription Buffer 25x dntp 10x Random Primer RT Enzyme 50U/uL RNA (0.1~10 ug) H2O 25 ul SYBR Chemistry 2x SYBR Master Mix 1x 12.5 ul F Primer optimized NA R Primer optimized NA Water NA cdna ng 5-10 ul 25 ul 62 Date AB CONFIDENTIAL

63 Applied Biosystems, Taiwan Accelerate the Pace of Discovery, Enable Sciences for Life :