Bio-Rad GradientBio-Rad Gradient Real-Time PCRReal-Time PCR溫度梯度定量溫度梯度定量 PCR PCR 第一品牌 第一品牌

美商伯瑞股份有限公司台灣分公司 賴智偉

What’s Real Time PCR?What’s Real Time PCR?

Real time PCR = QPCR = RT PCR ≠ Room Temperature PCR

主要應用領域主要應用領域

• 基因表現– 藥物作用– 腫瘤指標– 基因調控– 基因治療– 微陣列基因晶片確認– GMO

• 菌種偵測– 多重病原菌同時偵測– 適合大量檢體篩選 , 菌種活性– 定性 , 定量 同時完成– 藥物治療成效的監控

• SNP 分型– 唯一可用 Taqman, Molecular Beacon and FRET 三種方法的品牌

革命性的取代傳統定量方法的缺點革命性的取代傳統定量方法的缺點-- -- 競爭性 競爭性 PCRPCR ，，南南 (( 北北 )) 方墨點法 方墨點法 or cultureor culture

• 節省時間– 只需 1.5 小時儀器全自動 vs 2-3

天完全勞動 , 無須另外跑電泳或轉漬

• 更精確的數據– 自動計算精確的數據 vs 墨點濃淡的

比例

• 極高的操作成本

• 集多功於一平台– 定性篩選– 定量– 分型

Con. PCR vs QPCRCon. PCR vs QPCR

+Taq+dNTP+MgCl2+H2O+Buffer+Template+PrimerOn ice

• QL only• End point• Fuzzy data• Labor

+Master Mix+H2O+Template+PrimerRoom Temp

• QN/QL• On line monitor• Clear, Sensitive• Automatic

> 3 hrs(1.5 hrs labor)

<1.5 hrs(20 min labor)

Real Time PCR Real Time PCR 理論基礎 理論基礎

Baseline Log phase Plateau

Y= N0 2n

• Log phase 切線斜率

• Plateau 螢光強度

Ct Ct 與起始濃度的對數成反比 與起始濃度的對數成反比

高濃度低濃度

Real Time PCR Real Time PCR 壓力指數壓力指數

• Primer/Probe Design ( 壓力指數 : )

• NA Extraction ( 壓力指數 : )

• QPCR optimization ( 壓力指數 : )

• Data analysis ( 壓力指數 : )

• Trouble shooting ( 壓力指數 : )

• Report ( 壓力指數 : )

35%

2%

20%

10%

30%

3%

選擇 選擇 QPCRQPCR 時時 , , 您需要的是您需要的是……

• 高度整合的 Primer / Probe 設計軟體

• 多功能的操作軟體 / 硬體

• 完整試劑組合

• 完備的後勤支援

““ 快速” 的定義應是…快速” 的定義應是… ..

如何縮短測試時間如何縮短測試時間

QPCR QPCR 一個關鍵 三大課題一個關鍵 三大課題

• 如何縮短測試時間 : 關於 Primer / Probe 的效能

– Sensitivity• The difference from “Detection Limit”

– Dynamic Range• Serial dilution

• How to in-range

– PCR Efficiency• >100%

• <100%

唯一的解決方案唯一的解決方案

• 善用 Gradient ( 溫度梯度 ) 的功能10x 序列稀釋

65~55 度

Which one is better?Which one is better?

Gradient Gradient 抓條件抓條件 , , 一次搞定一次搞定

94C

65C

55C

72C

Series dilution

只須一次反應只須一次反應 , , 即可讓你知道即可讓你知道

• 定量偵測範圍 ( dynamic range) 有多寬 ?• 哪一個條件下靈敏度 (sensitivity) 最好 ? • 哪一個條件下可得最好的標準曲線 (best

PCR efficiency standard curve) ?

Extension

5’ 3’

5’3’5’ 3’

5’3’

Extension ContinuedApply ExcitationWavelength

5’ 3’

5’3’5’

5’

Taq

Taq

3’

5’3’

Taq

Taq5’

5’

SYBR Green I SYBR Green I dsDNA Binding DyesdsDNA Binding Dyes

DBD DBD

DBD DBDDBD

DBD DBD DBD

DBD DBD

l l l

ll

SYBR SYBR 注意事項注意事項

• Primer dimmer• Non-specific binding• Melting Curve• No need to adjust using Bio-Rad

5’ 3’5’3’5’3’

RQ

Extension Step

5’ 3’1. Strand DisplacementTaq

3’

Q

R

5’

5’ 3’3’

Q Taq

R

5’ 2. Cleavage

3. PolymerizationComplete5’ 3’

QTaqR

3’ 5’

4. Detection5’ 3’3’

QTaqR

5’

R

TaqMan (MGB) ProbeTaqMan (MGB) Probe

TaqMan TaqMan 注意事項注意事項

• More specific than SYBR Green• Multiplex able using different reporter dye• SNP genotyping application• More high cost than SYBR green I• Data collected in extension stage

Molecular BeaconsMolecular Beacons

5’ 3’5’3’

Extension Step5’ 3’

1. Strand Displacement2. Polymerization

Complete3. Probe Silent

Taq5’

5’3’

RQDetection

5’3’

RQ

5’ 3’3’ 5’

TaqR Q

MolecularBeacon

Molecular Beacons Molecular Beacons 注意事項注意事項

• The most specific• Higher cost• Not so popular in Taiwan• Data collected in annealing

stage

Bio-Rad Real-Time PCR Quick viewBio-Rad Real-Time PCR Quick view

Price

Features & FlexibilityFeatures & Flexibility

Price

Mini-OpticonMy iQ

Chromo4iQ5

Bio-Rad Real-time Bio-Rad Real-time PCR InstrumentsPCR Instruments

Dedicated Analytical Tool

• New software in a two phased release Q2 Improvements– 21 CFR Part 11 compliance– Integrated multifile gene

expression analysis– User profiles with new file

security options

• Improved costs of goods

• Modular, serviceable optical components

iQ5 Real-Time PCR detection system

Goal of Marketing positionGoal of Marketing position

• Gradient: – Primer / probe performance

• Complete Solution in qPCR– Primer probe design– qPCR optimization– Trouble shooting by everyone

The iQ5 Detection SystemThe iQ5 Detection System

Lamp • >20000 hrs• 400 – 700 nm• 更換光源 , 無需重新校正

激發光濾鏡 • 5 excitation filters

PCR 系統 • 96 well 同步收光 , no time delay• 6 peltiers, High uniformity and long life • Gradient, easy to optimized• Black block, no need optical tube , lower cost

• 15~100 ul, 10 ul demonstrated

吸收光濾鏡 • 5 emission filters• 光柵極窄 , low cross talk

CCD camera• 10 bit • 350,000 pixel

Position

Excitation Range Emission Range Dyes Detected

2 485 + 30nm 530 + 30nm FAM, SYBR Green I

3 530 + 30nm 575 + 20nm HEX, TET, VIC, JOE

4 545 + 30nm 585 + 20nm TAMRA, Cy3

5 575 + 30nm 625 + 30nm Texas Red, ROX

6 630 + 30nm 685 + 30nm Cy5, LC640

Filter Based DetectionFilter Based Detection

• Benefits of filter-based detection:– Flexibility in the selection of assays and fluorophores– Ease of evaluating and replacing optical component– Ease of exchange for alternate optical component

Light Source(488 nm)

Light Source(488 nm)

FAM HEX ROX Cy5

Fluorescent Intensity

WAVELENGTH (nm)

Light Source(400-700 nm)Light Source(400-700 nm)

Specific Excitation Filter

Specific Excitation Filter

FAM HEX ROX Cy5

490

530 575

635

Fluorescent Intensity

WAVELENGTH (nm)

Light Source(488 nm)

Light Source(488 nm)

Light Source(400-700 nm)Light Source(400-700 nm)

Specific Excitation Filter

Specific Excitation Filter

FAM HEX ROX Cy5

490

530 575

635

FAM HEX ROX Cy5

Fluorescent Intensity Fluorescent Intensity

WAVELENGTH (nm) WAVELENGTH (nm)

More specific Emission FilterMore specific

Emission FilterNormal Emission

FilterNormal Emission

Filter

光柵太寬的結果光柵太寬的結果

Accurate Mulitplexing With iCycler iQ

Fluo

roph

ore

mea

sure

d

Filter used

FAMHEX

Texas RedCy5

FAM

HEX

Texas Red

Cy5

FAM

HEX

Texas Red

Cy5

FA

M

VIC

TA

MR

A

RO

X

FA

M

VIC

TA

MR

A RO

X

02000400060008000

1000012000140001600018000

20000

FAM

VIC

TAMRA

ROX

Fluorophore measured

Filter used

Dye Bleed-Through in ABI 7000Filter 光柵太寬的結果

• Non-specific DNA binding dyes– SYBR™ Green I– SYBR™ Gold– Ethidium Bromide

• Specific Hybridization Probes– Cleavage probes (TaqMan®)

– Molecular beacons– ScorpionsTM

– AmplifluorTM

– LUXTM

– dual-oligo FRET pairs

本機適用的偵測方式本機適用的偵測方式 , , 文獻證明文獻證明Detection ChemistriesDetection Chemistries

Bio-Rad iQ5 Optical System SoftwareBio-Rad iQ5 Optical System Software

Applications vs. Analytic Functions

• 絕對定量• 相對定量• SNP 分析

可在反應過程中可在反應過程中 , , 增加或減少 增加或減少 Cycle Cycle 數數-- -- Modify PCR program when runningModify PCR program when running

可測 可測 Primer Dimmer Primer Dimmer 對 對 CtCt 值 的值 的影響影響

多重 多重 STD curve, STD curve, 獨立分析獨立分析

關於絕對定量

STD Curve: STD Curve: 鼠標所在處鼠標所在處 , , 數據立即呈現 數據立即呈現

多重多重 reference genereference gene ，符合趨勢，符合趨勢

Relative gene expression Normalized gene expression

多重多重 reference genereference gene ，符合趨勢，符合趨勢

軟體可修正軟體可修正 EEfficiencyfficiency以確保數據準確度以確保數據準確度

Multifile Gene Expression MFGE

• Over 50 data files can be imported into a Gene Study

– Over 5,000 wells of data can be analyzed in a Gene Study using the iQ5 Gene Expr module

– This is approx. 50 full plates (data files) of single color real-time PCR data

– Inter-run calibration can correct patch-to-patch variation

SNP analysis in iQ5SNP analysis in iQ5

光源及偵測系統壽命監控光源及偵測系統壽命監控

User ProfileUser Profile

Easy toEasy to Trouble Shooting Trouble Shooting

Primer Probe Design Software: Primer Probe Design Software:

Beacon DesignerBeacon Designer

唯一可設計四種 唯一可設計四種 Chemistries Chemistries 的軟體的軟體-- -- 定量 或 定量 或 SNPSNP 基因型分析基因型分析

• Molecular beacon design

• TaqMan® probe

• Primer design for SYBR Green

• FRET: Hybridization Probe

高度自動化操作軟體高度自動化操作軟體

預估可能形成的二級結構預估可能形成的二級結構

Double Check Your New Design Double Check Your New Design

多重偵測時的狀況模擬 – 多重偵測時的狀況模擬 – Cross reactionCross reaction

自動跨 自動跨 Exon-Exon Junction Exon-Exon Junction 設設計計

PCRiQ Supermix (100 / 500 / 1000)

iQ SYBR Green Supermix (100 / 500 / 1000 / 2000)

iScript cDNA Synthesis Kit

iTaq SYBR Green Supermix with ROX

iTaq Supermix with ROX

iQ MultiPlex Powermix (50 / 200)

RT PCRiScript One-Step RT-PCR Kit with SYBR Green (50 / 200)

iScript One-Step RT-PCR Kit for Probes (50 / 200)

Disposable223-9441 96-Well 0.2 ml Thin-Wall PCR Plates, 25

223-9444 Optical-Quality Sealing Tape, 100 sheets

223-9473 0.2 ml Thin-Wall Tubes, 1,000

• For all chemistry

• All reagents are 2x stock • All 3 mins Hot Start vs 10 or 20 mins Hot Start

• All reagent get 1yr Exp. Day in –20 and ½ yr in 4 degree

訓練剪影訓練剪影

2007Bio-Rad Laboratories

Certification of ParticipationCertification of Participation

Jeffrey LaiHas participated in

iCycler iQReal Time PCR Training

Anchor Chang, Product ManagerReal Time PCR

Ken Chien, Product SpecialistReal Time PCR

善用善用 Bio-Rad Web SiteBio-Rad Web Site

高規格硬體 20,000 hrs long life lamp, 96 well 同步激發 , no time delay 6 peltiers for “up to down” gradient 反應槽 無須特殊光學反應管 : Black thermal block 5 to 5 excitation to get the most specific and stronger signal Filter 光柵能盡量狹窄 , 避免 cross talk

符合使用者需求的操作軟體 可隨時增加 / 減少 cycle 數 可評估 Primer dimmer 對 Ct 值 的影響 PCR efficiency calculation

功能完備考慮週延的 Primer/Probe 設計軟體 SYBR, TaqMan, Beacon… Internet linkage, blasting Multiplex emulation

符合經濟效益的試劑耗材General thin wall tube Open reagents

完整的教育訓練及技術資源

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