Bio-Rad Gradient Real-Time PCR
溫度梯度定量PCR 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?
Real time PCR = QPCR = RT PCR ≠ Room Temperature PCR
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
-- 競爭性 PCR，南(北)方墨點法 or 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 QPCR
+Taq
+dNTP
+MgCl2
+H2O
+Buffer
+Template
+Primer
On ice
• QL only
• End point
• Fuzzy data
• Labor
> 3 hrs(1.5 hrs labor)
+Master Mix
+H2O
+Template
+Primer
Room Temp
<1.5 hrs(20 min labor)
• QN/QL
• On line monitor
• Clear, Sensitive
• Automatic
Real Time PCR 理論基礎
• Log phase
切線斜率
• Plateau
螢光強度
Y= N0 2n
Baseline
Log phase
Plateau
Ct 與起始濃度的對數成反比
低濃度
高濃度
Real Time PCR 壓力指數
• Primer/Probe Design
• NA Extraction
(壓力指數: 2% )
• QPCR optimization
• Data analysis
(壓力指數: 20% )
(壓力指數: 10% )
• Trouble shooting
• Report
(壓力指數: 35% )
(壓力指數: 30% )
(壓力指數: 3% )
選擇 QPCR時, 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….
如何縮短測試時間
QPCR 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
• 善用 Gradient (溫度梯度) 的功能
10x 序列稀釋
65~55 度
Which one is better?
Gradient 抓條件, 一次搞定
Series dilution
只須一次反應, 即可讓你知道
•
•
•
94C
72C
65C
55C
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) ?
SYBR Green I
dsDNA Binding Dyes
3’
5’
5’
3’
Extension
3’
DBD
5’
Taq
DBD
DBD
Taq
DBD
l
l
3’
DBD
l
DBD
3’
l
DBD
DBD
Taq
5’
DBD
l
DBD
Taq5’
3’
Extension Continued
Apply Excitation
Wavelength
SYBR 注意事項
•
•
•
•
Primer dimmer
Non-specific binding
Melting Curve
No need to adjust using Bio-Rad
TaqMan (MGB) Probe
Q
R
5’
3’
Extension Step
R
Q
R
Taq
5’
3’
1. Strand Displacement
R
Q
Taq
5’
3’
5’
Taq
R
5’
3’
5’
l
Taq
5’
2. Cleavage
3. Polymerization
Complete
R
5’
3’
4. Detection
TaqMan 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 Beacons
Detection
l
Q
R
5’
3’
Q
R
Taq
5’
3’
Extension Step
Molecular
Beacon
Taq
R Q
5’
5’
3’
1.
2.
3.
Strand Displacement
Polymerization Complete
Probe Silent
Molecular Beacons 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 view
Price
Price
Bio-Rad Real-time
PCR Instruments
iQ5
Chromo4
My iQ
Mini-Opticon
Features
& Flexibility
Features
& Flexibility
iQ5 Real-Time PCR detection system
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
Goal of Marketing position
• Gradient:
– Primer / probe performance
• Complete Solution in qPCR
– Primer probe design
– qPCR optimization
– Trouble shooting by everyone
The iQ5 Detection System
CCD camera
• 10 bit
• 350,000 pixel
吸收光濾鏡
• 5 emission filters
• 光柵極窄, low cross talk
激發光濾鏡
• 5 excitation filters
Lamp
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
• >20000 hrs
• 400 – 700 nm
• 更換光源, 無需重新校正
Filter Based Detection
Positi
on
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
•
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
(400-700 nm)
Light Source
(488 nm)
Specific Excitation
Filter
490
530
FAM HEX ROX Cy5
FAM
HEX
575
ROX
Fluorescent Intensity
Fluorescent Intensity
635
WAVELENGTH (nm)
WAVELENGTH (nm)
Cy5
光柵太寬的結果
Light Source
(400-700 nm)
Light Source
(488 nm)
Specific Excitation
Filter
490
635
530
FAM
HEX
ROX
Fluorescent Intensity
Fluorescent Intensity
FAM HEX ROX Cy5
575
WAVELENGTH (nm)
Normal Emission
Filter
WAVELENGTH (nm)
More specific
Emission Filter
Cy5
Accurate Mulitplexing With iCycler iQ
FAM
HEX
Texas Red
Cy5
Cy5
Texas Red
HEX
FAM
FAM
HEX
Texas Red
Cy5
Filter 光柵太寬的結果
Dye Bleed-Through in ABI 7000
20000
18000
16000
14000
12000
10000
8000
6000
4000
2000
0
ROX
TAMRA
VIC
FAM
ROX
TAMRA
VIC
FAM
Fluorophore
measured
FAM
VIC
TAMRA
ROX
Filter used
本機適用的偵測方式, 文獻證明
Detection Chemistries
•
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
Bio-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, 增加或減少 Cycle 數
-- Modify PCR program when running
可測 Primer Dimmer 對 Ct值 的影響
關於絕對定量
多重 STD curve, 獨立分析
STD Curve: 鼠標所在處, 數據立即呈現
多重reference gene，符合趨勢
Relative gene expression
Normalized gene expression
多重reference gene，符合趨勢
軟體可修正Efficiency
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 iQ5
光源及偵測系統壽命監控
User Profile
Easy to
Trouble Shooting
Primer Probe Design Software:
Beacon Designer
唯一可設計四種 Chemistries 的軟體
-- 定量 或 SNP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
預估可能形成的二級結構
Double Check Your New Design
多重偵測時的狀況模擬 – Cross reaction
自動跨 Exon-Exon Junction 設計
PCR
iQ 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 PCR
iScript One-Step RT-PCR Kit with SYBR Green (50 / 200)
iScript One-Step RT-PCR Kit for Probes (50 / 200)
Disposable
223-9441 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
2007
Bio-Rad Laboratories
Certification of Participation
Jeffrey Lai
Has participated in
iCycler iQ
Real Time PCR Training
Anchor Chang, Product Manager
Real Time PCR
Ken Chien, Product Specialist
Real Time PCR
善用 Bio-Rad Web Site
選擇 Bio-Rad QPCR, 您將擁有 ..
 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