Real-time PCR System ---Gentier
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Real-time PCR System ---Gentier PT. Interskala Medika Indonesia Green Sedayu Bizpark Jl. Daan Mogot KM. 18 Blok DM 9 No. 62 Kalideres, Jakarta Barat, DKI Jakarta, Indonesia 11840 Telp: 021-22527790 Email : [email protected] www.imimedika.com
Transcript of Real-time PCR System ---Gentier
Real-time PCR System ---Gentier
PT. Interskala Medika Indonesia Green Sedayu Bizpark
Jl. Daan Mogot KM. 18 Blok DM 9 No. 62Kalideres, Jakarta Barat, DKI Jakarta, Indonesia 11840
Telp: 021-22527790
Email : [email protected] www.imimedika.com
nurdian
Stamp
Contents
l Basic Information
l Products Introduction
l Competitors
Basic Information
PCR Basics
Polymerase chain reaction (PCR) is a method to amplify a target sequence from background nucleic acid . A way to make lots of copies of DNA
• Target DNA (example: environmental DNA) • 2 primers (20-30 nts long)• dNTP’s• Thermostable DNA polymerase• Mg2+ (cofactor for DNA polymerase)
Basic Components of PCR
dNTPsTaq polymerase
Primers
DNA template
Buffer
+ +
A C T G
Mg2+
What is PCR?
PCR Basic Steps
Quantitative Real Time PCR
Definition: Assay that monitors the accumulation of a DNA product from a PCR reaction in real time>> Fluorescence-based detection of amplification products through the use of a DNA-binding dye or hybridization probe>> PCR Real-time qPCR is used to quantify input nucleic acid by measuring the number of cycles required to reach a set level of product.>> Benefits over traditional PCR assaysl Sensitivity (as little a single copy)l Enhanced dynamic range of detection (7-8 magnitudes)l Increased reproducibilityl Increased throughputl Cost efficiency
l A linear increase follows exponential phase
l Eventually plateaus
Cycle #
Theoretical
Real Life
Log
Targ
et D
NA
Limitations of standard PCR
Amplification is exponential, but the exponential increase is limited:
In theory, the amount of DNA produced at every cycle should double,
Product(T) = (Template0) x 2n (n = # of cycles)
Standard PCR is as endpoint
96 identical reactions will have very different final amounts of fluorescence at endpoint
Real-Time PCR
Through the use of fluorescent molecules, real-time PCR has the ability to directly measure the reaction while amplification is taking place.
How is quantitative data collected?
Cycle #
Theoretical Real LifeLo
g Ta
rget
DN
A Detector
96 identical reactions will have almost identical CT values
Threshold Cycle, CT
l The point at which the fluorescence rises appreciably above backgroundl Threshold can be placed anywhere in the exponential (log-linear) phase
Threshold Cycle, CT
Threshold Setting
After baseline subtraction, a threshold line is set empirically or by a statistical calculation at a fluorescence value above background.
Threshold
Log View
• 1 CT Difference = 2 fold difference in starting template amount
• 3.3 CT Difference = 10 fold difference in starting template amount
ProductT=(Template0)2n
Where n=Number of Cycles
Mathematical Implications
Ideal PCR
Correlates strongly with the starting copy number
Threshold Cycle, CT
2n = 10 fold
n ln 2 = ln 10
n = ln10ln 2
n = 3.32
From CT values, we can determine the initial copy number
l Intercalation Dyes
l Hybridization Probes
Chemistries used in real time PCR
Intercalation (DNA binding) dyes
l DNA binding dyes are inexpensive compared to hybridization probes.
l EtBr is 25 times more fluorescent when bound to dsDNA
l SYBR Green I is 125 times more fluorescent brightly bound to dsDNA
Intercalation Dyes: SYBR Green I
3’
5’3’
Taq
Taq5’
5’
ID ID ID
ID ID
l l l
ll
l Advantages >> Experiment only requires
primers
l Disadvantages >> Potential contribution to fluorescence
from non-specific products (primer-dimers) >> No multiplexing
Hybridization Probes
l Cleavage-based assay >> TaqMan Assays >> Locked nucleic acids (LNA)
l Displaceable probe assays >> Molecular beacons >> Dual-oligo FRET probes
l Probes incorporated directly into the primers >> Amplifluor & Scorpions
Currently, hybridization probe strategies fall into three main categories
5’5’3’
3’
d.NTPs
Thermal Stable DNA Polymerase
Primers5’
3’5’
3’
5’
3’
5’
3’
5’
3’
5’
3’
5’
3’5’
3’
Add iQ Supermix,Hybridization Probe
and sample
Denaturation
5’
3’
5’
3’
5’
3’
5’
3’
5’
3’5’
3’
Annealing
Taq
l
5’ 3’
R Q
Probe
5’ 3’
R Q
Cleavage-based assay: TaqManTM
5’ 3’5’3’5’3’
RQ
5’ 3’
Taq3’
Q
R
5’
5’ 3’3’
Q Taq
R
5’
5’ 3’
QTaqR
3’ 5’
Extension Step
5’ 3’3’
QTaqR
5’
l
Cleavage-based assay: TaqManTM
TaqMan
l Advantages >> Target specific fluorescence >> Multiplexing
l Disadvantages >> High initial cost >> Assay design not trivial
Real-Time PCR Applications
Real-Time reaction monitoring provides information for relative or absolute
measurements of starting material.
l Gene Expression Studiesl Chromatin Immunoprecipitation (ChIP)l Methylation Specific PCR (HRM)l Microarray Validationl Transgenic Analysisl GMO Testingl Viral/Bacterial Load Studiesl Allelic Discrimination/SNP (HRM)
Products Introduction
Tianlong Real-time PCR System milestone
TL988-Ⅰ TL988-ⅡTL988-Ⅱ
（Revision）
TL988-Ⅳ（v1.02） TL988-Ⅳ（v3.0）
（2005） （2017）（2014）（2007）
（2017）（2014）（2013）
Gentier 48E
（2007）
CE Marked
Gentier 96E
TUV CE Marked
Real-time PCR System--Gentier Series
Your ideal choice for qPCR
Gentier96 Gentier48
Gentier Real-time PCR System Configurations
Gentier48
Model Sample Channel Gradient
Gentier48 E 48 4 Yes
Gentier48 S 48 4 No
Gentier48 R 48 2 Yes
Model Sample Channel Gradient
Gentier96 E 96 6 Yes
Gentier96 S 96 6 No
Gentier96 R 96 4 Yes
Gentier96 C 96 4 No
Gentier96
Covered low, middle and high end requirements
Seemless Upgrades
Gentier Series offer a seamless upgrades through a simply software Key without change the machine for a same serie.
Gentier48 R(2-Ch with Gradent)
Gentier48 S(4-Ch without Gradent)
Gentier48 E(4-Ch with Gradent)
Gentier96 C(4-Ch without Gradent)
Gentier96 R(4-Ch with Gradent)
Gentier96 S(6-Ch without Gradent)
Gentier96 E(6-Ch with Gradent)
Parameters--Gentier48
Gentier48E Gentier48R Gentier48S
Sample capacity 48
Consumables 0.2ml 8-strip tubes, 0.2ml PCR single tube (Optical flat cap, clear tube)
Gradient Range 1°C-40°C N/A 1°C-40°C
Gradient block 8 row 8 row
Special temperature
protocol
Gradient PCR, Long
PCR, Touch Down
PCR
Long PCR, Touch
Down PCR
Gradient PCR, Long
PCR, Touch Down PCR
Reaction Volume 5-100ul
Power usage AC 100 to 125V / 200 to 240V (50/60Hz)
Communication Port 1 ethernet and 2 USB
Footprint(W*D*H) 260mmX400mmX260mm
Gentier96E Gentier96R Gentier96S Gentier96C
Sample capacity 96
Consumables 0.2ml 96-well Plates (unskit); 0.2ml 8-strip tubes, 0.2ml PCR single tube
(Optical flat cap, forosted, white tube)
Gradient Range 1°C-40°C N/A 1°C-40°C N/A
Gradient block 12 row 12 row
Special temperature
protocol
Gradient PCR,
Long PCR, Touch
Down PCR
Long PCR, Touch
Down PCR
Gradient PCR, Long
PCR, Touch Down
PCR
Long PCR,
Touch Down
PCR
Reaction Volume 0-100ul
Power usage AC 100 to 125V / 200 to 240V (50/60Hz)
Communication Port 1 ethernet and 2 USB
Footprint(W*D*H) 355 mmX480mmX485mm
Parameters--Gentier96
Appearance Design
10.4 inch Touch screen
96 well heating block
Removable air vent cover
USB port7 inch Touch screen
USB port
Top lid handle
High sensitive optical system
Easy-to-use Software
Design for flexible use
Fast and uniform heating System
Quality data
Product Features
Design for flexible usel Standard with 10.4' or 7’’ LCD touch screen, functions quickly accessible through
simple, one-touch commandsl Wizard operation to lock and unlock the transport locker.l Message alarm indicate the status of the machine l USB port update software and firmwarel Unique power-off protection function can save all the setted configurations for
sudden power outages, and allow the experiment continues when power supply is restored.
Design for flexible use
System can be installed in three distinct configurations, providing unmatched flexibility and convenience.
1. Co-located configuration (PC control)2. Standalone (PC-free) configuration (Data can be export by USB and analysis on PC)3. Networked configuration (PC software automatically detect all systems in LAN and allow to remote monitoring one of experimental progress and downloading of the completed run file to the PC at your desk,one PC can max control 10 instuments)
Design for flexible use
Unified PC andf ARM software platform
PC software Interface Device software Interface
High sensitive optical system
Easy-to-use Software
Design for flexible use
Fast and uniform heating System
Quality data
Product Features
l Concealed lid with constant temperature and isopiestic pressurel Electrical controlled elevating & automatic locked platform l Thermo block is based on improved heat pipe technology, leading to extraordinary Iintra-well temperature homogeneity and maximized inter-well, inter-cycle reproducibility. l Highly efficient PID&DSP control algorithm ensures faster respond and stronger interference resistance, fast and uniform heating and heat sinking
Fast and uniform heating System
l Max heating rate: 8.0℃/sl Avg heating rate: 6.0℃/sl Max coolting rate: 5.5℃/sl Avg cooling rate: 4.0℃/sl Temperature accuracy: ±0.1℃l Temperature uniformity: ±0.1℃
l Max heating rate: 6.1℃/sl Avg heating rate: 4.5℃/sl Max coolting rate: 5.0℃/sl Avg cooling rate: 2.8℃/sl Temperature accuracy: ±0.1℃l Temperature uniformity: ±0.1℃l Support High Resolution Melting (HRM)
function, Min temperature resolution 0.06℃
High sensitive optical system
Easy-to-use Software
Design for flexible use
Fast and uniform heating System
Quality data
Product Features
High sensitive and precise optical system
lHigh-sensitivity and wide dynamic range of fluorescence detection system, sample dynamic range could be from 101 to 1010 copies lFast and precise mechanical scanning structure ensures the illumination uniformity of 96 wells, Eliminate edge effects, no need for ROX calibrationlFluorescence excitation by a high-intensity and long-life LED, Bulb Luminance will not attenuation over time, free maintenance optical system through the entire lifelProfessional designed fluorescence filters realize the highly fluorescence transmittance and meanwhile minimize the fluorescence channel crosstalk.
l LED-excitation and PD-detection,
l 2s lateral optical scanning
l LED-excitation and PD-detection
l 7s top optical scanning for 96 wells and 6 channels
CH1 CH2 CH3 CH4FAM
SYBR Green ISYTO 9
EvaGreenLC Green
HEXVICTETJOE
Texas RedROX Cy5
Gentier 48E/48S
Gentier 48R
1 465 /510 (FAM,SYBR Green I，SYTO9，EvaGreen，LC Green)2 527/ 563 (HEX,VIC,TET,JOE)3 580 /616 (ROX,Texas Red)4 632/ 664( Cy5)Channls are same with Roche LightCycler 96, Agilent Mx3000P
Gentier 48 channels
1 465 /510 (FAM,SYBR Green I，SYTO9，EvaGreen，LC Green)2 527/ 563 (HEX,VIC,TET,JOE)
CH1 CH2 CH3 CH4 CH5 CH6FAM
SYBR Green ISYTO 9
EvaGreenLC Green
HEXVICTETJOE
Texas RedROX Cy5 Alexa Fluor
680 FRET
Gentier 96E/96S
Gentier 96R/96C
Currently channls are same with Bio-rad CFX96,Qiagen RotorGene QCan change FRET to TAMRA/CY3 in future, it can be the seme with all ABI qPCR, Agilent Mx3005P
Channls are same with Roche LightCycler 96, Agilent Mx3000P,SLAN 96S/P,TL988 of Tianlong
TAMRACy3
Gentier 96 channels
High sensitive optical system
Easy-to-use Software
Design for flexible use
Fast and uniform heating System
Quality data
Product Features
Easy-to-use Software
l Friendly interface and clearly functional modules design, first-time users can easily start a new experiment.l All experiment data and setting options are shown conveniently on a single interface.
l Instrument Management allow you managing all the Gentiers in the WLANl More than thousands results can be stored in devicel USB or WLAN easily transfer experimments data anywhere you needed
Instrument Management
Instrument Management & Data Transmission
File Transmission
l Multi-options to start an experiment: New, New for Existed, Open data file, Double click data file
l User can define the experiments name for documentation l User can define the data storage directory themselfl Pre-installed templates can help user more easier to edit the protocals.
Experiments Management
Stage Types
l All running infoirmation in one screen, intuitivly and simplyl User can define each model to see what they like on the screen:Real-time Flurescence
,Heat map, Run info,Sample Setting, Sample info...l Run the experiment first by devie, later edit the sample setting in PC, when PC conencted
to machine, setting upload to machine automaticly.l After experiment, data download to PC automaticly.
Run monitoring
Data Analysisl Optional baseline setting: Automatic or Manuall Multi-option analytical method for different requirements: Automatic or Manual
threshold method, normalization methodl Reference Dye analysis
Data Analysis-most comprehensive data analysis
Absolute quantification Relative quantification Genotyping
End-point fluorescence Melting Curve Analysis High Resolution Melting
l One data can use different analysis method
Data Analysis-Others
l Statistics analysis: Replicate,Gene typesl Heating map options: Ct, Concentration, Flurescencel LIS connectivity, can export csv,Excel and Txt formatl Row data and results can export in Excel format
Statistics analysis Heating map
Report
Unique designed universal report(Research and Clinical) can be customized according to the requirements, report review and print function help clinical documentation as needed.
High sensitive optical system
Easy-to-use Software
Design for flexible use
Fast and uniform heating System
Quality data
Product Features
HBV DNA absolute quantitative uniformity
l 300IU HBV DNA absolute quantitative full plate test(with standard curves).l FAM for HBV DNAl HEX for internal control
Gradient diluted samples uniformity
l Gradient diluted sample amplification curves.l Can set different colors for each sample.
l 4 colors multiplex PCR gradient sample test l HEX channel is used for internal control, isn’t shown on this page.l A good performance even with multiplex PCR.
Multiplex PCR gradient samples uniformity
CA16/EV71/EU uniformity
l Multiplex PCR uniformity with CA16/EV71/EU assay.l Left and right half are low/high concentration duplicate samples.l FAM for CA16，Cy5 for EU.
HPV 16+2 uniformity
l HPV high risk genotyping assayl Multiplex PCR uniformity performance with HPV 16+2 high risk types assay.l Left and right half are low/high concentration duplicate samples.l HEX for HPV 16, ROX for HPV 18 , FAM for the rest of 16 high risk types.l Cy5 channel is for internal control, not shown on this page
Gentier 96E VS 7500
l The multiplex PCR amplification curves compared with ABI 7500, Same assay and samples.l Gentier 96E curves are more ideal than the competitor.
MTHFR C677T SNP genotyping
l MTHFR C677T SNP genotype test with genotyping analysis function.
l Temperature uniformity of melting curve with SYBR Green I dye.
l Multiplex PCR melting curves for SNP analysis. l FAM channel is for MTRR A66G，ROX channel is for MTHFR A1298C. The left melting peak is the wild type, the right melting peak is the mutation. And twin-peak means the sample is heterozygote.
l High Resolution Melting (HRM) analysis for MTHFR C667T SNP with saturation dye. l Group 1 is the wild type,Group 2 is the mutation, Group 3 is the heterozygote.
l A South Korea partner Seegene. Their special HPV high risk genotyping assasy requires 3 melting stages in one experiment. Each melting stage requires 5 colors scanning. This test could only be available on Bio-rad CFX96. One tube for 14 HPV high risk types rough quantitive detection.
l 5 colors multiplex melting curves.l Same positive control and clinical samples parallel test on Gentier 96E and CFX96.
l Melting 1
l Red peak（cy5 channel） is internal control, the blue peak（FAM） is HPV type 58, and the green peak (HEX) is HPV type 59.
l After 3 times amplification and melting, the type 59 can be detected. So roughly, we can know type 59 concentration is lower than type 58.
l Melting 2
l Melting 3
l Seegene’s STD assay. It requires 2 steps fluorescence reading in 3-step amplification cycle.l It can detect 7 different STI pathogens in one tube with 4 colors.l step 2 and step 3 fluorescence curves need to be plotted on 2 graphs.
l Step 2 amplification curves.
l Step 3 amplification curves.
Competitors
Specifications 7500 StepOne Plus
LightCycler480 II
CFX 96Touch
LightCycler96
Quant Studio 5
Gentier 96E
Manufacturer ABI ABI Roche Bio-rad Roche ABI TIANLONG
Sample Capacity 96 96 96/384 96 96 96/384 96
Consumable（mL） 0.2 0.1 0.1 0.2 0.1 0.2/0.1 0.2
Max Heating Rate（℃/s） 2.5 4.6 4.4 5.0 4.4 6.5 6.1
Temperature Accuracy（℃）
0.5 0.25 / 0.2 0.2 0.25 0.1
Temperature Uniformity（℃）
±1.0 ±0.5 ±0.1 ±0.4 ±0.2 ±0.4 ±0.1
Thermal Gradient × × × √ √ √ √
Flourescence Channels 5 4 5 6 4 6/5 6
Touch Screen NO 6 NO 7 10.4 7 10.4
Excitation Source Halogen LED LED LED LED LED LED
Launched Time 2004 2007 2008 2009 2012 2015 2017
Compare Chart
Gentier 96E vs 7500
l LED light source, free maintenancel No edge effects, no need for ROX
calibrationl Stand alone operationl LAN connectivetyl Better thermal block featuresl With Gradientl Alexa Flour 680 orFRET channel
morel HRM analysis function for free
Gentier 96E vs StepOne Plus
l Better thermal block featuresl With Gradientl Cy5 channell Alexa Flour 680 or FRET channel l HRM analysis function for free
Gentier 96E vs CFX 96 Touch
l Bigger touch screenl LAN connectivetyl Better thermal block featuresl HRM analysis function for free
Gentier 96E vs LightCycler 480 II
l Touch screenl Stand alone operationl LAN connectivetyl Better thermal block featuresl With Gradientl Texas Red/ROX channell Alexa Flour 680 channell Better consmable compatibility
Gentier 96E vs LightCycler 96
l Can save more than 1000 resultsl Better thermal block featuresl Alexa Flour 680 channell FRET orTAMRA/Cy3 channell Better consmable compatibility
Treatise(Compare with Roche)
Conclusion:The TL system has good analytical performance, and exhibits good agreement with the CAP/CTM system inclinical performance.
