Transcript Rotor-Gene 6000 System Introduction
1 Introducing the
Next-Generation Performance and More Capabilities corbett
LIFE SCIENCE www.corbettlifescience.com
2
What is the Rotor-Gene 6000?
An integrated device that: Maintains identical well-to-well conditions to monitor micro reaction tubes Illuminates and collects a wide range of optical signals Enables exquisite control of thermal conditions Provides an open platform for all chemistries Features a very fast data acquisition rate Capable of the broadest application set: Real-time analysis (e.g. quantitative amplification) End-point analysis (e.g. SNP genotyping) SYBR melt analysis HRM (high resolution melt) analysis Concentration analysis + future applications…
3
How does the rotary format work?
Samples spin continually during a run 400 RPM (heating or cooling) High-speed data collection all samples read in one revolution (0.15 sec) G-force keeps reagent at base of tube removes bubbles & condensation will not pellet components Continuous movement means no variation well-to-well thermal or optical
400 RPM >10 G
4
Why rotary?
Many advantages over other designs including: All wells are iso-thermal All wells are iso-illuminated Super fast data acquisition rate Minimal maintenance and calibration Simple and automated verification testing Enables the broadest application set
3
rd
Generation Rotary Design
The Rotor-Gene 6000 is based on proven technology 5 Rotor Gene 2000
2000
Rotor Gene 3000
2003
Rotor Gene 6000
2006
6
7
Optics
8
Cross-section of rotary optics
Reaction Chamber Tubes in Rotor Spin Past Optics Spindle/Motor Assembly Detection Filters Lens PMT Detector Assembly LED Light Source Assembly
9
Rotary optics 3D animation
rotor spins tubes at 400 rpm lens filter set (rotates for each channel) sensitive PMT (photomultiplier) detector LED light source (rotates for each channel)
10
Rotary Design means: low maintenance & maximum convenience
No optical normalization needed
(thus no special reagents either)
No passive reference dye needed
(e.g. ROX ™ )
No lamp replacement or maintenance
(lifetime guarantee on lightsource)
No block to clean
(because tubes hang in mid air)
No bubbles in reaction
(automatically removed by centrifugal rotor)
Tube caps can be labeled
(not possible with 96-well systems)
Supports several tube formats
(by simply swapping rotors)
11 Light-Emitting Diode (LED) Light Source LEDs last >100,000 hrs Low-power, low-temperature device LEDs have a focussed light-emitting area Separate LED used for each channel from infra-red (IR) to ultra-violet (UV) Estimated lifespan ~40 years (assuming 4 runs every working day)
Rotor-Gene LEDs are guaranteed for the life of the instrument!
12
UV IR Channel Excite/Detect (nm) Blue
365/460
Green Yellow Orange Red Crimson HRM
470/510 530/555 585/610 625/660 680/712 LP 460/510
Example fluorophores detected
BiosearchBlue ™ , Marina Blue ® , Bothell Blue ® , Alexa Fluor ® 350 FAM ™ , SYBR ® Green 1, Fluorescein, EvaGreen ™ , Alexa Fluor ® 488 JOE ™ , VIC ™ , HEX ™ , TET ™ , Yakima Yellow ® , Cal Flour ® Gold 540 ROX ™ , Cy3.5
® , Texas Red ® , Alexa Fluor ® 568, CAL Fluor ™ Red 610 Cy5 ® , Quasar 670 ™ , LightCycler Red 640 ® , Alexa Fluor ™ 633 Quasar705 ™ , LightCycler Red 705 ® , Alexa Fluor ® 680 SYTO ® 9, LC Green ® , LC Green ™Plus+ , EvaGreen ™
5 Models to suit different needs and budgets: 1.
2.
2-Plex 2-Plex HRM
Green/Yellow Green/Yellow + High Resolution Melt channel 3.
4.
5-Plex 5-Plex HRM
5.
6-Plex
Green/Yellow/Orange/Red/Crimson Green/Yellow/Orange/Red/Crimson + High Resolution Melt channel Blue/Green/Yellow/Orange/Red/Crimson NOTE:
ROX ™ normalization is not needed so all channels can be “plexed” for separate reactions
13
Thermal Control
Thermal Precision (uniformity) in a Block-based Cycler
± 0.50 º C (or more) across the 96 well block (thus > 1.00
º C variation is typical) Corner and Edge wells most affected NOTE: Fluorescence
1
/
Temp Localized “hotspots” as Peltier device junctions begin to fail The Rotor-Gene does not use Peltier devices —because they fail unpredictably and are expensive to repair
14
NOTE Rotor-Gene 6000 specifications: Uniformity: ±0.01°C, Resolution: ±0.02°C
15
Heating mechanism
Chamber vent seals to contain air Centrifugal fan drives air around chamber Heater elements switch on
Note:
holes in the rotor allow free airflow
16
Cooling mechanism
Chamber vent opens expelling hot air Centrifugal fan Drives air into chamber Centrifugal fan drives air around chamber Heater elements switch off
Note:
holes in the rotor allow free airflow Cool air in
17 Thermal Equilibration: Rotor vs. Block
72.5 Degrees 72 Degrees +/- 0.01
71.5 Degrees 96 ºC 72 ºC 60 ºC Equilibration time in Rotor-Gene is 0 sec (for ± 0.01 °C) Equilibration time in 96 well block is 15 sec (for ± 0.5 °C) Up to 50% faster run times with better uniformity between samples
18
Thermal Accuracy
Instrument Calibration and Performance Verification
19 For Checking and Calibrating Thermal Accuracy and Optical Performance Increasingly, laboratories require routine verification and validation of instrument performance and thermal accuracy The OTV Kit automates routine verification testing on the Rotor-Gene The OTV system comprises an OTV Disc, optical insert accessories and a CD With the kit, verification of instrument performance and thermal accuracy can be done at up to 30 times within the 6 month expiry date of the rotor consumable Automated verification testing in this manner is unique to the Rotor-Gene
20 OTV Mechanism The OTV system* uses the chemical properties of three different thermochromic liquid crystals (TLCs) as an absolute temperature reference.
When heated, a TLC changes from opaque to transparent at a very precise temperature.
But because TLCs do not fluoresce, a fluorescence scatter plate is inserted over the optics to enable detection by the Rotor-Gene. The Rotor-Gene measures the precise temperature transition of each TLC. This reported value is compared to the known calibrated value to verify the instrument is within specification. If not within specification, automatic re-calibration of the Rotor-Gene can be done at the press of a button.
OTV disc in rotor Rotor-Gene 6000 Scatter Plate Insert Rotor-Gene 3000 Scatter Plate Insert *patent pending
21 OTV Report The HTML report file can be saved, printed, e-mailed or exported to MS Word as a record of the verification test The report indicates the instrument was within specification when “No Adjustment Required” is stated (as shown here) Detailed analysis data is also reported
22
Software
More analysis options Raw data export For 3 rd party software analysis Unlimited user software license No additional license fees All users can copy & run the software to analyze files remotely Upgrades free (by web download)
Standard Curve Quantitation 2 Standard Curves Relative Quantitation Delta Delta Ct Relative Quantitation Comparative Quantitation Relative Expression Software Tool (REST) LinReg (Assumption-Free Analysis) Melt Analysis High Resolution Melt Analysis End-Point Analysis Allelic Discrimination Scatter Graph Analysis Concentration Analysis
23
Plug-and-Play Portability
Robust design suits transportability No optical alignment or calibration needed Self-configuring USB connection to computer Easy to carry Small: 370 mm (14.6") W, 420 mm (16.5") D, 275 mm (10.8") H Light: 14 kg (31 lbs)
24
Tube Formats and Sample Handling
Tube Formats
1.
36
0.2 mL PCR tubes Attached flat or domed caps NOTE: optical caps are not required since detection is through the base of the tube
25
2.
72
0.1 mL tubes - allow small reaction volumes (5 –10 µL) - in strips of 4 for ease of use - Frosted cap extensions allow write-on labelling + easy handling
Gene-Disc
™
Plates
3.
Gene-Disc ™ 72 0.1 mL tubes in a rotary “plate” design - Tubes oriented vertically (not angled) - Manual or automated loading - Heat-sealed in seconds
26
4.
Gene-Disc ™ 100 - For 96 sample workflow + 4 extra controls
-
30 µL wells in a rotary “plate” design - Manual or automated loading - Wells oriented vertically (not angled) - Heat-sealed in seconds
27
Direct Robotic Setup in Gene-Disc
™
Plates
Vertical tube orientation means a robot can set up all reactions directly into Gene-Disc tubes The new heat sealer provides a permanent or removable film seal (user selectable switch) Gene-Disc rotor ready for cycling
28
Amplification Performance
29
Replicates (full 72-well rotor)
No ROX normalization With optional ROX normalization
30
2-fold quantitative discrimination
2-fold discrimination
(=1 PCR cycle)
10 separate dilutions in triplicate No ROX normalization No data “smoothing” single-copy gene amplified from whole human genomic template Fast cycling
(40 cycles, 46 min)
Standard commercial master-mix Low probe
(60 nM = ¼ dilution)
256,000 copies 500 copies BCL-2 human gene target (68 bp amplicon) amplified from total genomic DNA template. Semi-log amplification plots shown of normalized fluorescence vs. cycle number with no smoothing applied and without ROX ™ normalization. Primer concentration 300 nM, dual labeled probe 60 nM, 40 cycle amplification completed in 46 min using standard Platimum ® qPCR SuperMix-UDG commercial master mix (Invitrogen Corp., Carlsbad, CA).
31
Concentration Measurement
32
DNA Concentration Measurement
1000 900
The Rotor-Gene is fully equipped to do DNA concentration measurement using fluorescent dyes
700 800
Using a standard run protocol and integrated analysis software, the concentration of unknown samples is determined from a standard curve.
No DNA controls 400 500 600 100 200 300
Concentration pg/µL
A DNA standard curve with replicates is shown. Curve interpolated using a spline curve fit (Rotor-Gene analysis software). Data was obtained using reagents in the Quant-iT ™ PicoGreen ® dsDNA Kit (Invitrogen Corp., Carlsbad CA). Standard Rotor-Gene concentration analysis run protocol was used. 10 µL PicoGreen ® (diluted1/200 in 1
TE buffer) was combined with 10 µL of each standard (diluted in 1
TE buffer). Final volume 20 µL.
33
SYBR Melt Analysis and HRM
™
(high resolution melt)
a new application for a new type of instrument
34
SYBR
™
Green I
Generic dsDNA intercalation dye Inexpensive & simple Used for real-time PCR product detection Used for DNA dissociation (melt) analysis Widely used
SYBR melt analysis of a DNA fragment
Raw data plot Fluorescence drops as DNA melts and SYBR is released 35 Derivative data plot This “rate” curve peaks at maximum dissociation rate which is indicative of the T m (temperature of melting)
36
SYBR
™
melts can reflect product size
500 bp fragment 250 bp fragment Raw data plot: fluorescence vs. temp.
250 bp fragment 500 bp fragment Derivative data plot: dF/dT vs. temp
SYBR
™
Green I melts can reflect sequence
detection of alleles Allele A Allele B Allele A Allele B Primer Dimer 37 Low primer conc (50 nM) Single band contains two species (alleles) High primer conc (900 nM) Primer-dimer appears as a third species
“Saturation” Hypothesis
SYBR ™ Green I is toxic to PCR, so concentration used is very low Theoretically, unsaturated binding may allow dye relocation during melts, making it less suitable for HRM 38
New dye technology for HRM Examples: EvaGreen, SYTO9
“Saturation” dyes are much less toxic, so concentration used can be higher This may reduce dye relocation events and improve HRM results Some dye can relocate as melting begins
SYBR ® Green I
Dye saturation leaves no room for relocation events during melting
LC Green ™ I
HRM on the Rotor-Gene 6000
39 To support HRM an instrument requires: high-intensity + high sensitivity optics high-speed data capture very precise temperature control extreme temperature resolution To support multiple wells: Superlative thermal and optical well-to well uniformity
82 83 84 85 86
Temperature ( °C)
87 88
•
Example SNP genotyping using HRM analysis. ACTN3 (R577X) SNP genotypes (C —T).
•
Ten replicates each genotype are shown.
•
Fragment pre-amplified using a 40 cycle fast protocol (46 min).
HRM workflow on the Rotor-Gene 6000
0.2 mL tubes 0.1 mL tubes Gene -Disc ™ 72 40 Gene -Disc ™ 100
Choose preferred tube Run PCR and HRM Autocall genotypes Up to 100 at a time
41
HRM Applications
Some HRM applications currently under investigation include: Mutation discovery Screening for loss of heterozygosity DNA fingerprinting SNP genotyping Characterization of haplotype blocks DNA methylation analysis DNA mapping Species identification Somatic acquired mutation ratios HLA compatibility testing Association (case/control) studies Alleleic prevalence in a population Identification of candidate predisposition genes
42
Automation
Precision Robotic Pipetting Improves Results
43
CAS-1200 ™
Precision Liquid Handling System
Rotor-Gene 6000 ™
Six Channel Multiplexing System
Rotor-Gene
™
results 10 µL reaction volume, 18 replicates
44
Hand pipetting
C T std dev 0.12
CAS-1200 robot
C T std dev 0.10
Rotor-Gene
™
results 5 µL reaction volume, 18 replicates
45
Hand pipetting
C T std dev 0.64
CAS-1200 robot
C T std dev 0.12
Robotic Workflow for Real-Time Analysis
46
“Extraction-to-Reaction”
47
Summary
Real-Time analysis is an exact science with many variables Through good design, the rotary format best minimizes these variables Precise and reproducible data is easier to achieve with the Rotor-Gene Future applications such as HRM and concentration measurement can only be achieved on the Rotor Gene, underlining it’s superior design Choose from more tube formats and data analysis options Verification testing and calibration is automated by the OTV system The robust design delivers minimum maintenance, lowest operating costs and maximum convenience
48
colors
49
Offices
Brisbane Australia
Corbett Robotics Pty Ltd 42 McKechnie Drive Eight Mile Plains, QLD 4113 T +61 7 3841 7077 F +61 7 3841 6077
Sydney Australia
Corbett Research Pty Ltd 14 Hilly Street Mortlake, NSW 2137 T 1 800 803 915 (Toll free) T +61 2 9736 1320 F +61 2 9736 1364
United Kingdom
Corbett Research UK Limited Unit 296 Cambridge Science Park Milton Road, Cambridge CB4 0WD T +44 (0)1223 424 288 F +44 (0)1223 424 144
USA
Corbett Robotics Inc 185 Berry Street, Suite 5200 San Francisco, CA 94107 T +1 866 380 1166 (Toll free) T +1 415 348 1166 F +1 415 348 1177
Web
www.corbettlifescience.com
All slides 2006 Corbett Life Science. All rights reserved