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Next-Generation Performance and More Capabilities

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LIFE SCIENCE

Introducing the

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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…

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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

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Why rotary?

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

Many advantages over other designs including:

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3rd Generation Rotary DesignThe Rotor-Gene 6000 is based on proven technology

Rotor-Gene 20002000

Rotor-Gene 60002006Rotor-Gene 3000

2003

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Optics

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Cross-section of rotary optics

Reaction Chamber

PMT DetectorAssembly

LED Light Source

Assembly

Tubes in Rotor Spin Past Optics

Lens

Detection Filters

Spindle/Motor Assembly

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Rotary optics 3D animation

LED light source(rotates for each channel)

rotor spins tubes at 400 rpm

lens

filter set(rotates for

each channel)

sensitive PMT (photomultiplier)

detector

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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)

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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!

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5 Models to suit different needs and budgets:

1. 2-Plex Green/Yellow

2. 2-Plex HRM Green/Yellow + High Resolution Melt channel

3. 5-Plex Green/Yellow/Orange/Red/Crimson

4. 5-Plex HRM Green/Yellow/Orange/Red/Crimson + High Resolution Melt channel

5. 6-Plex Blue/Green/Yellow/Orange/Red/Crimson

Channel Excite/Detect (nm) Example fluorophores detected

Blue 365/460 BiosearchBlue™, Marina Blue®, Bothell Blue®, Alexa Fluor® 350

Green 470/510 FAM™, SYBR® Green 1, Fluorescein, EvaGreen™, Alexa Fluor® 488

Yellow 530/555 JOE™, VIC™, HEX™, TET™, Yakima Yellow®, Cal Flour® Gold 540

Orange 585/610 ROX™, Cy3.5®, Texas Red®, Alexa Fluor® 568, CAL Fluor™ Red 610

Red 625/660 Cy5®, Quasar 670™, LightCycler Red 640®, Alexa Fluor™ 633

Crimson 680/712 LP Quasar705™, LightCycler Red 705®, Alexa Fluor® 680

HRM 460/510 SYTO®9, LC Green®, LC Green™Plus+, EvaGreen™

UV

IR

NOTE: ROX™ normalization is not needed so all channels can be “plexed” for separate reactions

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Thermal Control

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Thermal Precision (uniformity) in a Block-based Cycler

± 0.50 ºC (or more) across the 96 well block

(thus > 1.00 ºC 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

NOTERotor-Gene 6000 specifications: Uniformity: ±0.01°C, Resolution: ±0.02°C

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Centrifugal fan drives air around chamber

Chamber vent seals to contain air

Heating mechanism

Note: holes in the rotor allow

free airflow

Heater elements switch on

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Cool air in

Centrifugal fan Drives air into chamber

Centrifugal fan drives air around chamber

Chamber vent opens expelling hot air

Cooling mechanism

Heater elements switch off

Note: holes in the rotor allow

free airflow

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TIME

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

96 ºC

Thermal Equilibration: Rotor vs. Block

72 ºC

60 ºC

72.5 Degrees

71.5 Degrees

72 Degrees +/- 0.01

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Thermal Accuracy

Instrument Calibration andPerformance Verification

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For Checking and CalibratingThermal 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 instrumentperformance and thermal accuracy can bedone at up to 30 times within the 6 monthexpiry date of the rotor consumable

Automated verification testing in thismanner is unique to the Rotor-Gene

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OTV Mechanism

Rotor-Gene 3000 Scatter Plate Insert

OTV disc in rotor

The OTV system* uses the chemicalproperties of three different thermochromicliquid crystals (TLCs) as an absolutetemperature reference.

When heated, a TLC changes from opaqueto transparent at a very precise temperature.But because TLCs do not fluoresce, a fluorescencescatter 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.

*patent pending

Rotor-Gene 6000 Scatter Plate Insert

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The HTML report file can be saved, printed, e-mailed or exported to MS Word as a record of the verification test

OTV Report

The report indicates the instrument was within specification when “No Adjustment Required” isstated (as shown here)

Detailed analysis data is also reported

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Software

More analysis options

Raw data exportFor 3rd party software analysis

Unlimited user software licenseNo additional license fees

All users can copy & run the softwareto analyze files remotely

Upgrades free (by web download)

Standard Curve Quantitation2 Standard Curves Relative QuantitationDelta Delta Ct Relative QuantitationComparative QuantitationRelative Expression Software Tool (REST)LinReg (Assumption-Free Analysis)Melt AnalysisHigh Resolution Melt AnalysisEnd-Point AnalysisAllelic DiscriminationScatter Graph AnalysisConcentration Analysis

Standard Curve Quantitation2 Standard Curves Relative QuantitationDelta Delta Ct Relative QuantitationComparative QuantitationRelative Expression Software Tool (REST)LinReg (Assumption-Free Analysis)Melt AnalysisHigh Resolution Melt AnalysisEnd-Point AnalysisAllelic DiscriminationScatter Graph AnalysisConcentration Analysis

Relative Expression Software Tool (REST)

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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)

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Tube Formatsand

Sample Handling

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Tube Formats

36 0.2 mL PCR tubes Attached flat or domed capsNOTE: optical caps are not required since detection is through the base of the tube

1.

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

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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

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

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Gene-Disc rotor ready for cycling

The new heat sealer provides a permanent or removable film seal(user selectable switch)

Direct Robotic Setup in Gene-Disc™ Plates

Vertical tube orientation means a robot can set up all reactions directly into Gene-Disc tubes

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Amplification Performance

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Replicates (full 72-well rotor)

No ROX normalization

With optional ROX normalization

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2-fold quantitative discrimination

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).

256,000 copies

500 copies

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)

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Concentration Measurement

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DNA Concentration Measurement

The Rotor-Gene is fully equipped to do DNA concentration measurement using fluorescent dyes

Using a standard run protocol and integrated analysis software, the concentration of unknown samples is determined from a standard curve.

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.

Concentration pg/µL

Flu

ore

sce

nce

1000

900

800

700

600

500

400

300

200

100

No DNA controls

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SYBR Melt Analysis and

HRM™ (high resolution melt)

a new application fora new type of instrument

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Generic dsDNA intercalation dye

Inexpensive & simple

Used for real-time PCR product detection

Used for DNA dissociation (melt) analysis

Widely used

SYBR™ Green I

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SYBR melt analysis of a DNA fragment

Raw data plot

Fluorescence drops as DNA melts and SYBR is released

Derivative data plot

This “rate” curve peaks at maximum dissociation rate

which is indicative of the Tm (temperature of melting)

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SYBR™ melts can reflect product size

250 bp fragment

500 bp fragment

Raw data plot:fluorescence vs. temp.

Derivative data plot:dF/dT vs. temp

500 bp fragment

250 bp fragment

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SYBR™ Green I melts can reflect sequencedetection of alleles

Low primer conc (50 nM)

Single band contains two species (alleles)

High primer conc (900 nM)

Primer-dimer appears as a third species

Allele A

Allele B

Primer Dimer

Allele A

Allele B

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SYBR™ Green I is toxic to PCR,so concentration used is very low

“Saturation” Hypothesis

SYBR® Green I

Some dye can relocateas melting begins

New dye technology for HRMExamples: EvaGreen, SYTO9

LC Green™ IDye saturation leaves no room for

relocation events during melting

“Saturation” dyes are much less toxic, so concentration usedcan be higher

This may reduce dye relocation events and improve HRM results

Theoretically, unsaturated binding may allow dye relocation during melts, making it less suitable for HRM

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HRM on the Rotor-Gene 6000

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

Mutants(T allele)

Wild types(C allele)

Heterozygotes

Mutants(T allele)

Wild types(C allele)

Heterozygotes

Temperature (°C)

Flu

ore

sce

nce

82 83 84 85 86 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).

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HRM workflow on the Rotor-Gene 6000

Autocall genotypesUp to 100 at a

time

Choose preferred tube

0.1 mL tubes

Gene-Disc™ 72

0.2 mL tubes

Gene-Disc™ 100

Run PCR and HRM

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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

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Automation

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Precision Robotic Pipetting Improves Results

CAS-1200™

Precision Liquid Handling System

Rotor-Gene 6000™

Six Channel Multiplexing System

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Rotor-Gene™ results

10 µL reaction volume, 18 replicates

Hand pipettingCT std dev 0.12

CAS-1200 robotCT std dev 0.10

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Rotor-Gene™ results

5 µL reaction volume, 18 replicates

Hand pipettingCT std dev 0.64

CAS-1200 robotCT std dev 0.12

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Robotic Workflow for Real-Time Analysis

“Extraction-to-Reaction”

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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

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colors

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All slides 2006 Corbett Life Science. All rights reserved

E-mail info@corbettlifescience.com

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