1. 1 Next-Generation Performance and More Capabilities www.corbettlifescience.com corbett LIFE SCIENCE Introducing the

2. 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. 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. 4 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:

5. 5 3 rd Generation Rotary Design The Rotor-Gene 6000 is based on proven technology Rotor-Gene 2000 2000 Rotor-Gene 6000 2006 Rotor-Gene 3000 2003

6. 6

7. 7 Optics

8. 8 Cross-section of rotary optics Reaction Chamber PMT Detector Assembly LED Light Source Assembly Tubes in Rotor Spin Past Optics Lens Detection Filters Spindle/Motor Assembly

9. 9 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

10. 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. 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. 12 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 HRMGreen/Yellow/Orange/Red/Crimson + High Resolution Melt channel 5. 6-Plex Blue/Green/Yellow/Orange/Red/Crimson ChannelExcite/Detect (nm) Example fluorophores detected Blue365/460BiosearchBlue ™, Marina Blue ®, Bothell Blue ®, Alexa Fluor ® 350 Green470/510FAM ™, SYBR ® Green 1, Fluorescein, EvaGreen ™, Alexa Fluor ® 488 Yellow530/555JOE ™, VIC ™, HEX ™, TET ™, Yakima Yellow ®, Cal Flour ® Gold 540 Orange585/610ROX ™, Cy3.5 ®, Texas Red ®, Alexa Fluor ® 568, CAL Fluor ™ Red 610 Red625/660Cy5 ®, Quasar 670 ™, LightCycler Red 640 ®, Alexa Fluor ™ 633 Crimson680/712 LPQuasar705 ™, LightCycler Red 705 ®, Alexa Fluor ® 680 HRM460/510SYTO ® 9, LC Green ®, LC Green ™Plus+, EvaGreen ™ UV IR NOTE: ROX ™ normalization is not needed so all channels can be “plexed” for separate reactions

13. 13 Thermal Control

14. 14 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 NOTE Rotor-Gene 6000 specifications: Uniformity: ±0.01°C, Resolution: ±0.02°C

15. 15 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

16. 16 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

17. 17 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

18. 18 Thermal Accuracy Instrument Calibration and Performance Verification

19. 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. 20 OTV Mechanism Rotor-Gene 3000 Scatter Plate Insert OTV disc in rotor 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. *patent pending Rotor-Gene 6000 Scatter Plate Insert

21. 21 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” is stated (as shown here) Detailed analysis data is also reported

22. 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 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 Relative Expression Software Tool (REST)

23. 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. 24 Tube Formats and Sample Handling

25. 25 Tube Formats 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 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

26. 26 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

27. 27 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

28. 28 Amplification Performance

29. 29 Replicates (full 72-well rotor) No ROX normalization With optional ROX normalization

30. 30 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)

31. 31 Concentration Measurement

32. 32 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 Fluorescence 1000 900 800 700 600 500 400 300 200 100 No DNA controls

33. 33 SYBR Melt Analysis and HRM ™ (high resolution melt) a new application for a new type of instrument

34. 34 Generic dsDNA intercalation dye Inexpensive & simple Used for real-time PCR product detection Used for DNA dissociation (melt) analysis Widely used SYBR ™ Green I

35. 35 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 T m (temperature of melting)

36. 36 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

37. 37 SYBR ™ Green I melts can reflect sequence detection 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

38. 38 SYBR ™ Green I is toxic to PCR, so concentration used is very low “Saturation” Hypothesis SYBR ® Green I Some dye can relocate as melting begins New dye technology for HRM Examples: EvaGreen, SYTO9 LC Green ™ I Dye saturation leaves no room for relocation events during melting “Saturation” dyes are much less toxic, so concentration used can 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

39. 39 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 Temperature (°C) Fluorescence 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).

40. 40 HRM workflow on the Rotor-Gene 6000 Autocall genotypes Up 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

41. 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. 42 Automation

43. 43 Precision Robotic Pipetting Improves Results CAS-1200 ™ Precision Liquid Handling System Rotor-Gene 6000 ™ Six Channel Multiplexing System

44. 44 Rotor-Gene ™ results 10 µL reaction volume, 18 replicates Hand pipetting C T std dev 0.12 CAS-1200 robot C T std dev 0.10

45. 45 Rotor-Gene ™ results 5 µL reaction volume, 18 replicates Hand pipetting C T std dev 0.64 CAS-1200 robot C T std dev 0.12

46. 46 Robotic Workflow for Real-Time Analysis “Extraction-to-Reaction”

47. 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. 48 colors

49. 49 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 All slides  2006 Corbett Life Science. All rights reserved E-mail info@corbettlifescience.com 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 Brisbane Australia Corbett Robotics Pty Ltd 42 McKechnie Drive Eight Mile Plains, QLD 4113 T +61 7 3841 7077 F +61 7 3841 6077 Web www.corbettlifescience.com Offices