1、1,Next-Generation Performance and More Capabilities,,Introducing the,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 cond
2、itions Provides an open platform for all chemistries Features a very fast data acquisition rateCapable 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 an
3、alysis + 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 Co
4、ntinuous movement means no variation well-to-well thermal or optical,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 advantag
5、es over other designs including:,5,3rd Generation Rotary Design The Rotor-Gene 6000 is based on proven technology,Rotor-Gene 2000 2000,Rotor-Gene 6000 2006,Rotor-Gene 3000 2003,6,7,Optics,8,Cross-section of rotary optics,Spindle/Motor Assembly,9,Rotary optics 3D animation,LED light source (rotates f
6、or each channel),rotor spins tubes at 400 rpm,lens,filter set (rotates for each channel),sensitive PMT (photomultiplier) detector,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)N
7、o 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 roto
8、rs),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
9、are guaranteed for the life of the instrument!,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 HRM Green/Yellow/Orange/Red/Crimson + High Resolution Melt channel 5. 6
10、-Plex Blue/Green/Yellow/Orange/Red/Crimson,NOTE: ROX normalization is not needed so all channels can be “plexed” for separate reactions,13,Thermal Control,14,Thermal Precision (uniformity) in a Block-based Cycler, 0.50 C (or more) across the 96 well block (thus 1.00 C variation is typical) Corner an
11、d Edge wells most affected NOTE: Fluorescence 1/Temp Localized “hotspots” as Peltier device junctions begin to fail The Rotor-Gene does not use Peltier devicesbecause they fail unpredictably and are expensive to repair,NOTE Rotor-Gene 6000 specifications: Uniformity: 0.01C, Resolution: 0.02C,15,Cent
12、rifugal 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,Cool air in,Centrifugal fan Drives air into chamber,Centrifugal fan drives air around chamber,Chamber vent opens expelling hot air,Coolin
13、g mechanism,Heater elements switch off,Note: holes in the rotor allow free airflow,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 C,Thermal Equilibration: Roto
14、r vs. Block,72 C,60 C,72.5 Degrees,71.5 Degrees,72 Degrees +/- 0.01,18,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 pe
15、rformance and thermal accuracyThe OTV Kit automates routine verification testing on the Rotor-GeneThe OTV system comprises an OTV Disc, optical insert accessories and a CDWith the kit, verification of instrument performance and thermal accuracy can be done at up to 30 times within the 6 month expiry
16、 date of the rotor consumableAutomated verification testing in this manner is unique to the Rotor-Gene,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 temperatu
17、re 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.
18、 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,The HTML report file
19、can be saved, printed, e-mailed or exported to MS Word as a record of the verification test,OTV Report,22,Software,More analysis optionsRaw data export For 3rd party software analysisUnlimited user software license No additional license fees All users can copy & run the software to analyze files rem
20、otely Upgrades free (by web download),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 Forma
21、ts and Sample Handling,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.,26,Gene-Disc Plates,3.,Gene-Disc 72 - 0.1 mL tubes in a rotary “plate” design - Tubes oriented vertically (not angled) - Manua
22、l or automated loading - 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,28,Amplification Performance,29,Replicates (full 72-well rotor),No ROX normalization,With optional ROX normalizat
23、ion,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-labe
24、led 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” si
25、ngle-copy gene amplified from whole human genomic template Fast cycling (40 cycles, 46 min) Standard commercial master-mix Low probe (60 nM = dilution),31,Concentration Measurement,32,DNA Concentration Measurement,The Rotor-Gene is fully equipped to do DNA concentration measurement using fluorescent
26、 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
27、in the Quant-iT PicoGreen dsDNA Kit (Invitrogen Corp., Carlsbad CA). Standard Rotor-Gene concentration analysis run protocol was used. 10 L PicoGreen (diluted1/200 in 1 TE buffer) was combined with 10 L of each standard (diluted in 1 TE buffer). Final volume 20 L.,Concentration pg/L,Fluorescence,33,
28、SYBR Melt Analysis and HRM (high resolution melt) a new application for a new type of instrument,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,SYBR melt analysis of a DNA fragment
29、,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),36,SYBR melts can reflect product size,250 bp fragment,500 bp fragment,Raw data plot: fluorescence vs.
30、temp.,Derivative data plot: dF/dT vs. temp,500 bp fragment,250 bp fragment,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
31、,Allele A,Allele B,38,SYBR Green I is toxic to PCR, so concentration used is very low,“Saturation” Hypothesis,SYBR Green I,Theoretically, unsaturated binding may allow dye relocation during melts, making it less suitable for HRM,39,HRM on the Rotor-Gene 6000,To support HRM an instrument requires: hi
32、gh-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 analysi
33、s. ACTN3 (R577X) SNP genotypes (CT). Ten replicates each genotype are shown. Fragment pre-amplified using a 40 cycle fast protocol (46 min).,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 PC
34、R and HRM,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 mutat
35、ion ratios HLA compatibility testing Association (case/control) studies Alleleic prevalence in a population Identification of candidate predisposition genes,42,Automation,43,Precision Robotic Pipetting Improves Results,CAS-1200 Precision Liquid Handling System,Rotor-Gene 6000 Six Channel Multiplexin
36、g System,44,Rotor-Gene results,10 L reaction volume, 18 replicates,Hand pipetting CT std dev 0.12,CAS-1200 robot CT std dev 0.10,45,Rotor-Gene results,5 L reaction volume, 18 replicates,Hand pipetting CT std dev 0.64,CAS-1200 robot CT std dev 0.12,46,Robotic Workflow for Real-Time Analysis,“Extracti
37、on-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
38、 achieved on the Rotor-Gene, underlining its 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,Sydney
39、AustraliaCorbett Research Pty Ltd14 Hilly StreetMortlake, NSW 2137T 1 800 803 915 (Toll free)T +61 2 9736 1320F +61 2 9736 1364,United KingdomCorbett Research UK LimitedUnit 296 Cambridge Science ParkMilton Road, Cambridge CB4 0WDT +44 (0)1223 424 288F +44 (0)1223 424 144,All slides 2006 Corbett Lif
40、e Science. All rights reserved,E-mail ,USACorbett Robotics Inc185 Berry Street, Suite 5200San Francisco, CA 94107T +1 866 380 1166 (Toll free)T +1 415 348 1166F +1 415 348 1177,Brisbane AustraliaCorbett Robotics Pty Ltd42 McKechnie DriveEight Mile Plains, QLD 4113T +61 7 3841 7077F +61 7 3841 6077,Web ,Offices,
