GENETIC ANALYSIS FACILITY     The Genetic Analysis Facility of the Comparative Genomics Centre is housed in the Medical Genomics Laboratory and the Advanced Analytical Centre. It includes automated liquid handling systems (robots), plate based and high precision quantitative thermal cyclers and a 96 capillary automated sequencer.  AUTOMATED LIQUID HANDLING     The automated liquid handling systems available are the Corbett Robotics CAS-1200 and CAS-3800 table top units. The Corbett Robotics CAS-1200 precision liquid handling system automates quantitative PCR setup, including reagent preparation, dilution series and sample pipetting. The CAS-1200 has a single pipetting chamber (1 - 200ul) and six 96 or 384 well plate/tip rack housing positions. It is configured to automatically prepare DNA master mix and load up to 96 samples with high speed and precision. Level sensing is a standard feature that ensures reproducible volume delivery for all reagents. With the advent of real-time PCR it is now even more critical that pipetting steps are tightly controlled. As pipetting precision has a direct impact on the standard deviation of replicate samples that are used to create a standard curve, sample preparation with the CAS-1200 reduces these precision errors so fewer replicates need to be run.

PUTTING PROTOTYPES THROUGH THEIR PACES    The Comparative Genomics Centre assisted  Corbett Robotics in the development of two new Corbett Robotics DNA extractors. The Corbett Robotics CAS-3810 liquid handling system automates repetitive large scale pipetting actions, such as PCR setup for the genetic typing of linkage analysis. The CAS-3800 features an eight chamber pipetting head (1 - 200ul) and twelve 96 or 384 well plate/tip rack housing positions. It also houses a 96 well magnetic extraction station with integral heating unit, ideal for DNA clean up prior to sequencing or automated genotyping.      The second DNA extractor being tested is the Corbett Robotics CAS-1820, dubbed the "X-Tractor Gene". This automated liquid handling station features an eight chamber pipetting head (1 - 200ul) and four 96 or 384 well plate/tip rack housing positions. It also houses a vacuum extraction manifold, with integral computer driven vacuum pump system. This unit was designed to extract up to 10ug genomic DNA/well from whole blood, tail tips or ear punches.     Comparative Genomics Centre staff Alan Baxter and Margaret Jordan played a critical role in developing the chemistry used by the CAS-1820 for DNA extraction from solid tissues. The resulting kit is sold by Sigma-Aldrich as the Corbett Robotics X-tractor Pack 2 (Product number 045K6158).

GENE MICROARRAYS    The Comparative Genomics Centre has a state-of-the-art Affymetrix GeneChip system, including fluidics station and scanner. The CGC's GeneChip Scanner 3000 7G scans high density arrays, enabling new applications for genotyping with up to 500,000 SNPs, tiling arrays for transcription, and all-exon arrays for whole-genome analysis. It incorporates advanced design improvements with space-saving, speed, superior performance, and lower scanner-to-scanner variation to dramatically improve throughput and efficiency in your genetic analysis. This new scanner provides improved auto-focus, more accurate gridding, and more consistent scanner-to-scanner biological performance, improving data integrity and data sharing between researchers. Additionally, the GeneChip Scanner 3000 7G has been designed to accommodate future advancements in GeneChip probe array technology.    The GeneChip Fluidics Station 450, incorporates advanced design improvements that provide improved ease-of-use and true walk-away freedom to dramatically automate hybridisation and washing of Affymetrix GeneChips.

QUANTITATION AND QUALITY CONTROL    The NanoDrop ND-1000 Spectrophotometer is a full spectrum (220-750 nm) spectrophotometer that measured 1ul samples with high accuracy and reproducibility. It utilises surface tension to precisely localise the sample at the assay point and to provide a 1mm light path through the sample, eliminating the need for cuvettes or cassettes. The light source is a xenon flash applied to the sample via a fibre optic light path, providing a dynamic range far higher than that of conventional spectrophotometers. The NanoDrop ND-1000 Spectrophotometer is suited for measuring: Nucleic acid concentrations up to 3700 ng/ul without dilution Fluorescent dye labeling density of nucleic acid microarray samples Purified protein up to 100 mg/ml Expanded spectrum analysis of fluorescent dye labeled samples Bradford assays Lowry assays Cell density measurements (including bacteria) General UV and visual spectral densities

AUTOMATED SEQUENCING AND GENOTYPING    MegaBACE 1000 DNA Analysis System is a high-throughput, fluorescence-based DNA system utilizing capillary electrophoresis with up to 96 capillaries operating in parallel. The system performs sample injection, gel matrix replacement, DNA separation, detection, and data analysis. It is configured for both sequencing and genotyping applications. The minimal sample loading volume is 5 µl, with the samples introduced to the system in a 96-well format.The MegaBACE has dual lasers (488 and 532 nm) for genotyping (only the 488 nm laser is used for sequencing), confocal optics with multiple filter sets and 4 channels with 2 photomultiplier tubes.

CORBETT SUPPORT FILES Introduction to the CAS1200 Introduction to the RG6000 High resolution melt temperature (HRMT) analysis HRMT assay and primer design Quantitative PCR

REAL-TIME QUANTITATIVE PCR     The Comparative Genomics Centre houses both a Corbett Research Rotor-Gene 3000 for the analysis of gene expression levels and a Corbett Research Rotor-Gene 6000 for the analysis of gene expression levels and single nucleotide polymorphism detection.      The Corbett Research Rotor-Gene 3000 uses a world first centrifugal design to provide the researcher with improved sensitivity and precision with reduced run times. An improved software interface includes new functionality developed in conjunction with Rotor-Gene users.  The Rotor-Gene multi-filter system can detect all available real-time chemistries including Sybr-Green, dual-labelled and MGB probes, FRET and Molecular Beacons. Most DNA amplification enzymes/ buffers can be used on the system to generate Quantitation/Melt data and it is not necessary to use expensive kits that are specific to the instrument. SNP ANALYSIS     In addition to supporting 5-colour multiplexing, the Rotor-Gene 6000 has been engineered for high resolution melt temerature analysis (HRM). HRM characterizes nucleic acid samples based on their disassociation (melting) behavior. Samples can be discriminated according to their sequence, length, GC content or strand complementarity. Even single base changes such as SNPs (single nucleotide polymorphisms) can be readily identified. The Rotor-Gene 6000 incorporates a specially tuned high-intensity optical channel, high-speed data capture (up to 1000 data collection points per °C transition), extreme thermal resolution (0.02°C) and dedicated analysis software. Dyes such as SYTO® 9 EvaGreen™ and LC Green® provide optimal results since they can be used at higher concentrations to provide increased resolution over traditional dyes like SYBR® Green 1.     Typical uses of HRM 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 Association (case/control) studies Alleleic prevalence in a population Identification of candidate predisposition genes

ACKNOWLEDGEMENTS:     The CGC is grateful to the Australian Research Council, the Federal Department of Education, Science and Training, the Russell McKimmin Charitable Trust and the Clive and Vera Ramaciotti Foundations for their support of the Genetic Analysis Facility. MANUFACTURERS' LINKS ABI Prism 7700 Corbett Robotics OTHER LINKS: Comparative Genomics Centre home page Comparative Genomics Centre News James Cook University home page

Corbett Robotics engineer, Bruce Harrison, gives the "thumbs up" to the prototype CAS-1820p, dubbed the "X-Tractor Gene" and the custom chemistry reagent pack for tissue extraction, developed by the CGC and sold by Sigma.

Comparative Genomics Centre, James Cook University, Key words: Autoimmune diabetes, Type 1 diabetes mellitus, childhood diabetes, lupus, systemic lupus erythematosus, haemolytic anaemia, hemolytic anemia, Coombs' test, antinuclear antibodies, renal failure, glomerulonephritis, gastritis, type A gastritis, pernicious anemia.