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The LightScanner ® System

The LightScanner ® System. Achieve High Throughput Mutation Discovery and Genotyping. The LightScanner System. The LightScanner System enables High Resolution Melting (Hi-Res Melting™). Hi-Res Melting is a post PCR technique for homogenous mutation discovery and scanning.

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The LightScanner ® System

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  1. The LightScanner® System Achieve High Throughput Mutation Discovery and Genotyping

  2. The LightScanner System • The LightScanner System enables High Resolution Melting (Hi-Res Melting™). • Hi-Res Melting is a post PCR technique for homogenous mutation discovery and scanning. • FAST – results in less than 5 minutes. • SIMPLE – no post PCR processing. • ECONOMICAL – costs significantly less than competing technologies.

  3. Scanning by Hi-Res Melting • Closed-tube • dsDNA dye before PCR • No processing, additions, or separations • No exposure to the environment • Rapid • 5-10 min for 96/384 samples • Non-destructive • Downstream processing if necessary • Minimal Maintenance • No expensive waste disposal

  4. Essential components of Hi-Res Melting • dsDNA binding dye • LCGreen Plus, designed specifically for detection of heteroduplexes formed during PCR, manufactured exclusively by Idaho Technology • High resolution instrumentation • LightScanner instrument, designed specifically for Hi-Res melting applications

  5. LCGreen® Plus Dye • Heteroduplex detection is a feature not shared with other dyes traditionally used in real-time PCR, such as SYBR Green I.

  6. LC Green –a “saturation” dye Non-Saturating Dye Saturating dye

  7. LightScanner Instrument • Available in 96- or 384- well format • Uniformity across plate +/- 0.15°C • 100 data points collected per °C at 0.10 °C/second

  8. Hi-Res Melting Workflow • Amplify samples with LCGreen dye • 2 hrs. including PCR setup • Melt Samples • 5-10 minutes • Data Analysis • 5-10 minutes • Recover samples for sequencing (if necessary) • 30-60 minutes

  9. Observed Combination of 4 Duplexes Heterozygote Amplification Two Homoduplexes Two Heteroduplexes

  10. Melting Analysis

  11. Melting Analysis

  12. Melting Analysis SNP 1 homozygote SNP 1 + 2 SNP 1 SNP 2

  13. Melting Analysis SNP 1 + 2 SNP 1 SNP 2 SNP 1 homozygote

  14. 100 bp Product C/C Homozygote C/G Heterozygote C/T Heterozygote C/A Heterozygote • Homozygotes are easily distinguished from heterozygotes • Different heterozygotes trace unique melting paths

  15. Detection of Homozygous Variants

  16. Blinded Tumor Samples

  17. Target Dosing

  18.  X  Use of a DNA “toolbox” as a model system for mutation scanning • Highsmith et al., Electrophoresis (1999) • Constructed plasmids of 40%, 50%, and 60% GC content with A, C, G, or T at one position • PCR primers on each side spaced 50 bp apart Reed and Wittwer Clin. Chem. 2004, 50(10): 1748-54

  19. Sensitivity and Specificity • Dependence on Product Size • N=1248, each instrument • Three different targets • All possible SNPs and WTs Clin Chem. 2004;50:1748-54.

  20. Benefits of Hi-Res Melting Platform • What about Hi-Res Melting on real-time PCR instruments? • Commercially available real-time PCR instruments lack the precise temperature control, the data density acquisition and the analysis software required for successful implementation of the method.

  21. Benefits of Hi-Res Melting Platform • The power of DNA melting analysis depends directly on the resolution of the melting instrument. • Precision of the melt curves enables identification of different sample genotypes. Normalized melting curves of 110-bp amplicon in the presence of LCGreen Plus. Figures demonstrate the advantage of Hi-Res Melting Platform. True Hi-Res Melting profiles enable discrimination of wild type (AA) in green, heterozygotes (AT) in blue and homozygous mutants (TT) in red.

  22. Sample Throughput Approximate time required to process 96 samples Approximate total elapsed time Assumptions: DHPLC at 2 temperatures Sequencing in 1 direction using 48 capillary analyser & software detection Data Taken from Dr C Taylor: Cancer Research UK Clinical Centre, St James's University Hospital, Leeds, UK

  23. Project Cost DHPLC, FSSCP and sequencing costs based on actual costs incurred for samples processed 2002-2003. Hi-Res Melting costs based on actual consumables cost and projected use. Data Taken from Dr C Taylor: Cancer Research UK Clinical Centre, St James's University Hospital, Leeds, UK

  24. ~ 90% Normal Wild type ~10% Abnormal Unlabeled probe genotyping of known variants ~ 9% Identified Variant genotyped ~1% Not identified DNA sequencing Eliminate 99% of Sequencing? Scanning of PCR fragments for variants

  25. Conclusions • The LightScanner System includes all components necessary for successful Hi-Res Melting Applications • LCGreen Plus Dye • LightScanner Master Mix • LightScanner Instrument • Software available for assay design, mutation discovery and genotyping • Assays Online Protocols • Save Time and Money • Achieve High Throughput Mutation Discovery and Gene Scanning

  26. Q & A For more information about the LightScanner System please visit www.idahotech.com

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