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

6. Non-Saturating Dye Saturating dye LC Green –a “saturation” 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 1.Amplify samples with LCGreen dye 2 hrs. including PCR setup 2.Melt Samples 5-10 minutes 3.Data Analysis 5-10 minutes 4.Recover samples for sequencing (if necessary) 30-60 minutes

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

10. Melting Analysis

12. SNP 1 homozygote SNP 2 SNP 1 SNP 1 + 2

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

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. 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 Use of a DNA “toolbox” as a model system for mutation scanning X       Reed and Wittwer Clin. Chem. 2004, 50(10): 1748-54

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

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. Instrument FeatureLightScannerReal-time platforms ResolutionHighLow Analysis software specific for Hi-Res Melting YesNo Fluorescent CompatibilityYesSome, not all compatible with LCGreen dye Time to result5 minutes>30 minutes

21. 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. 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. Benefits of Hi-Res Melting Platform

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

23. 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 Project Cost

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

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.comwww.idahotech.com