SNP technologies

1. SNP technologies GENERAL REVIEW

2. detection methods for SNP assays • Fluorescence Polarization • Fluorescence Intensity • Absorbance • Others (Sequencing, MALTI-TOF Mass Spectrometry, etc)

3. fluorescence polarization (FP) • Single Base Extension (SBE) requires three steps: • PCR reaction • Amplifies the SNP target to increase specificity and signal strength. • SAP/EXO reaction • ‘Inactivates’ the PCR reagents. • Terminal Dye Incorporation • A single base extension from the SNP primer which incorporates a fluorescent tagged nucleotide • Purely additive (homogeneous) chemistry, no precipitations, washes, tube changes, etc. • Examples are SNP-it (Orchid) & AcycloPrime (PerkinElmer) • FP requires an Analyst AD, HT or Acquest instrument.

4. fluorescence intensity • FRET based assays (PCR required) • PCR reaction • Amplifies the SNP target to increase specificity and signal strength. • Allele specific hybridization • One of the more tricky steps to perform (may require individual optimization) • Additive chemistry • Examples are TaqMan (Applied Biosystems) & Amplifluor (Intergen) • TaqMan requires specific fluorescent labeled primers for each genotype • Amplifluor uses two universal fluorescent labeled primers • Fluorescent intensity can use a Gemini, Analyst AD/HT or Acquest instrument.

5. fluorescence intensity • FRET based assays (no PCR required) • Allele specific hybridization • One of the more tricky steps to perform (may require individual optimization) • Additive chemistry • Each genotype needs optimization by Third Wave Tech which currently has approx 35k total available. • Example is Invader (Third Wave Technologies) • Fluorescent intensity can use a Gemini, Analyst AD/HT or Acquest instrument.

6. absorbance (colorimetric) assay • Horse-radish peroxidase reporter • Requires PCR reaction • Requires biotin labeled primers • Requires subsequent random hybridization step (easy to perform) • 60 min for results (after PCR step) • Read absorbance at 450nm • Example is IMBP (Gene Check Inc.) • Absorbance assays can use Emax, Vmax, VERSAmax, Spectramax 340PC, Spectramax 190, SpectramaxPLUS, and of course the Analyst and Acquest.

7. others - sequencing • Straight sequencing reactions (Sanger) • Requires running gels • Requires costly (~$250k) sequencing instruments (ABI 3700 etc) • Altered sequencing reactions (Pyrosequencing) • Must run four ‘reactions’ per nucleotide sequenced • Requires their instrument • Requires a significant licensing fee • MDC does not support this type of instrument

8. others -MALTI-TOF mass spectrophotometer • Extremely expensive to implement in house ($millions$) • Large number of technical support personnel required • Fast – very fast!! • Great for large number of samples but few SNPs (not as good for large number of SNPs, each one has to be set up and optimized, that costs $$) • Therefore, primarily a service type business model. (Sequenom) • MDC does not support this type of instrumentation

9. Survey Says….. Bio techs and Core facilities

10. gov’t commitment to lab instrumentation Source: FASEB assessment and recommendations for future funding (Dec 2000) 51% of 1000 surveys returned

11. commitment to shared facilities

12. allocation of funds available

13. Key Selling Points SNP Genotype Detection with Fluorescence Polarization

14. SNP Genotyping by Analyst HEFPKey Selling Points • Unique Confocal Optics • Proprietary Dichroic Filter • Seamless scalability from 96-well to 384-well format • Highest Sensitivity of any FP Reader • Low cost/assay

15. Seamless scalability from 96 to 384-well format The Sensed Volume remains constant regardless of assay volume or plate density

16. FP™ Genotype Detection with Double Dichroic R110 Em Tamra Em R110 Ex. Tamra Ex

17. Double Dichroics Increase the Signal:Background Ratio Dye R110 Tamra 50/50 Beamsplitter 4.3 7.6 Double Dichroic* 13.3 71.9 *Patents Pending

18. Highest sensitivity Competitive Profile • Analyst AD/HT = <10 mP STDEV @ 100 pm fluorescein • Victor2V = <10mP STDEV @ 1 nM fluorescein • Tecan Ultra = <10 mP STDEV @ 1 nM fluorescein • SmartOptics makes HEFP precise • low mP noise increases the dynamic range of FP assays • SmartOptics’ high energy light source reduces FP noise, expressed as mP Standard Deviation • Analyst has the lowest noise of any commercially available FP instrument

19. Highest sensitivity means lowest cost per assay Using Single Base Extension Chemistry e.g. Acycloprimetm Post-PCR cost per genotype • Analyst $0.25 or less 15ul assay volume 96-well plate • Analyst/competitors $0.40 or less using 50:50 beamsplitter 15ul assay volume 96-well plate • Analyst $0.10 or less 5 ul assay volume 384-well plate • Competitors $0.20 (est.) Using 50:50 beamsplitter 5 ul assay volume 384-well plate

20. Lowest cost per assay yields greatest return on investment Laboratory throughput = 2 thousand 96-well plates/yr Analyst AD Victor2V ($59,500 List) ($30,000 List) @ $0.25/assay= $48,000/yr @ $0.40/assay= $76,800/yr Savings in yearly assay costs= $28,800 Analyst List @ $59,500 – savings @ $28,800 = $30,700 @ Cost of Victor2V Thus: Savings in assay cost pays for the price difference between Analyst and Victor in just 1 year Or: The customer will be buying the equivalent of a new Victor2V year after year after year in lost savings!

21. what are we doing for you? • More App notes – covers the major SNP chemistries • Different tradeshow presence – provides a broader market • Training – online and sales updates as appropriate • Sales support – Dave and Curtis are available for sales calls (includes individualized SNP training)

22. RUN the SNP Gauntlet

23. Run the SNP question gauntlet…….. • Rules: • 1) You have 45 seconds to complete the gauntlet • 2) There are 10 questions • 3) Read question on table, place the Transgenic mouse by the correct answer (multiple guess!!) • 4) The person with the most correct answers wins the grand prize. In case of ties, the quickest to answer wins. • 5) There are prizes for 1st 2nd 3rd and a couple honorable mentions (in other words, for the SNP challenged)