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SNP technologies. GENERAL REVIEW. detection methods for SNP assays. Fluorescence Polarization Fluorescence Intensity Absorbance Others (Sequencing, MALTI-TOF Mass Spectrometry, etc). fluorescence polarization (FP). Single Base Extension (SBE) requires three steps: PCR reaction
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SNP technologies GENERAL REVIEW
detection methods for SNP assays • Fluorescence Polarization • Fluorescence Intensity • Absorbance • Others (Sequencing, MALTI-TOF Mass Spectrometry, etc)
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.
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.
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.
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.
others - sequencing • Straight sequencing reactions (Sanger) • Requires running gels • Requires costly (~$250k) sequencing instruments (ABI 3700 etc) • Altered sequencing reactions (Pyrosequencing) • Must run four ‘reactions’ per nucleotide sequenced • Requires their instrument • Requires a significant licensing fee • MDC does not support this type of instrument
others -MALTI-TOF mass spectrophotometer • Extremely expensive to implement in house ($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
Survey Says….. Bio techs and Core facilities
gov’t commitment to lab instrumentation Source: FASEB assessment and recommendations for future funding (Dec 2000) 51% of 1000 surveys returned
Key Selling Points SNP Genotype Detection with Fluorescence Polarization
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
Seamless scalability from 96 to 384-well format The Sensed Volume remains constant regardless of assay volume or plate density
FP™ Genotype Detection with Double Dichroic R110 Em Tamra Em R110 Ex. Tamra Ex
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
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
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
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!
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)
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)