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

commitment to shared facilities

allocation of funds available

Key Selling Points SNP Genotype Detection with Fluorescence Polarization

SNP Genotyping by Analyst HEFP Key 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 Ex.

R110 Em

Tamra Ex

Tamra Em

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

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

Using Single Base Extension Chemistry e.g. Acycloprimetm

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 Gauntlet

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)