Custom Assay Development Process

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Brooke Singleton
6 years ago
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1 Custom Assay Development Process Unravelling DNA to Enable Your Discovery Ben Jackson PhD Global Marketing Manager

2 <4 weeks 4-20 weeks <1 week Assay Development Process Project Scoping 1. Enquiry / Qualification 2. Consultation Sales Agreement 3. Proof of Concept Assay Development Production 4. Development 5. Validation 6. Custom Packaging / Kitting Project Manager Performance Report Support 7. Manufacturing 8. Technical Services Final Report Out Full SOP; Planning, Concept, Research, Design & Development, Process Validation & Commercialization Stages (68 steps)

3 Assay Optimization & Validation

4 Ct Value Ct Value [dutp] Buffer [dutp] vs Ct Value, Buffer [dutp] vs Ct Value, Amplicon 1 Amplicon ,14 34,59 35, ,24 32,43 33, ,34 28,66 29, ,83 26,64 27,02 25 Dilution ,15 23,19 23,59 Dilution ,89 20,56 20,56 Dilution 3 20 Dilution 2 15 Dilution 5 15 Dilution Control Low [dutp] Buffer High [dutp] Buffer 0 Control Low [dutp] Buffer High [dutp] Buffer dutp is an inhibitor of Taq, often influencing activity and sensitivity

5 [dutp] Amplicon F * * * Ratio 1 Ratio 2 Ratio 3 Ratio 4 * * * * Ratio 5 Ratio 6 Ratio 7 Ratio 8 Prioritized for further study The ratio & concentrations of dntp and Mg can influence baseline, end-point fluorescence and Ct values

6 [dutp] Amplicon G * * * Ratio 1 Ratio 2 Ratio 3 Ratio 4 * * * * Ratio 5 Ratio 6 Ratio 7 Ratio 8 Prioritized for further study The ratio & concentrations of dntp and Mg can influence baseline, end-point fluorescence and Ct values

7 Freeze-Thaw Stability Testing Fast Freeze-Thawing Template concentration 2 Slow Freeze-Thawing Template concentration 2 Control 0 cycles Test 20 cycles Control 0 cycles Test 20 cycles Template concentration 1 Template concentration 1 Following optimization, freeze-thawing of the master mix is shown to have little impact on assay performance

8 Ct Value Buffer ph Buffer ph vs Ct Value ,07 37,61 36,01 35,86 35,62 35,50 34, ,94 28,80 28,29 29,14 28,70 28,80 28, Amplicon 1 Amplicon Control Buffer A Buffer B Buffer C Buffer D Buffer E Buffer F Buffer ph ph can influence Ct values, S-shape in amplification plots, starting and end-point fluorescence

9 Reaction Volume Amplicon X Amplicon Y Reaction volume 1 Reaction volume 2 Reaction volume 1 Reaction volume 2 Improved performance as judged by curve profile and end-point fluorescence at very low target concentration

10 Ct Value Thermal Cycling Protocol Length ,18 30, ,06 28,94 28,6 28,53 Amplicon 1 Amplicon 2 28 Amplicon h 56m 1h 6m Total protocol length Assay performance holds steady when the cycling protocol is reduced by 50min, enabling fast quantitation

11 [Salt] Amplicon 1 Conc 1 Conc 2 Conc 3 Conc 4 Conc 5 Conc 6 Conc 7 Conc 8 Prioritized for further study * * Salt concentration can affect the Ct, end-point fluorescence and repeatability

1,00E+00 1,00E-01 1,00E-02 1,00E-03 1,00E-04 GAPDH Dilution 0,000 1,00E+00 1,00E-01 1,00E-02 1,00E-03 1,00E-04 GAPDH Dilution

12 Average Ct Value 21,54 21,14 24,97 24,49 28,40 27,93 Ct Value, Standard Deviation 0,100 0,103 0,128 0,103 0,125 0,131 0,318 0,491 1,112 31,44 31,28 35,53 35,44 2,284 Assay Format 40,00 2,500 35,00 30,00 2,000 25,00 1,500 20,00 15,00 1,000 10,00 0,500 5,00 0,00 1,00E+00 1,00E-01 1,00E-02 1,00E-03 1,00E-04 GAPDH Dilution 0,000 1,00E+00 1,00E-01 1,00E-02 1,00E-03 1,00E-04 GAPDH Dilution Standard qpcr Mix Lyophilized qpcr Mix Standard qpcr Mix Lyophilized qpcr Mix Lyophilization of the optimized master mix yields comparable results

13 End Point Fluorescence, Average Fluorescent Dye Assay Fluorophore vs. End Point Fluorescence 4,50 4,00 4,02 3,50 3,43 3,00 2,50 2,54 2,43 2,00 1,50 1,00 0,81 0,50 0,00 Control Dye 1 Dye 2 Dye 3 Dye 4 Assay Fluorophore Fluorescent dye can influence end-point fluorescence and reproducibility

14 Relative Fluoresence Relative Fluoresence Relative Fluoresence Assay Format & Fluorescent Dye 4 FAM WetMix Lyo 2,5 VIC WetMix Lyo 6 Cy5 WetMix Lyo 3, ,5 2 1,5 3 1, ,5 0, ,1 0,01 0,001 0,0001 Serial Dilution 1 0,1 0,01 0,001 0,0001 Serial Dilution 0 1 0,1 0,01 0,001 0,0001 Serial Dilution Fluorophore dye choice can influence the repeatability of fluorescent signal in different formats

15 Ct Value Assay Format & Stability Testing 35 32,058 32,33 32,679 32, ,214 28,391 27,98 28,145 Standard qpcr Mix ,494 21,815 21,349 21,445 Standard qpcr Mix + 12 Months 15 Lyophilized qpcr Mix 10 Lyophilized qpcr Mix + 12 Months ,01 0,0001 g-actin Serial Dilution Negligible variation in Ct value following lyophilization & storage at room temperature

16 Thermal Stability Testing GAPDH / FAM Arrival +1 week +1 month +3 months B2mg / VIC Arrival +1 week +1 month +3 months Storage at room temperature Storage at 37 o C Stability testing demonstrates lyophilized assays are stable to up 1 month at 37 C

17 To learn more.

Guidelines for Developing Robust and Reliable PCR Assays

Guidelines for Developing Robust and Reliable PCR Assays Leta Steffen, PhD Applications Scientist Promega Corporation Outline 1) PCR reaction components What is in the reaction? How does it affect assay