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Extending the Armored RNA Technology to DNA Cindy WalkerPeach, Ph.D. Director, Development and Operations Ambion Diagnos

Extending the Armored RNA Technology to DNA Cindy WalkerPeach, Ph.D. Director, Development and Operations Ambion Diagnostics. BSI EN ISO9001:2000 Certified BSI Registration Number: FM 57081 Quality System Regulations (21CFR§820) Medical Device Manufacturing Owner/Operator Number: 9049048.

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Extending the Armored RNA Technology to DNA Cindy WalkerPeach, Ph.D. Director, Development and Operations Ambion Diagnos

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  1. Extending the Armored RNA Technology to DNACindy WalkerPeach, Ph.D.Director, Development and OperationsAmbion Diagnostics BSI EN ISO9001:2000 Certified BSI Registration Number: FM 57081 Quality System Regulations (21CFR§820) Medical Device Manufacturing Owner/Operator Number: 9049048

  2. Armored RNA Quant™ Armored RNA -> Armored RNA QuantPackaging System Armored RNA®

  3. Armored RNA Quant™ Armored DNA Armored DNA Proof of Principle – Concept Armored DNA

  4. DNA Controls & StandardsCommonly Used • DNA plasmids • Infectious or attenuated / inactivated viruses • Patient samples • PCR amplicons

  5. 371bp PCR product 239bp PCR product 900bp 900bp ArmoredDNA Armroed RNA control Ladder ArmoredDNA Armroed RNA control Ladder Armored DNAProof of Principle – DNA Packaging of two Targets Packaging of two unrelated sequences

  6. Armored DNAProof of Principle - Resistant to DNase DNA DNA Marker Armored Armored DNA DNA No DNase +DNase No DNase +DNase 1 ug of PCR target or 5 ug of Armored DNA Internal Control was treated with and without Turbo DNase for 1 hour

  7. 371bp 239bp AD (Packaged,Extracted) Extracted DNA fragment DNA fragment Ladder AD (Packaged,Extracted) Extracted DNA fragment DNA fragment Ladder Armored DNAProof of Principle – DNA Protection DNA protected from nuclease degradation

  8. Armored DNAProof of Principle - QC HPLC Assay Phosphate Assay DNA Integrity Functional Testing (PCR)

  9. Phosphate Assay (copies/uL) qRT-PCR HBV Assay (copies/uL) HBV 8.0 x1010 (±0.65 x 1010) 8.6 x1010 Armored DNA Proof of Principle – Quantification 7% Difference

  10. Armored DNAHeat Stability Profile AD Internal Control Control 50 C 60 C 70C ARQ Armored DNA ARQ Armored DNA ARQ Armored DNA ARQ Armored DNA

  11. Armored DNADetection Over a Linear Range Parallel assays of AD HBV serial curve (42 to 0.042 pg) Test with two sets of primers Direct input of samples, 2 ul per reaction in duplicate

  12. Armored DNAInitial Stability in Plasma • Stable 7 days at 37°C • Stable 14 days at 25°C • Stable 1x freeze-thaw • CV < 4% 4copies/uL (AD HBV 239bp) incubated in plasma (1 x 103 copies extracted in duplicates)

  13. Armored TechnologyArmored DNA summary • Armored Technology compatible with DNA • Packaging of 239 and 371bp double stranded DNA up to an undefined limit • Stable in Plasma • 7 days at 37°C • 14 days at 25°C • 1x freeze-thaw • Stable in TSMIII • 7 days at 37°C • 7 days at 25°C • 14 days at 4°C • 1x freeze-thaw

  14. Armored DNA Ideal Controls and Standards • Stable – Nuclease resistant • Quantifiable – Armored DNA referenced to a NIST standard • Homogeneous – single, sequence defined target • Compatible – compatible with broad range of assay technologies • Flexible – allows for addition to human matrix of choice • Customizable – user defined sequences, genotyping • Non-infectious – ease in handling and shipping

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