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Confidential 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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Confidential Armored RNA -> Armored RNA Quant Packaging System Armored RNA ® Armored RNA Quant ™
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Confidential Armored DNA Proof of Principle – Concept Armored DNA Armored RNA Quant ™ Armored DNA
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Confidential DNA Controls & Standards Commonly Used DNA plasmids Infectious or attenuated / inactivated viruses Patient samples PCR amplicons
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Confidential Armored DNA Proof of Principle – DNA Packaging of two Targets Packaging of two unrelated sequences 900bp Armored DNA Armroed RNA control Ladder 239bp PCR product Armored DNA Armroed RNA control Ladder 371bp PCR product 900bp
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Confidential Armored DNA Proof of Principle - Resistant to DNase 1 ug of PCR target or 5 ug of Armored DNA Internal Control was treated with and without Turbo DNase for 1 hour DNA DNA Marker Armored Armored DNA DNA No DNase +DNase
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Confidential Armored DNA Proof of Principle – DNA Protection DNA protected from nuclease degradation AD (Packaged,Extracted) Extracted DNA fragment DNA fragment Ladder 239bp AD (Packaged,Extracted) Extracted DNA fragment DNA fragment Ladder 371bp
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Confidential Armored DNA Proof of Principle - QC Phosphate Assay HPLC Assay DNA Integrity Functional Testing (PCR)
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Confidential Armored DNA Proof of Principle – Quantification Phosphate Assay (copies/uL) qRT-PCR HBV Assay (copies/uL) HBV 8.0 x10 10 (±0.65 x 10 10 ) 8.6 x10 10 7% Difference
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Confidential Armored DNA Heat Stability Profile AD Internal Control Control 50 C 60 C 70C ARQ Armored DNA ARQ Armored DNA
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Confidential Armored DNA Detection 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
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Confidential Armored DNA Initial Stability in Plasma 4 copies/uL (AD HBV 239bp) incubated in plasma (1 x 10 3 copies extracted in duplicates) Stable 7 days at 37°C Stable 14 days at 25°C Stable 1x freeze-thaw CV < 4%
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Confidential Armored Technology Armored 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
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Confidential 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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