Post on 25-Feb-2016
description
Confidential
Extending the Armored RNA Technology to DNA
Cindy WalkerPeach, Ph.D.Director, Development and Operations
Ambion Diagnostics
BSI EN ISO9001:2000 CertifiedBSI Registration Number: FM 57081
Quality System Regulations (21CFR§820)Medical Device Manufacturing
Owner/Operator Number: 9049048
Confidential
Armored RNA -> Armored RNA QuantPackaging System
Armored RNA®
Armored RNA Quant™
Confidential
Armored DNA Proof of Principle – Concept
Armored DNA
Armored RNA Quant™Armored DNA
Confidential
DNA Controls & StandardsCommonly Used
• DNA plasmids
• Infectious or attenuated / inactivated viruses
• Patient samples
• PCR amplicons
Confidential
Armored DNA Proof of Principle – DNA Packaging of two Targets
Packaging of two unrelated sequences900bp
Armored D
NA
Armroed RNA control
Ladder
239bp PCR product
Armored D
NA
Armroed RNA control
Ladder
371bp PCR product
900bp
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 DNANo DNase +DNase No DNase +DNase
Confidential
Armored DNA Proof of Principle – DNA Protection
DNA protected from nuclease degradation
AD (Packaged,Extracted)
Extracted DNA fragm
ent
DNA fragment
Ladder
239bpAD (Packaged,Extracted) Extracted DNA fragm
entDNA fragm
entLadder
371bp
Confidential
Armored DNAProof of Principle - QC
ARQ Phosphate Standard Curve
y = 0.0171x + 0.2215R2 = 0.9984
0
0.1
0.2
0.3
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1
0 5 10 15 20 25 30 35 40 45
ng Phosphate
OD2
60
Phosphate Assay HPLC Assay
DNA Integrity Functional Testing (PCR)
Confidential
Armored DNA Proof of Principle – Quantification
Phosphate Assay
(copies/uL)
qRT-PCR HBV Assay
(copies/uL)
HBV 8.0 x1010 (±0.65 x 1010)
8.6 x1010
7% Difference
Confidential
Armored DNA Heat Stability Profile AD Internal Control
Control 50 C 60 C 70C ARQ Armored DNA ARQ Armored DNA ARQ Armored DNA ARQ Armored DNA
Confidential
Armored DNA Detection Over a Linear Range
Syber Green Assay of Armored DNA HBV
y = -1.6067Ln(x) + 18.27R2 = 0.9993
y = -1.5763Ln(x) + 18.055R2 = 0.9998
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35.00
0.001 0.01 0.1 1 10 100
Armored DNA Input (picograms)
Ave
Ct
Log. (Primer pair B)
Log. (Primer pair A)
Parallel assays of AD HBV serial curve (42 to 0.042 pg)Test with two sets of primersDirect input of samples, 2 ul per reaction in duplicate
Confidential
Armored DNA Initial Stability in Plasma
4 copies/uL (AD HBV 239bp) incubated in plasma(1 x 103 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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0.50
1.00
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2.50
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3.50
4.00
Baseline 2 days, 37C 7 days, 37C 7 days, 25C 14 days, 25C 1X Freeze-Thaw
Log
copi
es/rx
n
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
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