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Bead Deposition. Load DNA & Enzyme beads into PicoTiter™ Plate. N’. DNA. DNA. DNA Polymerase. (1). +. Pyrophosphate (PPi). N. dNTP. (2). PPi. ATP Sulfurylase. ATP. + SO 4 2-. Adenosine Phosphosulfate (APS). Firefly Luciferase. Light. (3). ATP. - PowerPoint PPT Presentation
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Proprietary
Bead DepositionBead Deposition
Load DNA & Enzyme beads into PicoTiter™
Plate
Proprietary
Pyrophosphate Sequencing ChemistryPyrophosphate Sequencing Chemistry
Sequencing by synthesis – Cyclic additions of 4 nucleotides: dTTP → dATP → dCTP → dGTP → dTTP …
DNA Polymerase
dNTP
DNA
N Pyrophosphate(PPi)
DNA+(1)
Adenosine Phosphosulfate (APS)
ATP SulfurylasePPi + SO4
2-ATP(2)
dNTP (or ATP)Apyrase
dNMP (or AMP) + 2Pi(4)
N’
ATP CO2 + Oxyluciferin + AMP + PPi +Firefly LuciferaseD-luciferin + O2
(3) Light
Proprietary
Simultaneous Enzymatic Reactions in Simultaneous Enzymatic Reactions in Hundreds of Thousands of Picoliter-size WellsHundreds of Thousands of Picoliter-size Wells
Proprietary
Physico-Chemical Processes on PTP Physico-Chemical Processes on PTP A. Nuc Flow
Nuc Diffusion
Convective transport of nucleotides, APS & D-lucInto flow chamber; diffusive transport into wells
B. Nuc Flow
PPi/ATPCyclic
Reactions
Nuc incorporation on DNA → PPi production → Diffuses to Enzyme beads → Cyclic ATP & PPi generation reactions start → light generation starts
C. Apyrase Flow
Reaction decay
Apyrasediffusion
PPi, ATPdiffusion
PPi, ATP start diffusing out of well; Apyrase flows into chamber & diffuses into well → Neutralizes unincorporated nucs & ATP → Cyclic reaction starts to decay → signal starts to decay
D. Wash Buffer
Reaction ends
Apyrasediffusion
PPi, ATPdiffusion
Wash Buffer flows into chamber; Unused apyrase diffuses out of well → PPi, ATP continue to diffuse out of well → Reaction comes to an end → signal decays to baseline
Proprietary
T (sec)
Co
nce
ntr
atio
n (M
)
0.1*[dNTP]
[PPI]
[ATP]
[DNA]
107 DNA
Reagent Flow
10 Million copies of DNA in a wellReaction time ~ 30 seconds
Simulation: Enzyme Kinetics & Reagent Transport Simulation: Enzyme Kinetics & Reagent Transport