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Bead DepositionBead Deposition

Load DNA & Enzyme beads into PicoTiter™

Plate

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Pyrophosphate Sequencing ChemistryPyrophosphate Sequencing Chemistry

Sequencing by synthesis – Cyclic additions of 4 nucleotides: dTTP → dATP → dCTP → dGTP → dTTP …

DNA PolymerasedNTP

DNAN 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

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Simultaneous Enzymatic Reactions in Simultaneous Enzymatic Reactions in Hundreds of Thousands of Picoliter-size WellsHundreds of Thousands of Picoliter-size Wells

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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

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T (sec)

Con

cent

ratio

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