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Cyclic DNA Sequencing with Enzyme Beads in PicoTiter™ Plate: A Novel Approach

This innovative method utilizes a PicoTiter™ plate to facilitate cyclic DNA sequencing through a series of proprietary enzymatic reactions. By loading DNA and engineered enzyme beads, we enable the production of pyrophosphate (PPi) and ATP, which drive light generation in real-time. The process includes cyclic additions of nucleotides and a specialized diffusion mechanism for efficient nucleotide incorporation. Apyrase enzyme is employed to neutralize unincorporated nucleotides, thereby ensuring accurate signal processing. This technology enhances sequencing speed and accuracy.

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Cyclic DNA Sequencing with Enzyme Beads in PicoTiter™ Plate: A Novel Approach

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  1. Bead Deposition Load DNA & Enzyme beads into PicoTiter™ Plate Proprietary

  2. N’ DNA DNA DNA Polymerase (1) + Pyrophosphate (PPi) N dNTP (2) PPi ATP Sulfurylase ATP + SO42- Adenosine Phosphosulfate (APS) Firefly Luciferase Light (3) ATP CO2 + Oxyluciferin + AMP + PPi + D-luciferin + O2 Apyrase (4) dNTP (or ATP) dNMP (or AMP) + 2Pi Pyrophosphate Sequencing Chemistry • Sequencing by synthesis – Cyclic additions of 4 nucleotides: dTTP → dATP → dCTP → dGTP → dTTP … Proprietary

  3. Simultaneous Enzymatic Reactions in Hundreds of Thousands of Picoliter-size Wells Proprietary

  4. Physico-Chemical Processes on PTP A. Nuc Flow Nuc Diffusion B. Nuc incorporation on DNA → PPi production → Diffuses to Enzyme beads → Cyclic ATP & PPi generation reactions start → light generation starts Nuc Flow PPi/ATP Cyclic Reactions 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 C. Apyrase Flow Apyrase diffusion PPi, ATP diffusion Reaction decay 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 D. Wash Buffer Apyrase diffusion PPi, ATP diffusion Reaction ends Convective transport of nucleotides, APS & D-luc Into flow chamber; diffusive transport into wells Proprietary

  5. Simulation: Enzyme Kinetics & Reagent Transport 0.1*[dNTP] Concentration (mM) Reagent Flow [DNA] 107 DNA [PPI] 10 Million copies of DNA in a well Reaction time ~ 30 seconds [ATP] T (sec) Proprietary

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