How does Countable PCR work?
Countable PCR is a next-generation solution that brings single-molecule precision to the benchtop. By combining physical isolation of single target molecules, standard thermal cycling, and 3D light sheet imaging, it enables direct, absolute molecule counting — without the complexity of NGS or digital PCR (dPCR).
The platform is built on three core principles:
- Single-molecule isolation of DNA targets
- A transparent matrix chemistry
- High-precision detection through light sheet imaging
Single-molecule isolation is the foundation of Countable PCR
Unlike conventional PCR or dPCR, which rely on curve fitting or statistical estimation, Countable PCR performs direct counting by imaging physically isolated single molecules. There’s no need for Poisson correction, thresholding, or extrapolation — each count reflects a real molecule.
This isolation step is what enables Countable PCR’s precision. Single DNA targets are physically confined in a gel matrix in a PCR tube. After amplification, each fluorescent signal corresponds to exactly one original molecule — enabling absolute quantification without assumptions.
The result is precision that conventional PCR and digital PCR can’t match. With Countable PCR, you get tiny error bars (~1% CV), enabling confident detection of rare variants and subtle expression changes—all without competition from more abundant targets. This precision also reduces the need for technical replicates and focuses on what matters: biological variability.
A transparent matrix enables single-tube workflows
Achieving single-molecule precision doesn’t have to require complex instrumentation or microfluidics. With Countable PCR, the entire process takes place in a single PCR tube:
- Prepare and load the PCR reaction mix into a spin tube
- Centrifuge to encapsulate targets in a structured gel matrix
- Perform thermal cycling directly in the same tube. The transparent matrix maintains spatial separation, enabling downstream imaging without transfer steps.
The matrix keeps each DNA molecule spatially isolated for amplification and detection — all in the same PCR tube.
Light sheet imaging enables precise molecule detection
After PCR, the sealed tube is directly placed into the Countable System for counting:
- Thin optical “light sheet” slices of the PCR tube are sequentially illuminated and captured
- Fluorescent signals from amplified targets are imaged in 3D
- Automated software reconstructs the reaction volume and counts each signal as an individual molecule — no thresholding or manual gating required
Because amplification occurs from single molecules and spatial separation is preserved, the data are inherently clean — free of ambiguous 'rain' often seen in digital PCR. The software provides unbiased, reproducible results — no manual thresholding or gating required.
With Countable PCR, single-molecule resolution is no longer limited to complex platforms. This benchtop system unlocks high sensitivity, low variability, and true multiplexing in a single, streamlined assay.
