Bulk amplification narrows your assay scope and introduces bias
In most genomics workflows that use qPCR, dPCR, and even NGS, multiple target sequences are amplified simultaneously in the same reaction. All the amplification reaction components are mixed — primers, polymerase, and templates — and share the same environment.
This simultaneous amplification approach forces tradeoffs and imposes limitations:
- Short amplicons tend to amplify more efficiently, long or GC-rich targets struggle, and rare variants get lost in the amplification
- Multiplexing takes a lot of work to get right
- Quantification relies on estimation — applying thresholding, standard curves, or correction algorithms rather than direct counting
- PCR bias affects even NGS, since many library preparation workflows use bulk amplification before sequencing begins
Bulk amplification also introduces variability and limits sensitivity. When quantification is the backbone of your assay, compromises have a cascading effect: you’re less confident in your results, assay optimization is more challenging, and you miss signals from low-abundance targets that often are the ones that matter most to you.
Free your assays from bulk amplification constraints with Countable PCR
As a result of these limitations, you have no choice but to design your assays around the chemistry — what the PCR reaction tolerates — instead of focusing on the biology — the questions you need your assay to answer.
The Countable PCR approach to amplification is category-defining and transformative.
It starts with isolating single molecules before amplification. This enables each molecule to be amplified independently, which results in practical advantages:
- The competition or the bias that comes from working in an environment of shared reaction components disappears
- Long and short targets amplify equally well
- Rare molecules and other difficult-to-amplify sequences no longer get drowned out.
Another rather transformative advantage that comes from physically separating single molecules is the ability to count them directly — there’s no longer a need for curve fitting, thresholding, or molecular barcodes required.
Expand your assay design capabilities while keeping assays simple
With Countable PCR, you’re not limited by what the PCR reaction tolerates. Instead, you’re free to focus on what your assay needs, and you’re finally more successful at:
- Amplifying 2 kb or longer amplicons without extra assay optimization
- Designing and optimizing reliable higher-plex panels without having to figure out a technical workaround because the system itself removes the underlying constraints
Count on precise quantification from single-molecule isolation
At the core of the many challenges experienced with quantification and assay development in PCR is the fact that amplification happens in bulk. Once you separate the molecules before amplification, many of those issues disappear.
Countable PCR enables true single-molecule resolution and direct counting with the speed and familiarity of legacy PCR.
The result is fundamentally different data outputs, like:
- Smaller error bars, even for rare molecule detection
- Ability to detect subtle changes in copy number or expression levels with confidence
- Sensitivity to reliably detect rare molecules missed by bulk amplification
- Co-amplification and detection of rare and housekeeping genes in the same reaction
Countable PCR represents a practical shift toward more accurate quantification and a more flexible approach to building assays that reflect the complexity of biology without the complexity of traditional methods.
