Choosing a droplet pcr machine: how digital pcr counts molecules instead of comparing to a standard curve, what a digital pcr instrument buys on rare targets and in inhibited samples, and the partitioning and threshold questions that decide the number

What the method actually does

1. Partitioning turns concentration into a count. The reaction is split into many thousands of droplets or wells, each amplifying independently. Counting positives and applying Poisson statistics gives an absolute concentration with no standard curve and no reference material, which is the whole point.
2. Rare targets are where it wins clearly. A variant present at a small fraction of a background is hard to distinguish by cycle threshold and straightforward to count when it occupies its own partitions. Rare mutation detection and copy number differences are the established applications.
3. It tolerates inhibition better. Because the readout is endpoint presence or absence rather than amplification efficiency, partial inhibition that would shift a cycle threshold has less effect on a count. That matters for difficult matrices where quantitative PCR is unreliable.
4. The dynamic range is set by the partition count. Too few target molecules and precision is poor; too many and every partition is positive and the count saturates. The usable range follows from how many partitions the instrument makes, so samples must be diluted into it rather than run as they arrive.
5. Thresholding is a decision, and it should be a rule. Separating positive from negative partitions on a fluorescence amplitude plot is where the result is actually made. Set the rule in advance, include a no-template control on every plate, and record the threshold with the data.

Assay design, which is not the same as for qPCR

Amplicons should be short, and a restriction digest of genomic DNA before partitioning separates tandem copies that would otherwise share a partition and be counted once. Both are routine steps that people carry over from quantitative PCR and omit.

Probe-based chemistry is the norm and multiplexing is possible by amplitude as well as by colour, which lets a single well carry more targets than the detector count suggests.

Throughput, cost per sample and whether an automated pcr machine helps

Droplet generation, amplification and reading are separate steps and the whole cycle is slower than a quantitative PCR plate. Consumables are proprietary and considerably more expensive per sample.

For routine quantification of an abundant target, quantitative PCR remains faster and cheaper. Digital methods earn their cost where the counting is what makes the answer possible.

Contamination control, which matters more here

An endpoint count of rare events is exquisitely sensitive to a trace of amplicon in the laboratory. Separate pre and post amplification areas, dedicated pipettes and filter tips, and a no-template control on every plate.

Investigate a positive no-template control rather than repeating around it. On a rare variant assay, a low level contamination produces a plausible result rather than an obviously wrong one.

Reading a digital droplet pcr cost per sample

The consumable is the cost and it is per WELL rather than per sample, so the arithmetic follows how many targets and replicates each sample needs. Cartridges or plates, oil, and the tips that generate the droplets are the recurring items, and a run that is not full still spends the consumable it loaded. Batch the samples, and cost a realistic batch rather than a full plate.

The other half is what the technique replaces. Where absolute quantitation without a standard curve is the point, or where a rare variant has to be separated from a large background, the per sample price buys something a plate of quantitative PCR cannot. Where a relative expression answer would do, it is an expensive way to get one.

A pcr master mix 2x, and what the concentrate contains

A two times mix carries buffer, magnesium, nucleotides, the polymerase and usually a dye or a loading agent at twice the working concentration, so it is diluted one to one with template and primers. The convenience is fewer pipetting steps and less variation; the constraint is that the magnesium and buffer are fixed, so an assay needing more magnesium or a different additive has to be built from components. Check whether the mix is hot start, whether it carries a passive reference dye, and what it does with uracil carryover.

Common questions

What does a droplet pcr machine do differently from real time pcr machines?

It partitions the reaction into thousands of independent amplifications and counts the positives, giving an absolute concentration by Poisson statistics rather than a value read off a standard curve.

When is a digital pcr instrument worth it over a qrt pcr machine?

For rare variant detection against a background, for small copy number differences, for absolute quantification without a reference standard, and for samples where inhibition makes quantitative PCR unreliable.

What limits the dynamic range?

The number of partitions. Below a few target molecules the counting statistics are poor, and above the point where nearly every partition is positive the count saturates. Samples have to be diluted into the usable window.

Is it more accurate than an rt pcr machine?

For absolute quantification and small differences, generally yes, because it removes the standard curve and the amplification efficiency assumption. It is also slower, more expensive per sample and has a narrower dynamic range.

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