Shear methodologies in use today result in a variety of droplet sizes that follow a normal distribution curve – resulting in only 1% of droplets that are the desired size. cyphoPrep’s technology results in consistently, correctly sized droplets that can achieve the theoretical maximum of usable material per assay.


WHY SIZE MATTERS

Emulsion PCR (ePCR) is a commercially significant variation of standard PCR in which the reagents have been reformatted into very small droplets suspended in oil. By dividing and isolating the individual components of a PCR reaction into separate microscale droplets as they undergo PCR, the reaction is effectively being performed thousand- or million-fold in parallel. This simple reformatting of a standard PCR reaction opens the door to a multitude of new array types.

However, the full potential of ePCR is limited by the inability of commercial devices to create uniform emulsions. Most systems employ shaking or stirring steps that result in a wide distribution of droplet sizes.

Droplets that are too small will not contain adequate amplicons—if any template is present at all. Droplets that are too large are statistically likely to contain more than one bead or template. Either instance constitutes a failure mode that inhibits accurate sequencing.

The greater the variation in droplet size, the smaller the volume of usable material in any given assay. In ePCR, uniform droplets are key to cutting processing times, reducing reagent consumption, and improving assay accuracy.

Droplets that are too large are statistically likely to contain more than one bead or template. Either instance constitutes a failure mode that inhibits accurate sequencing. The greater the variation in droplet size, the smaller the volume of usable material in any given assay. In ePCR, uniform droplets are key to cutting processing times, reducing reagent consumption, and improving assay accuracy.

Every ePCR assay design anticipates an optimal droplet number and volume that reflects the total volume of the reaction, the number of templates or beads, and the portion of droplets that are likely to contain a single DNA template or bead. The better the emulsion can conform to the ideal droplet dimensions, the more likely they are to contain a single bead or template. Only uniform droplets offer sample yields that approach the theoretical maximum.

Where reagent concentration is matched perfectly to droplet volume, there is a 36% chance of a single template present in a given droplet. In other words, even in ideal circumstances, about 64% of ePCR reagents do not contribute to assay results. Additional variation, such as droplet diameter, can substantially reduce reagent efficiency.

Commercial emulsion preparations typically produce emulsions with a coefficient of variation (CV) of about 50%. Only about 1% of the total reaction volume—sample sequences and PCR reagents—contributes to usable data. In fact, standard commercial ePCR workflows remove 99% of PCR reagents downstream of the droplet generation step. Creating a small-scale emulsion is a difficult engineering challenge that cannot be resolved through minor improving to existing methods.

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