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The science of microfluidic liquid handling for droplet digital PCR (ddPCR)
During the last decade digital-PCR (dPCR) has become one of the most prominent assays for this class of analytical methods dPCR carries out a single reaction within a sample as standard PCR, however, the sample is separated into a large number of partitions, and the reaction is carried out in each partition individually. This separation allows for more reliable collection and sensitive measurement of nucleic acid amounts. It has already been demonstrated that microfluidic dPCR offers a new level of precision compared to quantitative PCR (qPCR)76. Droplet-based microfluidic is an excellent solution to partition a sample. dPCR technology that makes use of droplet microfluidics is usually called droplet digital PCR (ddPCR). For performing the assay, the sample volume is split into multiple droplets in such a way that each droplet contains either one or none of the target DNA molecules. Due to the small droplet volume, the PCR reaction runs very efficiently even from a single molecule. During amplification, a fluorescent dye is formed or activated. The positive droplets become fluorescent. Absolute quantitation of the number of target molecules is simplified to the count of fluorescence active droplets in the generated droplet collection. ddPCR is an excellent example of a transition of a microfluidic system from the academic field to industry.
- Higher accuracy
- Higher sensitivity
- Absolute quantitation