ddPCR stands for digital droplet polymerase chain reaction. ddPCR is a relatively new type of PCR with several applications in clinical, plant, animal, and environmental studies. ddPCR employs a water-oil emulsion system that divides the template nucleic acid in the target sample into 20000 nanoliter-sized droplets. Each of these droplets contains the target sequence.
Instead of a single sample PCR, each droplet in ddPCR analysis becomes a separate PCR sample. At the end of the reaction process, researchers assess individual droplets to identify whether they have a sequence in them or not. ddPCR analysis has numerous advantages over other variants, such as qPCR assays. With several applications, such as pharmacokinetic studies, ddPCR analysis is rapidly employed in drug development studies. The current article compares ddPCR analysis with other quantitative PCR methods.
ddPCR analysis versus qPCR services
qPCR is a relatively quantitative assessment tool. It employs a standard curve to calculate the initial concentrations of target samples. However, it lacks absolute quantification and hence is an analog measurement of the target sample. Fluorescence intensity at specific time intervals is crucial for qPCR assessments. Typically, researchers measure these levels at the end of every amplification site. This measure is called the threshold per cycle and helps calculate the initial DNA concentration.
On the other hand, ddPCR analysis does not require any reference standard curves or extrapolation. This method provides absolute quantification once the experiment is complete. Hence, ddPCR is a digital measurement as each sample fluorescence is determined at the end of the cycle. Besides, ddPCR is more precise than qPCR. It can effectively distinguish between differences in gene expression or copy number variations.
Digital PCR assays have several benefits compared to traditional PCR assays. However, they do have limitations. dPCR assays aren’t scalable and require manual serial dilutions. This manual pipetting will lead to errors. Besides, chip-based systems are complex and time-consuming. However, ddPCR assays overcome all these limitations. A robust droplet generator effectively partitions the samples into individual droplets. Besides, 20000 unique droplets generate tens of thousands of individual data points. Moreover, the entire ddPCR analysis consists of fewer steps and is easy to master. Following are some of the most significant advantages of ddPCR analysis.
- Absolute quantification: ddPCR analysis is ideal for viral load testing, microbial quantification, and target DNA measurements.
- Superior partitioning and accuracy: ddPCR can accurately produce 20000 droplets per sample and deliver more data points at the end of the amplification run.
- Unparalleled precision: ddPCR analysis can accurately measure minute differences in target copy numbers
- Eliminate bias: ddPCR analysis significantly reduces error rates during experimental protocols.
- Improve signal-to-noise ratio: ddPCR assays are highly sensitive. They can detect analytes and rare targets present in low abundance.
- Cost-effective sample volume: ddPCR analysis ranges from nano to picomolar liters. This reduction in reaction values saves valuable biological samples and reagents, further reducing the experimental costs. Besides, ddPCR analysis can be easily conducted on standard thermal cyclers without any specific microfluidic technology or chip-based systems.
ddPCR analysis has numerous advantages over other quantitative PCR methods and is increasingly being employed in drug discovery and development studies.