Introduction
The polymerase chain reaction (PCR) has undergone modifications through three generations. The first generation of PCR relies on gel electrophoresis for the analysis of PCR products, yet it faces limitations due to its inadequate detection threshold, labor-intensive procedures, and its singular application scope (qualitative). The subsequent generation of PCR, commonly referred to as real-time quantitative PCR (RT-qPCR), is capable of quantifying products using a standard curve; however, it also exhibits low tolerance to the presence of interfering substances. Digital PCR (dPCR), representing the third generation of PCR, facilitates absolute quantification by partitioning the reaction into discrete compartments.
Characterized by its higher sensitivity and precision in molecular identification, this technology has exhibited applicability in domains such as trace DNA identification, the detection of infrequent mutations, and the assessment of copy number variations.
How the technology Works
Digital Droplet PCR is a refined method of absolute nucleic acid quantification. Unlike qPCR, which gives relative quantification based on fluorescence curves, ddPCR works by partitioning a sample into thousands of nanoliter-sized droplets—each acting as its own PCR micro-reaction.
At the end, you count how many droplets were positive or negative, and from this, you directly calculate the number of target molecules, no standard curve required!
Step-by-Step
Sample Partitioning
Your DNA or cDNA sample is mixed with PCR reagents and divided into ~20,000 droplets using oil emulsion.
PCR Amplification
Each droplet undergoes thermal cycling. Some droplets will contain the target sequence—others won’t.
Fluorescence Detection
After amplification, droplets are passed through a reader that detects fluorescence, telling you which droplets were positive.
Quantification
Using Poisson statistics, ddPCR gives you absolute copy number per microliter of reaction.
Your DNA or cDNA sample is mixed with PCR reagents and divided into ~20,000 droplets using oil emulsion.
Each droplet undergoes thermal cycling. Some droplets will contain the target sequence—others won’t.
After amplification, droplets are passed through a reader that detects fluorescence, telling you which droplets were positive.
Using Poisson statistics, ddPCR gives you absolute copy number per microliter of reaction.
🎥 Watch the Full Breakdown
Be sure to check out our YouTube video:
📌 [Digital Droplet PCR Explained | 3rd Generation PCR]
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