Digital PCR (dPCR) is a revolutionary technology that has emerged as a powerful tool for precise quantification of nucleic acids. It offers several advantages over traditional PCR methods, such as increased sensitivity, accuracy, and reproducibility. In this article, we will provide you with a comprehensive guide to digital PCR, including its principle, applications, advantages, limitations, and future prospects.
Introduction
PCR (Polymerase Chain Reaction) is a widely used technique in molecular biology that amplifies a specific DNA sequence. However, conventional PCR methods have limitations in terms of sensitivity, accuracy, and reproducibility. Digital PCR (dPCR) has overcome these limitations by partitioning a PCR reaction into thousands of tiny droplets or chambers, each containing a single DNA molecule. This allows for absolute quantification of nucleic acids with high precision and accuracy.
Request A Sample PDF Brochure: https://www.fortunebusinessinsights.com/enquiry/request-sample-pdf/digital-pcr-market-102014
What is Digital PCR?
Digital PCR (dPCR) is a technique that partitions a PCR reaction into thousands of droplets or chambers, each containing a single molecule of DNA. The amplification of each molecule occurs independently in its own droplet or chamber. After PCR amplification, the endpoint fluorescence of each droplet or chamber is measured, and the number of positive droplets or chambers is used to determine the absolute quantity of the target molecule in the original sample.
How Does Digital PCR Work?
Digital PCR involves three main steps: partitioning, amplification, and detection.
Partitioning
The sample containing the target molecule is first partitioned into thousands of droplets or chambers, each containing a single molecule of DNA. This can be achieved using microfluidics or emulsion PCR methods.
Amplification
After partitioning, PCR amplification is performed in each droplet or chamber independently, using standard PCR reagents and cycling conditions.
Detection
After PCR amplification, the endpoint fluorescence of each droplet or chamber is measured using a fluorescence reader. The number of positive droplets or chambers is used to determine the absolute quantity of the target molecule in the original sample.
Types of Digital PCR
There are two main types of digital PCR: droplet digital PCR (ddPCR) and digital PCR in wells (dPCR-wells).
Droplet Digital PCR (ddPCR)
Droplet digital PCR (ddPCR) is the most widely used digital PCR technique. It partitions a PCR reaction into thousands of droplets, each containing a single molecule of DNA. After PCR amplification, the endpoint fluorescence of each droplet is measured, and the number of positive droplets is used to determine the absolute quantity of the target molecule in the original sample.
Digital PCR in Wells (dPCR-wells)
Digital PCR in wells (dPCR-wells) partitions a PCR reaction into thousands of wells, each containing a single molecule of DNA. After PCR amplification, the endpoint fluorescence of each well is measured, and the number of positive wells is used to determine the absolute quantity of the target molecule in the original sample.
Applications of Digital PCR
Digital PCR has several applications in research, clinical diagnostics, and other fields.
Research Applications
- Absolute quantification of nucleic acids
- Rare mutation detection
- Copy number variation analysis
- Gene expression analysis
- Next-generation sequencing library quantification
- Environmental DNA analysis
