Introduction:
- Digital polymerase chain reaction (digital PCR, DigitalPCR, dPCR, or dePCR) is a biotechnology advancement that can be used to directly quantify and clonally amplify nucleic acids such as DNA, cDNA, or RNA. dPCR provides for the accurate quantification of nucleic acids, making it possible to measure minor percentage differences and identify rare mutations.
- Digital PCR (dPCR) is a method used to quantitatively measure the amount of DNA or RNA in a sample. It is similar to traditional PCR (polymerase chain reaction), but instead of amplifying the target DNA or RNA in a sample and measuring the amplification using a fluorimeter or other instrument, dPCR divides the sample into many small partitions and performs PCR on each partition. The number of partitions that contain the target DNA or RNA is then used to calculate the concentration of the target in the original sample. The term “digital” refers to the use of discrete partitions and the representation of the amplified target as either present or absent, rather than as a continuous signal as in traditional PCR.
- Polymerase chain reaction (PCR) has been modified by three generations. Gel electrophoresis is the primary method used by the first generation of PCR for product analysis, although it has limitations due to its low detection limit, lengthy operation, and single application (qualitative). The second generation of PCR, also called real-time quantitative PCR (RT-qPCR), can quantify the products with standard curves, but also show low tolerance to interfering substances.
- Digital PCR (dPCR) is the third generation of PCR that enables absolute quantification through partitioning the reaction. Highly sensitive and accurate in molecular detection, this technology has demonstrated applications like trace DNA detection, rare mutation detection and copy number variation.
History:
The concept of digital PCR (dPCR) was first described in a 1996 paper by Scherer and colleagues, who proposed using microfluidic devices to partition a sample into thousands of small compartments and perform PCR on each compartment. The first commercial dPCR instrument, the Bio-Rad iCycler iQ, was introduced in 2003. Since then, dPCR has become an important tool in molecular biology, with a number of commercial instruments now available on the market. In addition to its use in quantifying DNA and RNA, dPCR has also been used for a variety of applications, including the detection of mutations and single nucleotide polymorphisms (SNPs), the analysis of gene expression, and the identification of viral pathogens.
Principles:
The principle of digital PCR (dPCR) is based on the partitioning of a sample into many small compartments, each of which contains a small volume of sample and reagents. The sample is then amplified using PCR, and the presence or absence of the amplified target is determined for each compartment.
There are two main types of dPCR: absolute quantification dPCR and relative quantification dPCR.
Absolute quantification dPCR
It involves the use of a standard curve to determine the concentration of the target in the original sample. The standard curve is generated by amplifying known concentrations of the target DNA or RNA and measuring the amplification in each compartment. The concentration of the target in the sample is then determined by comparing the number of positive compartments in the sample to the standard curve.
Relative quantification dPCR
It involves the comparison of the amplification of the target DNA or RNA in the sample to a reference sample or a control. The reference sample or control is used to normalize the amplification in the sample, and the relative concentration of the target in the sample is calculated based on the ratio of the amplification in the sample to the amplification in the reference or control.
Both absolute quantification dPCR and relative quantification dPCR rely on the fact that the amplification of the target DNA or RNA in each compartment is either present or absent. This binary information is used to calculate the concentration of the target in the original sample.
Steps:
Prepare the sample:
The sample is usually prepared by extracting the DNA or RNA from a biological sample using a kit or protocol specific to the type of sample (e.g. blood, tissue, cells). The purity and concentration of the sample should be checked using a spectrophotometer or other instrument.
Design the PCR assay:
The PCR assay involves the selection of primers and probes that specifically amplify the target DNA or RNA in the sample. The primers are short DNA sequences that bind to the target DNA or RNA, and the probes are labeled with a fluorescent dye or other marker that allows them to be detected after amplification. The PCR assay is typically optimized using a series of experiments to determine the optimal concentrations of the reagents and the optimal cycling conditions.
Fig: Steps of dPCR
Partition the sample:
The sample is divided into many small compartments, each of which contains a small volume of sample and reagents. This can be done using a variety of techniques, including microfluidic devices, droplet-based systems, and bead-based systems.
Perform PCR:
The sample is amplified using PCR, with each compartment containing a separate PCR reaction. The amplification is typically monitored using a fluorimeter or other instrument, which detects the fluorescence emitted by the labeled probes.
Analyze the results:
The presence or absence of the amplified target in each compartment is determined, and this binary information is used to calculate the concentration of the target in the original sample. This can be done using software or by manually counting the number of positive compartments.
Interpret the results:
The results of the dPCR assay are interpreted in the context of the experimental goals and the specific application of the assay. The results may be used to confirm or refute a hypothesis, to make decisions about the sample or the study, or to inform the design of future experiments.
Digital PCR vs. Real-Time PCR vs. Traditional PCR
Polymerase Chain Reaction (PCR) is a powerful technique used to amplify small amounts of DNA or RNA into much larger amounts that can be easily detected and analyzed. In summary, traditional PCR is the most basic form of PCR, real-time PCR allows for the quantification of DNA during the reaction, and digital PCR is a more precise but labor-intensive method for quantifying DNA.
Traditional PCR: This is the most basic form of PCR and involves denaturing DNA, annealing primers to the template, and then extending the primers using a DNA polymerase enzyme.
Real-time PCR: Also known as quantitative PCR (qPCR), this method allows for the real-time detection of amplified DNA during the PCR reaction. This is done by using fluorescent probes or dyes that bind to the amplified DNA and emit a signal that can be detected by a machine. This allows for the quantification of the amount of DNA present in the sample.
Digital PCR: This method involves partitioning a sample into many small reactions, each containing a small number of copies of the target DNA molecule. The number of positive reactions is then counted, and the original concentration of the target DNA can be calculated. Digital PCR is more precise than real-time PCR because it does not rely on the amplification of the target DNA. However, it is also more labor-intensive and requires specialized equipment.
Fig: Digital PCR vs. Real-Time PCR vs. Traditional PCR
Advantages:
Digital PCR (dPCR) has several advantages over traditional PCR and real-time PCR. Overall, dPCR is a powerful tool for accurate and precise DNA and RNA quantification, and is particularly useful for detecting and quantifying low abundance targets.
Increased precision: dPCR is more precise than traditional PCR and real-time PCR because it does not rely on the amplification of the target DNA. This makes it ideal for quantifying low copy number targets or rare mutations.
Greater dynamic range: dPCR has a wider dynamic range than real-time PCR, meaning it can accurately quantify a wider range of target concentrations.
Improved accuracy: dPCR is less prone to errors caused by variations in amplification efficiency, which can occur with traditional PCR and real-time PCR.
Ability to detect rare or low abundance targets: dPCR is able to accurately detect and quantify very low abundance targets, such as rare mutations or pathogens.
Greater sensitivity: dPCR is more sensitive than traditional PCR and real-time PCR, allowing for the detection of very small amounts of DNA or RNA.
Applications:
dPCR is a powerful tool for accurate and precise DNA and RNA quantification, and has a wide range of applications in various fields. Nucleic acid quantification is made possible by the ground-breaking technology known as digital PCR. Allelic variations (SNPs), targets with complicated backgrounds, low abundance targets, and the monitoring of changes in target levels are all applications of this approach.
Genetic testing: dPCR is used to detect and quantify rare genetic mutations, such as those associated with inherited diseases.
Pathogen Detection and Microbiome Analysis: dPCR is used to detect and quantify the presence of pathogens, such as viruses and bacteria, in clinical samples.
DNA methylation analysis: dPCR can be used to accurately quantify the levels of DNA methylation at specific loci, allowing for the study of epigenetic changes.
Gene expression analysis: dPCR can be used to accurately measure the levels of specific mRNA transcripts in a sample, allowing for the study of gene expression.
Fertility testing: dPCR can be used to quantify the number of sperm in a sample, allowing for the diagnosis of male infertility.
Environmental monitoring: dPCR is used to detect and quantify microorganisms in environmental samples, such as water or soil.
Oncology: dPCR is used to detect and quantify the presence of cancer-associated mutations, such as those found in the BRCA1 and BRCA2 genes.
Gene Expression: Measurement and detection of genomic DNA methylation and absolute quantification employed in this technique increases the sensitivity of transcriptional analyses.
References:
- Mao X, Liu C, Tong H, Chen Y, Liu K. Principles of digital PCR and its applications in current obstetrical and gynecological diseases. Am J Transl Res. 2019 Dec 15;11(12):7209-7222. PMID: 31934273; PMCID: PMC6943456.
- Pomari E, Piubelli C, Perandin F, Bisoffi Z. Digital PCR: a new technology for diagnosis of parasitic infections. Clin Microbiol Infect. 2019 Dec;25(12):1510-1516.
- Quan, P.L., Sauzade, M. and Brouzes, E., 2018. dPCR: a technology review. Sensors, 18(4), p.1271.