What is Digital PCR? How Does It Work?

The most important application of PCR is either to amplify DNA, quantify it, or do both in the same reaction. In conventional PCR, the DNA of interest is amplified to take more than one copy because single or double DNA molecules are not sufficient for downstream analyzes. However, gene measurement using conventional PCR is not possible, so gene expression and nucleic acid measurement cannot be done with conventional PCR.

An advanced PCR machine called a real-time thermocycler is needed to quantify nucleic acid, viral load, and pathogen. Using fluorescence chemistry, the amount of nucleic acid present in a sample can be measured by quantitative PCR. By using fluorescent dye or hydrolysis probe, relative quantification can be done in real-time PCR for gene expression, microRNA studies, and pathogen load measurement.

Real-time PCR is now the gold standard method for the quantification of cytokines and even the most reliable quantification machine for TB detection. However, due to several limitations of real-time PCR, an improved, robust, and more accurate method for quantification of nucleic acid is needed. Using real-time PCR, scientists can estimate the amount of DNA or RNA contained in a sample. However, it can also lead to the quantification of unwanted nucleic acids due to the large volume used during reaction preparation. In addition, several molecules are not quantified, although the excess is not very large. Digital PCR was invented to overcome this problem. The digital PCR principle is based on absolute quantification by dividing a sample into smaller micro-volumes. The general idea of ​​dividing the sample into a smaller volume is to measure a single molecule contained in that micro-volume, thus increasing the accuracy of the measurement.

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What is Digital PCR?

Digital PCR, commonly known as dPCR or dePCR, is used for the absolute measurement of gDNA, cDNA or RNA. In 1999, Bert Vogelstein and Kenneth Kinzler discovered “digital PCR” by analyzing rare genotypes of cancer mutations. The concept of digital PCR was introduced in 1992 by Sykes et al. The method is widely used for the detection of rare alleles and for point mutations. It is also used in clonal amplification during NGS.

The current gold standard method for quantification of nucleic acid is real-time PCR based on fluorescent chemistry. A hydrolysis-based fluorescent probe or DNA intercalation dye is used to determine the amount of nucleic acid. In summary, when the fluorescently labeled probe finds its complementary strand in a sample where it binds to the array, the hydrolyzed probe emits fluorescence detected by the machine. In the layered dye-based method, fluorescent dye binds only to dsDNA and emits fluorescence. The amount of fluorescence emitted is measured by the machine. However, “traditional qPCR” implementation requires standards. Standards are used as the basis for measuring the sample, also the sample volume must be the same amount.

In addition, performing the multiplexing quantification test requires expertise. Performing multiplication assays is not an easy task due to the competitive assay nature. Real-time PCR is generally not applicable to the detection of rare alleles or heterogeneous samples because the quantification limit between two samples is 2-fold. These problems are overcome using the next-generation nucleic acid quantification method called digital PCR, which counts the total number of DNA molecules in digital format by dividing a sample into micro-volumes. Digital PCR’s workflow is similar, qPCR uses the same reagents and similar thermal cycler conditions. Chemistry relies on the use of fluorescent molecules for detection (same as qPCR). In the preparation of the PCR reaction, dNTPs, primer set, PCR reaction buffer, template DNA, Taq DNA polymerase, and other PCR enhancers are used.

Digital PCR Principle

In quantitative PCR, the first PCR cycles are not exponential, so nucleic acid cannot be quantified accurately. In DPCR, after the PCR reaction mix has been prepared, the PCR reaction mix (with template DNA) is divided into thousands of smaller droplets where the separate PCR reaction occurs as individual droplets.

Processing mode, fluorescence (PCR reaction for Drop PCR: dNTPs, hot-start DNA polymerase, MgCl2, optimum reaction buffer, primers, fluorescent probe and quencher probes). Real-time PCR as the labeled probe emits machine-measured light when coupled to DNA It is similar to. In each micro-volume reaction (droplet), the amount of amplification and the number of nucleic acid molecules are measured using the Poisson distribution method. The fluorescence emitted by the droplet without a template is called “0” and is used as a baseline or zero for calculation (PCR-negative reaction). The amount of fluorescence emitted by the droplet with a single pattern is called “1” and recorded individually (PCR-positive reaction).

Absolute measurement of target DNA is made using the Poisson distribution method, using the number of positive and negative reactions in each droplet. After the partitioning, the PCR reaction proceeds to the endpoint of polymerization. The reaction drop making machine divides a sample into more than 20,000 nano-trash-sized droplets. Therefore, no reference, calibration curve, or endogenous control is required to quantify the DNA. In DPCR, the reaction mixture is divided into smaller micro-volume reactions so that there is only one or zero target molecules per micro-volume reaction.

In the final calculation, the total number of positive reactions (with 1 template) is equal to the total number of target DNA contained in the original volume of a sample. The absolute concentration of the total volume is calculated using this equation:

Absolute concentration = a total number of template DNA counted / total volume measured in each droplet (or well or microvolume reaction).

The DPCR method is based on TaqMan chemistry, where the fluorescent dye-labeled probe is used as a complement to the template. Dye FAM (fluorescent dye) is used in conjunction with HEX reporter dye. Sample compartment creation methods; oil emulsion, micro-hollow plates, non-fluid chips, and capillaries.

Digital PCR Applications

There are many areas where digital PCR applications are used. Some of these areas are:

• Rare allele detection as well as rare sequence detection.
• Correct copy number variation studies
• Accurate NGS library measurement

Viral load detection

Pathogen detection

Single-cell gene expression analysis

• Confirmation of low-frequency mutations identified by sequencing.

Gene expression and microRNA research

One of the biggest advantages of DPCR is that digital PCR can detect the difference in gene expression less than 30%, only a single copy number variation can be accurately distinguished. In addition, it can identify alleles with allelic frequencies of less than 0.1%.

Advantages of Digital PCR

• A simple setup does not require higher expertise as in quantitative PCR.

Higher precision

• Higher repeatability
• Measure target in low samples

Improved multiplexing capacity

High tolerance capacity against PCR inhibitors

• Early and accurate detection of low-frequency genotypes.

Droplet Digital PCR

During the water-oil emulsion technique process, an oil droplet of nano volume size is created to split the reaction. Quantification of DNA using this technique is called droplet digital PCR. However, the basic principle of droplet digital PCR is similar to dPCR. DdPCR produces more than 20,000 micro-volume sections for absolute measurement. If the reaction is split, the more micro-volumed reactions, the higher the chance of having zero or one template in the micro-volume reaction chamber, so the counting becomes more accurate. Therefore, ddPCR (using a water-oil emulsion technique) is recommended for rare alleles and low-frequency genotype detection.

Digital PCR is one of the most accurate methods used for quantitation of nucleic acid. Its power to detect even a single allele provides results with greater accuracy in the detection of rare mutations and rare alleles in cancer research and cancer development.

References:

• https://www.ncbi.nlm.nih.gov/pubmed/26843045
• https://www.sciencedirect.com/science/article/abs/pii/S0956566319304889