Abstract
Digital Polymerase Chain Reaction (dPCR) is a novel method for the absolute quantification of target nucleic acids. Quantification by dPCR hinges on the fact that the random distribution of molecules in many partitions follows a Poisson distribution. Each partition acts as an individual PCR microreactor and partitions containing amplified target sequences are detected by fluorescence. The proportion of PCR-positive partitions suffices to determine the concentration of the target sequence without a need for calibration. Advances in microfluidics enabled the current revolution of digital quantification by providing efficient partitioning methods. In this review, we compare the fundamental concepts behind the quantification of nucleic acids by dPCR and quantitative real-time PCR (qPCR). We detail the underlying statistics of dPCR and explain how it defines its precision and performance metrics. We review the different microfluidic digital PCR formats, present their underlying physical principles, and analyze the technological evolution of dPCR platforms. We present the novel multiplexing strategies enabled by dPCR and examine how isothermal amplification could be an alternative to PCR in digital assays. Finally, we determine whether the theoretical advantages of dPCR over qPCR hold true by perusing studies that directly compare assays implemented with both methods.
Highlights
Quantification of Nucleic Acids by Quantitative polymerase chain reaction (PCR) and Digital PCRwe present the basic concepts that underlie the quantification of nucleic acids by digital and quantitative real-time PCR
Despite the relatively limited number of thorough studies that compare the performance of Digital Polymerase Chain Reaction (dPCR) and quantitative real-time PCR (qPCR) side-by-side, a few key points emerge: (1) small partition volume contributes to dPCR resilience to a large variety of inhibitors; (2) dPCR is more precise for quantifying relative abundance (e.g., Copy Number Variant (CNV), mutant allele burden); (3) dPCR suffers from lower sensitivity for absolute quantification, which is attributed to its smaller reaction volume
Conclusions dPCR reduces the quantification of a target sequence to the enumeration of a series of positive and negative amplification reactions, converting a continuous or analog signal into a series of binary or digital signals. dPCR has been enabled by advances in microfluidics that provide efficient methods to create many independent reactors
Summary
Polymerase Chain Reaction (PCR) is an in vitro technique that amplifies DNA, generating several millions of copies of a specific segment of DNA from a minute amount of starting material [1]. Different phases a real-time PCR amplification plot on a linear (1) In early PCR cycles, the fluorescence signal due to amplification product remains at background level. The cycle of quantification (Cq) is the cycle number at which the amplification plot intersects the the amount of PCR product doubles with each cycle in perfect reaction conditions, i.e., if amplification threshold line that is set significantly above the baseline. A typical real-time PCR amplification plot displays a sigmoidal-shaped curve (on a linear scale) and includes a baseline phase, followed by an exponential phase that reaches a plateau via a linear phase (Figure 1b). Sensors 2018, 18, x FOR PEER REVIEW relative to a standard curve generated from a sample of known quantity or copy number (Figure 2) This method implies that amplificationand efficiencies of thelimited samplereproducibility and the standards areitequivalent.
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