The threshold (dotted lines) is set above the background within the exponential phase. (2) During the exponential phase, the amount of PCR product doubles with each cycle in perfect reaction conditions, i.e., if amplification efficiency is 100%. The baseline is set to eliminate the background fluorescent signal. (1) In early PCR cycles, the fluorescence signal due to amplification product remains at background level. A typical PCR amplification plot displays a sigmoidal-shape curve with 4 distinct phases. Each cycle can theoretically result in a doubling of the number of molecules of the target sequence ( b) Different phases of a real-time PCR amplification plot on a linear scale. A typical PCR reaction is cycled 20–40 times. Each PCR cycle includes three steps: (1) Denaturation of double-stranded DNA by heat (2) Annealing of primers to their complementary target DNA sequences (3) Extension of primers by a thermostable DNA polymerase. ( a) Principles of the polymerase chain reaction (PCR). Conventional PCR analyzes amplified products at the end of the reaction using gel electrophoresis (end-point measurement). In practice, the amplification process saturates and reaches a plateau as PCR reagents are depleted and accumulated PCR products self-anneal, preventing any further amplification. Each cycle results in a doubling of the number of target DNA molecules (exponential amplification) and 2 n copies can, in theory, be produced after n cycles. Each cycle includes the denaturation of DNA duplexes, the hybridization of two DNA oligonucleotides (primers) flanking the target sequence, and the elongation of those primers by a DNA polymerase ( Figure 1a). PCR typically consists of a series of temperature cycles repeated 20 to 40 times. Its specificity relies on sequence hybridization and its sensitivity depends on enzyme-based amplification. 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. Finally, we determine whether the theoretical advantages of dPCR over qPCR hold true by perusing studies that directly compare assays implemented with both methods. We present the novel multiplexing strategies enabled by dPCR and examine how isothermal amplification could be an alternative to PCR in digital assays. We review the different microfluidic digital PCR formats, present their underlying physical principles, and analyze the technological evolution of dPCR platforms. We detail the underlying statistics of dPCR and explain how it defines its precision and performance metrics. In this review, we compare the fundamental concepts behind the quantification of nucleic acids by dPCR and quantitative real-time PCR (qPCR). Advances in microfluidics enabled the current revolution of digital quantification by providing efficient partitioning methods. The proportion of PCR-positive partitions suffices to determine the concentration of the target sequence without a need for calibration. Each partition acts as an individual PCR microreactor and partitions containing amplified target sequences are detected by fluorescence. Quantification by dPCR hinges on the fact that the random distribution of molecules in many partitions follows a Poisson distribution. Digital Polymerase Chain Reaction (dPCR) is a novel method for the absolute quantification of target nucleic acids.
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