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. 2019 Sep 12;9(3):44. doi: 10.3390/jpm9030044

Table 2.

PCR methodologies at a glance [33,34,35].

Classical PCR qPCR Digital PCR (ddPCR)
Overview Measures the amount of accumulated PCR product at the end of the PCR reaction, at the plateau.
Semiquantitative—through comparing the intensity of the amplified band on the gel to standards of a known concentration.		 Measures the PCR amplification at the end of each cycle at the exponential phase.
Relative quantification—the data are collected during the exponential (log) phase of PCR when the quantity of the PCR product is directly proportional to the amount of template nucleic acid. It is necessary to have DNA from reference genes or standards.		 Partitioning a sample into many individual qPCR reactions that run in parallel; some of these reactions contain the target molecule (positive) while others do not (negative). Measures the fraction of negative replicates to determine absolute numbers of copies.
Quantitative—the fraction of positive versus negative PCR reactions is used to count the number of target molecules.
Application examples Amplification of DNA for:

  • Sequencing;

  • Genotyping;

  • Cloning.

  • Quantitative gene expression analysis;

  • Microarray verification;

  • Single nucleotide polymorphisms (SNP) genotyping;

  • Copy number variation;

  • MicroRNA analysis.

  • Absolute quantification of gene expression;

  • Absolute quantification of next generation sequencing (NGS) libraries;

  • Rare allele detection;

  • Gene copy number.
Advantages/Disadvantages

  • Poor Precision;

  • Low sensitivity;

  • Low resolution;

  • Lower dynamic range (<2 logs);

  • Size-based discriminatory only;

  • Post-PCR processing.

  • Higher precision;

  • Higher sensitivity;

  • Requires gene references or standard curves;

  • No post-PCR processing;

  • Large dynamic range;

  • Higher throughput, automation compatibility;

  • Highly flexible (users can change reaction volume, throughput, and detection method).

  • Improved precision and lower errors;

  • Greater discrimination between similar sequences;

  • No need to rely on references or standards—absolute measurements;

  • Desired precision can be achieved by increasing total number of PCR replicates;

  • Capable of analyzing complex mixtures;

  • Allows for small fold change differences to be detected;

  • Greater sensitivity for rare mutation detection;

  • Very low sample volume.