Introduction

Real-Time PCR—also called quantitative polymerase chain reaction (qPCR)—is one of the most powerful and sensitive gene analysis techniques available and is used for a broad range of applications including quantitative gene expression analysis, genotyping, SNP analysis, pathogen detection, drug target validation, and for measuring RNA interference. Frequently, real-time polymerase chain reaction is combined with reverse transcription to quantify messenger RNA (mRNA) and microRNA (miRNA) in cells or tissues.

As the name suggests, real-time PCR measures PCR amplification as it occurs. This completely revolutionizes the way one approaches PCR-based quantitation of DNA and RNA. In traditional PCR, results are collected after the reaction is complete, making it impossible to determine the starting concentration of nucleic acid.

Digital PCR is a new approach to nucleic acid detection and quantification, which is a different method of absolute quantification and rare allele detection relative to conventional qPCR, because it directly counts the number of target molecules rather than relying on reference standards or endogenous controls.

Real-time PCR vs. traditional PCR vs. digital PCR at a glance

Digital PCR Real-time PCR Traditional PCR
Overview Measures the fraction of negative replicates to determine absolute copies. Measures PCR amplification as it occurs. Measures the amount of accumulated PCR product at the end of the PCR cycles.
Quantitative? Yes, the fraction of negative PCR reactions is fit to a Poisson statistical algorithm. Yes, because data is collected during the exponential growth (log) phase of PCR when the quantity of the PCR product is directly proportional to the amount of template nucleic acid. No, though comparing the intensity of the amplified band on a gel to standards of a known concentration can give you 'semi-quantitative' results.
Applications
  • Absolute quantification of viral load
  • Absolute quantification of nucleic acid standards
  • Absolute quantification of next-gen sequencing Libraries
  • Rare allele detection
  • Absolute quantification of gene expression
  • Enrichment and separation of mixtures
  • Quantitation of gene expression
  • Microarray verification
  • Quality control and assay validation
  • Pathogen detection
  • SNP genotyping
  • Copy number variation
  • MicroRNA Analysis
  • Viral quantitation
  • siRNA/RNAi experiments

Amplification of DNA for:

  • Sequencing
  • Genotyping
  • Cloning
Summary

Advantages of digital PCR:

  • No need to rely on references or standards
  • Desired precision can be achieved by increasing total number of PCR replicates
  • Highly tolerant to inhibitors
  • Capable of analyzing complex mixtures
  • Unlike traditional qPCR, digital PCR provides a linear response to the number of copies present to allow for small fold change differences to be detected

Adavantages of real-time PCR:

  • Increased dynamic range of detection
  • No post-PCR processing
  • Detection is capable down to a 2-fold change
  • Collects data in the exponential growth phase of PCR
  • An increase in reporter fluorescent signal is directly proportional to the number of amplicons generated
  • The cleaved probe provides a permanent record amplification of an amplicon

Disadvantages of traditional PCR:

  • Poor Precision
  • Low sensitivity
  • Short dynamic range < 2 logs
  • Low resolution
  • Non-automated
  • Size-based discrimination only
  • Results are not expressed as numbers
  • Ethidium bromide for staining is not very quantitative
  • Post-PCR processing

 To understand why traditional PCR is limiting, it is important to understand what happens during a PCR reaction. A basic PCR run can be broken up into three phases:

Exponential

Exact doubling of product is accumulating at every cycle (assuming 100% reaction efficiency). The reaction is very specific and precise. Exponential amplification occurs because all of the reagents are fresh and available, the kinetics of the reaction push the reaction to favor doubling of amplicon.

Linear (high variability)

As the reaction progresses, some of the reagents are being consumed as a result of amplification. The reactions start to slow down and the PCR product is no longer being doubled at each cycle.

Plateau (End-point: gel detection for traditional methods)

The reaction has stopped, no more products are being made and if left long enough, the PCR products will begin to degrade. Each tube or reaction will plateau at a different point, due to the different reaction kinetics for each sample. These differences can be seen in the plateau phase. The plateau phase is where traditional PCR takes its measurement, also known as end-point detection.

 Figure 1: PCR phases.

Traditional PCR measures at the plateau, giving you variable results

In Figure 2, three replicate samples, which had same amount of DNA in the beginning of the reaction, have different quantities of PCR product by the plateau phase of the reaction (due to variations in reaction kinetics). Therefore, it will be more precise to take measurements during the exponential phase, where the replicate samples are amplifying exponentially.

Figure 2: Identical samples produce different quantities of reaction product by the plateau phase of PCR.

Real-time PCR measures at the exponential phase for more accurate quantitation

Real-time PCR focuses on the exponential phase because it provides the most precise and accurate data for quantitation. Within the exponential phase, the real-time PCR instrument calculates two values. The Threshold line is the level of detection at which a reaction reaches a fluorescent intensity above background. The PCR cycle at which the sample reaches this level is called the Cycle Threshold, Ct. The Ct value is used in downstream quantitation or presence/absence detection. By comparing the Ct values of samples of unknown concentration with a series of standards, the amount of template DNA in an unknown reaction can be accurately determined.

Figure 3: The PCR cycle at which the sample reaches a fluorescent intensity above background is the Cycle Threshold or Ct.

Digital PCR counts individual molecules for absolute quantification

Digital PCR works by partitioning a sample into many individual real-time PCR reactions; some portion of these reactions contain the target molecule (positive) while others do not (negative). Following PCR analysis, the fraction of negative answers is used to generate an absolute answer for the exact number of target molecules in the sample, without reference to standards or endogenous controls.

Figure 4:  Digital PCR uses the ratio of positive (black) to negative (white) PCR reactions to count the number of target molecules.

TaqMan® Probe- and SYBR® Green-based detection

Every real-time PCR reaction contains a fluorescent reporter molecule—a TaqMan® probe or SYBR® Green dye, for example—to monitor the accumulation of PCR product. As the quantity of target amplicon increases, so does the amount of fluorescence emitted from the fluorophore.

Figure 5:  Advantages of real-time PCR vs. traditional PCR.

Applied Biosystems® has a full range of real-time PCR products for routine and challenging applications

Applied Biosystems offers a comprehensive set of products for real-time PCR-based gene expression, miRNA, copy number variation, and SNP genotyping analysis, from off-the-shelf gene-specific probe and primer sets, to everyday reagents and plastics, instrument systems, software, and everything in between.