qpcr vs. digital pcr vs. traditional pcr

8/11/2019 QPCR vs. Digital PCR vs. Traditional PCR

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qPCR vs. Digital PCR vs. Traditional PCR

IntroductionReal-time PCR vs traditional PCR vs digital PCR at a glance

Traditional PCR measures at the plateau, giving you variable resultsReal-time PCR measures at the exponential phase for more accurate quantitationDigital PCR counts individual molecules for absolute quantificationTaqMan probe- and SYBR Green-based detectionApplied Biosystems has a full range of real-time PCR products for routine and challenging applications

ntroduction

Real-Time PCRalso called quantitative polymerase chain reaction (qPCR)is one of the most powerful and sensitive gene analysis techniquesvailable and is used for a broad range of applications including quantitative gene expression analysis, genotyping, SNP analysis, pathogen detection,rug target validation, and for measuring RNA interference. Frequently, real-time polym era se chain reaction is combined with reverse transcription touantify 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-baseduantitation of DNA and RNA. In traditional PCR, results are collected after the reaction is complete, making it impossible to determine the startingoncentration 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 alleleetection relative to conventional qPCR, because it directly counts the number of target molecules rather than relying on reference standards or ndogenous controls.

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Rea l-time PCR vs. traditional PCR vs. digital PCR at a glance

Digital PCR Real-time PCR Traditional PCR

Overview Measures the fraction of negativereplicates to determine absolute copies.

Measures PCR amplification as it occurs. Measures the amount of accumulated PCR product at the endof the PCR cycles.

Quantitative? Yes, the fraction of negative PCRreactions is fit to a Poisson statisticalalgorithm.

Yes, because data is collected during theexponential growth (log) phase of PCR whenthe quantity of the PCR product is directlyproportional to the amount of template nucleicacid.

No, though comparing the intensity of the amplified band on a gel tostandards of a known concentrationcan give you 'semi-quantitative'results.

Applications Absolute quantification of viral load Absolute quantification of nucleicacid standards

Absolute quantification of next-gensequencing LibrariesRare allele detection

Absolute quantification of geneexpressionEnrichment and separation of mixtures

Quantitation of gene expressionMicroarray verificationQuality control and assay validationPathogen detectionSNP genotypingCopy number variationMicroRNA AnalysisViral quantitationsiRNA/RNAi experiments

Amplification of DNA for:

SequencingGenotypingCloning

Summary Advantages of digital PCR:

No need to rely on references or standardsDesired precision can be achievedby increasing total number of PCRreplicatesHighly tolerant to inhibitorsCapable of analyzing complex

Adavantages of real-time PCR:

Increased dynamic range of detectionNo post-PCR processingDetection is capable down to a 2-foldchangeCollects data in the exponential growthphase of PCR

An increase in reporter fluorescent signal is

Disadvantages of traditional PCR:

Poor PrecisionLow sensitivityShort dynamic range < 2 logsLow resolutionNon-automatedSize-based discrimination onlyResults are not expressed as
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mixturesUnlike traditional qPCR, digital PCRprovides a linear response to thenumber of copies present to allowfor small fold change differences tobe detected

directly proportional to the number of amplicons generatedThe cleaved probe provides a permanentrecord amplification of an amplicon

numbersEthidium bromide for staining isnot very quantitativePost-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 upnto three phases:

Exponential

Exact doubling of product is accumulating at every cycle (assuming 100% reaction efficiency). The reaction is very specific and precise. Exponentialmplification 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 PCRroduct is no longer being doubled at each cycle.

lateau (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

lateau at a different point, due to the different reaction kinetics for each sample. These differences can be seen in the plateau phase. The plateauhase is where traditional PCR takes its measurement, also known as end-point detection.

Figure 1: PCR phases.

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Traditional PCR measures at the plateau, giving you variable results

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

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

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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 exponentialhase, the real-time PCR instrument calculates two values. The Threshold line is the level of detection at which a reaction reaches a fluorescentntensity above background. The PCR cycle at which the sample reaches this level is called the Cycle Threshold, Ct. The Ct value is used inownstream quantitation or presence/absence detection. By comparing the Ct values of samples of unknown concentration with a series of standards,he amount of template DNA in an unknown reaction can be accurately determined.

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

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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 moleculepositive) while others do not (negative). Following PCR analysis, the fraction of negative answers is used to generate an absolute answer for the exactumber of target molecules in the sample, without reference to standards or endogenous controls.

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

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TaqMan Probe- and SYBR Green-based detection

Every real-time PCR reaction contains a fluorescent reporter moleculea TaqMan probe or SYBR Green dye, for exampleto monitor theccumulation of PCR product. As the quantity of target amplicon increases, so does the amount of fluorescence emitted from the fluorophore.

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

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Real-time PCReducational resources

Real-time PCRwebinarsReal-time PCRHandbookDigital vs. real-time PCRinfographic poster

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 SNPenotyping analysis, from off-the-shelf gene-specific probe and primer sets, to everyday reagents and plastics, instrument systems, software, andverything in between.

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qpcr vs. digital pcr vs. traditional pcr

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