Digital PCR and Real-Time PCR (qPCR) Choices for Different Applications

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Overview

Both quantitative PCR (qPCR) and digital PCR (dPCR) provide sensitive and specific detection, and precise quantification of nucleic acids. Both technologies have similarities, but they have differences that make one or the other the more adapted choice for specific applications. Find out how qPCR and dPCR compare for different types of research.

What are qPCR and dPCR?

Real-time PCR or quantitative PCR is a well-established technology that has become the tool of choice for the rapid, sensitive quantification of nucleic acid in various biological samples. qPCR measures the accumulation of DNA during a PCR reaction. The increase in quantity of DNA at each cycle is measured by the change in intensity of a fluorescent signal. Comparison to a reference sample determines the number of original copies of template DNA in the reaction.

Digital PCR is a highly precise approach to sensitive nucleic acid detection and quantification. Each sample is partitioned into thousands of individual reactions (droplets for Droplet Digital™ PCR technology). Each partition is analyzed after end-point PCR cycling for the presence or absence of a fluorescent signal, and the absolute number of molecules present in the sample is calculated. dPCR does not require a standard curve for quantification.

For more information, see the Applications and Technologies pages for qPCR and dPCR.

What are the Strengths of dPCR and qPCR?

Digital PCR Real-Time PCR
Quantifies rare targets in complex backgrounds Broad dynamic range — measures low and high expression levels in same reaction
Detects small fold changes — 10% Can detect as little as 2-fold changes
Up to 96-well throughput High throughput with 384-well format and automation
Robust quantification (high tolerance to PCR inhibitors) Economical running costs
Rapid, simple optimization Rapid time to results
Not reliant on standard curve Well-established technology

Sample partioning and cycling to end point makes dPCR less impacted by changes in PCR efficiency (including partial polymerase inhibition), which does not affect results significantly. For qPCR, without using an inhibitor-tolerant supermix such as SsoAdvanced™ Universal Inhibitor-Tolerant SYBR® Green Supermix, the presence of PCR inhibitors in experimental samples will impact the results.

Major Applications

The table indicates the suitability of each technology, the level of fitness is reflected by the number of dots
( equates to higher capability), for major applications.

Application Digital PCR Real-Time PCR
Mutation/SNP Detection
Genome Edit Detection
Copy Number Variation
Standards Validation
NGS Validation
Haplotyping
Microarray Validation
siRNA, miRNA, lncRNA Detection
Environmental Studies
Pathogen Detection
Gene Expression

Mutations, SNP Detection, and Allelic Discrimination

Due to its high level of specificity, dPCR is recommended for detection of mutations and SNPs, as well as allelic discrimination. dPCR provides the required sensitivity to detect rare mutations on wild-type backgrounds, haplotypes, and low-abundance targets in liquid biopsy (such as circulating tumor cells and cell-free DNA). For samples with rare targets where amplification bias may otherwise occur, dPCR provides accurate and absolute quantification. qPCR is suitable when the mutation frequency is known to be above 1%, and for high resolution melt analysis (HRM).

Leading technology: dPCR

  • Detect mutation rates <1%
  • Rare SNP detection
  • Haplotyping
  • Allelic discrimination
  • Precise and sensitive, ideal for liquid biopsy

Supporting technology: qPCR

  • High throughput
  • Reliable detection >1%
  • Economical for large screening studies
  • HRM
  • Broad dynamic range

Gene Expression

qPCR is recommended for studying gene expression, because of its wide dynamic range, with the ability to quantify different expression levels and discriminate splice variants, the capability for high throughput applications and automation, and the ability to multiplex. Additionally, qPCR is more economical when processing a large number of samples. In some cases, for instance, when low fold-changes (<2-fold) need to be detected, the precision of dPCR may be required.

Leading technology: qPCR

  • Broad dynamic range — measure low and high expressors in same well
  • High throughput with 384 well format, automation compatible
  • Multiplexing capabilities
  • Protocols available for single cell studies, simultaneous protein expression, quantification, etc.
  • Assays available: 12 organisms in probe or SYBR Green Supermix/SsoFast™ EvaGreen Supermix

Supporting technology: dPCR

  • Detection of small fold changes — 10%
  • Direct quantification of gene expression
  • Allelic discrimination
  • Assays available: human, rat, mouse in probe or primer pairs for QX200™ ddPCR™ EvaGreen Supermix

Gene Editing

dPCR is recommended for analysis of gene editing due to the low frequency of edits, both desired and off-target events. Additionally, dPCR permits analysis of very low levels of gDNA. qPCR is a method frequently used to screen large cell populations, and can provide rapid genotyping by HRM.

Leading technology: dPCR

  • Detect CRISPR/Cas1, ZFN2 and TALEN3 edits
  • Precise and sensitive NHEJ4 and HDR5 quantification
  • Detection of off-target events

Supporting technology: qPCR

qPCR is a method frequently used for large screens of cells populations, and can provide rapid genotyping by HRM.

  • High throughput
  • Reliable detection >1%
  • Economical for large screening studies
  • Multiplexing capabilities
  • HRM

1 Clustered regularly interspaced short palindromic repeats/ CRISPR associated, 2 zinc finger nuclease, 3 transcription activator-like effector nuclease, 4 non-homologous end joining, 5 homology directed repair

Copy Number Variation

dPCR is recommended for analysis of copy number variation, since it can accurately detect copy changes with fewer replicates than qPCR. With qPCR, as the difference between copy numbers decreases, the number of replicates required increases rapidly. dPCR can detect small fold changes such as a change in copy number, a change from 5 to 6 copies (see Bulletin 6475). qPCR is useful for high throughput applications, and where there are bigger differences in copy number.

Leading technology: dPCR

  • Detection of small copy number changes — expressed as 10% precision; the fold change changes across the range of the experiment
  • Precise and sensitive copy number discrimination
  • Few replicates needed

Supporting technology: qPCR

  • Suited for detection of large variations
  • High throughput screens
  • Broad dynamic range

Pathogen Detection and Microbiome Analysis

qPCR is recommended for both pathogen detection and microbiome analysis, due to its ability to accommodate a broad range of concentrations of targets in a single well, and the capability to screen a large number of samples simultaneously. qPCR is increasing in importance as PCR continues to replace traditional methods of screening for pathogens. dPCR is suited for quantification of low level pathogens, such as pathogens detected in HIV/AIDs studies involving a combination of antiretroviral therapies to help improve standard of care.

Leading technology: qPCR

  • Sensitivity
  • High throughput
  • Time to result
  • Economical
  • Broad dynamic range

Supporting technology: dPCR

  • Precise quantification of low viral and bacterial loads
  • No standard curve required
  • Ideal for monitoring persistent infection
  • Multiplex closely related species (e.g., co-infection analysis)

Conclusion

Though both qPCR and dPCR provide sensitive detection and precise quantitation, their distinct strengths provide different advantages for different applications. qPCR provides high throughput and wide dynamic range, which is useful for screening large numbers of samples. dPCR offers unparalleled sensitivity for fractional abundance (mutant/wild-type ratio) and exquisite precision. Both technologies have complementary capabilities, and the combination of both methods offers researcher a wide palette of solutions for various genomic applications.

Related Content

Literature
Number Description Download
6311 QX200™ Droplet Digital™ PCR System Brochure, Rev C Click to download
6093 CFX96 Touch™ Real-Time PCR Detection System Brochure Click to download
6096 CFX384 Touch™ Real-Time PCR Detection System Brochure Click to download
6105 CFX Connect™ Real-Time PCR Detection System Brochure Click to download
6407 Droplet Digital PCR Applications Guide, Rev A Click to download
5279 Real-Time PCR Applications Guide, Rev B Click to download
6475 Droplet Digital PCR: High-Resolution Copy Number Variation Analysis Application Note, Rev A Click to download
6712 Ultra-Sensitive Quantification of Genome Editing Events Using Droplet Digital™ PCR Application Note, Ver B Click to download
6554 Droplet Digital™ PCR: Detection of DNA Methylation Application Note, Rev A Click to download
5990 Gene Expression Analysis Solutions Brochure, Rev C Click to download
6004 A Practical Guide to High Resolution Melt Analysis Genotyping, Rev A Click to download
6629 High-Throughput Real-Time PCR Solutions Brochure, Ver B Click to download
6634 A Fast Semi-Automated Quantitative PCR Method for Monitoring Differential Gene Expression for Drug Target Discovery, Ver B Click to download
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