Digital PCR

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Overview

Digital PCR (dPCR) enables precise, highly sensitive quantification of nucleic acids. Traditional PCR is an end-point analysis that is semi-quantitative at best — the amplified product is detected by agarose gel electrophoresis after the reaction is finished. Real-time PCR (or qPCR) uses fluorescence-based detection to allow the measurement of accumulated amplified product as the reaction progresses. qPCR requires normalization to controls (either to a reference or to a standard curve), allowing only relative quantification. Furthermore, variations in amplification efficiency may affect qPCR results. Digital PCR builds on traditional PCR amplification and fluorescent-probe–based detection methods to enable highly sensitive absolute quantification of nucleic acids without the need for standard curves. This section provides an overview of digital PCR technology and methods and its application.


What is Digital PCR?

The concept of digital PCR was first described in 1992 by Sykes et al., who recognized that the combination of limiting dilution, end-point PCR, and Poisson statistics could yield an absolute measure of nucleic acid concentration (Sykes et al. 1992). Subsequently, Vogelstein and Kinzler at Johns Hopkins University developed a method whereby a sample can be diluted and partitioned to the extent that single template molecules can be amplified individually, each in a separate partition, and the products detected using fluorescent probes (Vogelstein and Kinzler 1999). This approach transformed qPCR into digital-format qPCR because the results are binary — either positive or negative — and thus the new term “digital PCR” was coined for this generation of PCR methods.

Digital PCR represents a third generation of PCR that enables absolute quantification of target sequences

Digital PCR represents a generation of PCR that enables absolute quantification of target sequences.

How Does Digital PCR Work?

Digital PCR improves upon the sensitivity of qPCR and enables the detection of rare events such as rare single-nucleotide mutations in a population of wild-type sequences. In conventional qPCR, the signal from wild-type sequences can dominate and obscure the signal from rare mutants. Digital PCR overcomes the difficulties inherent in amplifying rare sequences, enabling exquisitely sensitive and precise absolute quantification of nucleic acids.

A critical step in digital PCR is sample partitioning — the separation of each nucleic acid sample into discrete partitions prior to amplification by PCR. The sample is prepared in a manner similar to that for real-time PCR but is then either diluted extensively or separated into hundreds or even thousands of partitions, each ideally containing either zero or one (or, at most, a few) template molecules. Individual PCR amplification reactions are then conducted within each partition and, as with real-time PCR, fluorescent probes  are used to identify amplified target DNA, so each partition can be readily analyzed after amplification to determine whether or not it contains the target sequence. Samples containing amplified product are considered positive (1, fluorescent), and those without product, and thus with little or no fluorescence, are negative (0), the ratio of positives to negatives in each sample is the basis of quantification. Unlike real-time qPCR, digital PCR does not rely on the number of amplification cycles to determine the initial amount of template nucleic acid in each sample; rather, it relies on Poisson statistical analysis to determine the absolute template quantity.

The unique sample partitioning step of digital PCR, paired with Poisson statistical data analysis, allows higher precision than traditional PCR and qPCR methods. Accordingly, digital PCR is particularly well suited for applications that require the detection of small amounts of input nucleic acid or finer resolution of target amounts among samples, for example, rare sequence detection, copy number variation (CNV) analysis, and gene expression analysis of the rare targets.

Sample partitioning is the key to digital PCR

Sample partitioning is the key to digital PCR. Template molecules are distributed randomly among partitions, such that some partitions have no template molecules and others have one or more. Each partition undergoes PCR amplification and analysis separately, and the partitions with and without amplified product are individually counted.

Digital PCR Methods

The methods described by Sykes et al. (1992) and Vogelstein and Kinzler (1999) have been modified and made commercially available in different formats. For example, microfluidics can be used to partition one nucleic acid sample into hundreds or even thousands of individual chambers for subsequent PCR amplification on a microfluidic chip (Warren et al. 2006, Ottesen et al. 2006, Fan and Stephen 2007). Other systems involve separation onto microarrays (Morrison et al. 2006) or spinning microfluidic discs (Sundberg et al. 2010) and droplet techniques based on oil-water emulsions (Hindson et al., 2011). These systems vary in the volume and number of samples required and partitions generated. Therefore, the different methods offer different resolution, precision, and cost per sample.

Bio-Rad’s Droplet Digital™ PCR (ddPCR™) system combines water-oil emulsion droplet technology with microfluidics to partition a standard 20 µl real-time PCR reaction mixture into thousands of nanoliter-sized droplets. This technique requires smaller sample volumes than other commercially available digital PCR systems, helping to reduce experimental costs and preserve precious samples, all while offering high sensitivity and precise quantification.

Emerging Applications of Digital PCR

Sample partitioning allows the sensitive, specific detection of single template molecules and precise quantification while mitigating the effects of target competition, making PCR amplification less sensitive to inhibition and greatly improving the discriminatory capacity of assays that differ by only a single nucleotide. Digital PCR offers the benefits over other PCR methods of absolute quantification and greatly enhanced sensitivity and dynamic range. Therefore, applications for digital PCR have been increasing in the following areas:

  • Copy number variation (CNV) CNVs result in too few or too many dosage-sensitive genes responsible for phenotypic variability, complex behavioral traits, and disease. Digital PCR enables the measurement of 1.2-fold (±10%) differences in gene copy numbers
  • Rare sequence detection — rare event detection requires the amplification of single genes in a complex sample, for example, a few tumor cells in a wild-type background. Digital PCR is sensitive enough to detect rare mutations or sequences present at frequencies as low as 1 in 100,000
  • Gene expression and miRNA analysis — digital PCR provides absolute quantification of expression levels, especially low-abundance miRNAs, with sensitivity and precision
  • Single cell analysis — the high degree (10–100-fold) of cell-cell variation in gene expression levels among homogeneous single-cell populations and stem cells drives the need for the analysis of single cells; digital PCR enables low copy number target quantification
  • Pathogen detection — pathogens carry unique genomic signatures, but they may be present in very small amounts in biological samples; digital PCR enables the high-precision detection and quantification of pathogens in samples quickly and precisely
  • Next-generation sequencing (NGS) — digital PCR allows the quantification of NGS sample preparations to increase sequencing accuracy and reduce run repeats. ddPCR also enables the validation of sequencing results with absolute quantification

Citations

Fan HC and Quake SR (2007). Detection of aneuploidy with digital polymerase chain reaction. Anal Chem 79, 7576–7579. PMID: 17715994

Hindson BJ et al. (2011). High-throughput droplet digital PCR system for absolute quantitation of DNA copy number. Anal Chem 83, 8604–8610. PMID: 22035192

Morrison T et al. (2006). Nanoliter high-throughput quantitative PCR. Nucleic Acids Res 34, e123. PMID: 17000636

Ottesen EA et al. (2006). Microfluidic digital PCR enables multigene analysis of individual environmental bacteria. Science 314, 1464–1467. PMID: 17138901

Pohl G and Shih IeM (2004). Principle and applications of digital PCR. Expert Mol Rev Diagn 4, 41–47. PMID: 14711348

Sundberg SO et al. (2010). Spinning disk platform for microfluidic digital polymerase chain reaction. Anal Chem 82, 1546–1550. PMID: 20085301

Sykes PJ et al. (1992). Quantitation of targets for PCR by use of limiting dilution. BioTechniques 13, 444–449. PMID: 1389177

Vogelstein B and Kinzler KW (1999). Digital PCR. Proc Nat Acd Sci USA 96, 9236–9241. PMID: 10430926

Warren L et al. (2008). Transcription factor profiling in individual hematopoietic progenitors by digital RT-PCR. Proc Natl Acad Sci USA 103, 17807–17812. PMID: 17098862

Further Reading

Baker M (2012). Digital PCR hits its stride. Nat Methods 9, 541–544.

Huggett J and Scott D (2010). Digital polymerase chain reaction: New diagnostic opportunities. European Pharmaceutical Review, Industry Focus 2010.

Kubista M (2008). Emerging real-time PCR applications. Drug Discovery World Summer 2008, 57–66.

Kubista M and Stahlberg A (2011). DNA diagnostics gets digitized. Drug Discovery World Fall 2011, 77–82.

McCaughan P and Dear PH (2010). Single-molecule genomics. J Pathol 220, 297–306. PMID: 19927313

Related Content

Literature
Number Description Download
6277 Probing Copy Number Variations Using Bio-Rad's QX100 Droplet Digital PCR System, Rev A Click to download
6451 Droplet Digital PCR: Guidelines for Multiplexing Using Bio-Rad’s QX100 Droplet Digital PCR System Application Note, Rev A Click to download
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