Digital PCR (dPCR) has significantly advanced molecular biology by enabling accurate and sensitive nucleic acid quantification. Its utility spans various domains, including cancer research, infectious disease diagnostics, and genetic testing. With its ability to provide precise absolute quantification and detect rare mutations, dPCR has become a crucial and reliable tool for molecular analysis.
Copy number variation
Copy number variation (CNV) analysis measures the number of sequence repeats for a specific genomic location with respect to a normal or reference genome. qPCR is an established tool for standard copy number variation analysis, with sufficient resolution for variants with 0 to 5 genomic copies. However, digital PCR provides more sensitivity when quantifying small fold copy number changes and discriminating between complex somatic copy number variations.
Rare mutation detection
Rare mutation detection aims to identify a very low fraction of mutant targets in abundant wild-type targets. Detection of rare mutations can be particularly challenging because probes for mutants usually only differ from wild type by a single base. Digital PCR overcomes the challenge of probe-sequence similarities by digitizing into micro-reactions, thereby enriching for the targets. Also, dPCR is 100 times more sensitive than conventional methods for this type of analysis, with the ability to pool samples for even higher sensitivity.
Monitoring biomarkers via liquid biopsy is a promising tool in clinical research. In clinical cancer research liquid biopsy is used to measure circulating tumor DNA (ctDNA) as an indicator of therapeutic response, residual tumor burden, and potential therapeutic resistance. With its high precision and sensitivity for quantitative detection of rare targets, digital PCR can effectively discriminate ctDNA among high levels of background DNA in liquid biopsy samples.
Cell and gene therapy research
Measure common viral titer backbones, custom genes of interest, or the presence of residual DNA without the use of a standard curve for faster, simpler, more precise quantification.
Accurately measure viral load and pathogens or other biomarkers in environmental samples such as wastewater by absolute quantification with improved performance compared to qPCR in the face of common PCR inhibitors.
NGS library quantification and verification
Digital PCR provides precise quantification of next-generation sequencing libraries to help maximize data quality and output. Because no other samples or standard curves need to be run, digital PCR removes a source of variability to help ensure accurate results.
Detect genome editing events in a fast and reliable way, especially those created using nucleases that cause double-stranded breaks, such as CRISPR-Cas9. Precise quantification of these nucleic acid editing events is crucial to determine the activity and efficiency of these enzymes in cells.
Both qPCR and dPCR use similar fluorescent probe-based technology to quantify specific nucleic acid targets. However, qPCR and dPCR use different methods to calculate quantity, and these differences can confer application-specific advantages.
Because qPCR measures target amplification against a reference, it is often used for relative quantification, such as gene expression analysis. Using dPCR, absolute quantification is possible without a standard curve.
By removing variability associated with standard curve quantification, dPCR offers greater precision for applications that require absolute quantification or detection of low-level targets.
Digital PCR is a specialized approach to nucleic acid detection and quantification that estimates absolute numbers of molecules through statistical methods. The digitization process distributes the PCR mix across thousands of microreactions so that each microreaction will effectively either contain one, zero, or just a handful of the target nucleic acid molecules. In effect, the original reaction is turned into many yes-or-no reactions. After counting the positive microreactions, simple statistics can be used to then determine the “absolute” quantity of the target molecule rather than a quantity estimated by comparing to a standard of known concentration.
As the name suggests, real-time PCR measures PCR amplification as it occurs. It focuses on the exponential phase because it provides the most precise and accurate data for quantitation. 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.
Digital PCR works by digitizing a sample into many individual real-time PCR reactions; some portion of these microreactions 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 the need for Ct values or reference to standards.
dPCR allows you to simply count the number of microchambers that have a specific or set of specific target molecules. This provides a mechanism for absolute quantification, which lets you determine the number of molecules in your sample without the need for standard curve as would be typical in a qPCR reaction. This standard curve–free absolute quantification is especially beneficial for laboratories in need of highly precise and consistent quantification methods. Since dPCR also allows you to identify handfuls of rare molecules in an overwhelming number of normal or background molecules, it is excellent for applications such as detecting rare oncogenic mutations in circulating free DNA. Multiplexing enables you to perform absolute quantification to several target molecules in parallel.
Additionally, dPCR takes an endpoint measurement in order to determine concentration and is overall less sensitive to some common inhibitors that can interfere with qPCR reaction kinetics and efficiency. This improved performance in the face of inhibitors is an important attribute for research labs working with environmental or precious samples.
dPCR is well suited to performing rare allele detection, measurement of copy number variation, viral titer measurement, quantification of next-generation sequencing libraries, and detection of rare targets from environmental samples such as wastewater. Specific applications available for absolute quantification include:
Learn how scientists are using the QuantStudio Absolute Q Digital PCR System for real-world applications. With a simple workflow that delivers consistently accurate results, researchers are using the QuantStudio Absolute Q system for applications ranging from inherited disease research to oncology research and environmental surveillance