Droplet Digital PCR (ddPCR)
Our ddPCR service provides absolute quantification of target DNA or RNA molecules for probe-based digital PCR applications.
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Polymerase Chain Reaction (PCR) allows the in vitro amplification of DNA fragments starting with tiny amounts of biological sample and oligonucleotide primers derived from sequence data.
Subsequently, PCR has given researchers the ability to detect and identify mutations for inherited diseases as well as study gene expression and detect the presence of pathogens in samples. While most researchers are familiar with real-time quantitative PCR (qPCR), droplet digital PCR (ddPCR) is much more sensitive, precise, reproducible, and overcomes some of the drawbacks to the commonly used qPCR assays.
What is droplet digital PCR (ddPCR)? How does it work?
ddPCR is an offshoot to digital PCR, which is based on a water-oil emulsion system. The main principle behind ddPCR involves massive partitioning of the target sample – dividing a 20 µl sample into 20,000 nanoliter-sized droplets. This partitioning takes place inside a microfluidic device, whereby droplets are formed in a water-oil emulsion to form the partitions that separate the template DNA molecules. After partitioning is complete, PCR amplification of the template molecules occurs in each droplet (using TaqMan probe-based assays). After the thermocycler has completed each run, droplets from each sample are streamed in a file on a Droplet Reader to count positive and negative reactions, providing absolute quantification in digital form.
Since each sample droplet may contain multiple target molecules, instead of performing one PCR analysis on a single sample, every droplet becomes a PCR sample of its own. After amplification, the droplets are measured to see which ones contain the target sequence (positive droplets) and which do not (negative droplets). Using a Poisson distribution, the fraction of positive droplets helps determine the concentration of the template in the original sample.
ddPCR vs qPCR
Although qPCR is the current gold standard method for detecting mRNA expression changes, genotypes, or viral loads, ddPCR can offer superior sensitivity and reproducibility especially for low abundant targets, compared to qPCR. ddPCR has also been shown to perform better than qPCR in monitoring patients’ disease progression by reducing false negative rates.
Furthermore, when employing qPCR, the intensity of fluorescence reaching a plateau either early or late in the amplification process, depending on the number of target molecules in the sample, can lead to inaccuracies. This is because fluorescence levels are typically measured at the end of every amplification cycle. The value derived from these measurements is called cycle threshold (CT), and it serves to calculate the relative amount of the initial DNA concentration, compared to standards. In ddPCR, no extrapolation, standard curves, or references are required. ddPCR gives absolute quantification at the end of the amplification when the experiment is completed. Therefore, ddPCR is considered a “digital” measurement. Because each PCR reaction occurs in a diluted sample, ddPCR isn’t as sensitive to the presence of PCR inhibitors or primer-template mismatch as qPCR.
Additionally, ddPCR is more precise than qPCR. This is because qPCR can distinguish between copy number variations or differences in gene expression that are two-fold or smaller. Whereas ddPCR can detect differences in gene expression of less than 30% and can also identify alleles that are less frequent than 0.1% and distinguishes between copy number variations that are different by only one copy.
Advantages of ddPCR
ddPCR has a smaller sample volume requirement (reaction volumes are in the pico to nanoliter ranges in ddPCR), reducing cost and preserving precious samples.
Unlike other PCR methods, ddPCR does not involve serial dilution of the sample and therefore isn’t reliant on standard curves and extrapolations it is ideal for microbial quantification, viral load testing, and measuring DNA concentrations. It provides an absolute count of target nucleic acid copies per sample volume, most commonly copies per µl.
Increased accuracy and powerful statistical analysis
ddPCR massively partitions the sample in the fluid phase in one step, thus generating tens of thousands of data points from one sample and increasing the power of statistical analysis.
ddPCR has a huge advantage over qPCR as it can precisely measure small fold differences in target sequence copies between samples, which is crucial in genetic testing.
No more PCR bias
Regardless of the sample’s origin, there is a dramatic reduction in error rates from ddPCR samples compared to qPCR and other traditional PCR methods
Improved signal-to-noise ratio
Higher sensitivity afforded by ddPCR allows researchers to more accurately detect rare targets and typically boasts precision of ±10% . These targets would typically have a low frequency in a wild-type background.
Sampled can accept samples from a variety of sample sources, such as blood, saliva, or FFPE tissues. Our experts work with BioRad’s QX200 Droplet Reader to analyze droplets using a two-color detection system (set to detect FAM and either HEX or VIC), enabling multiplexed analysis for different targets in the same sample. The droplet reader and its bundled QX Manager™ software count the PCR-positive and PCR-negative droplets.
This allows researchers to use our ddPCR capabilities for the development of:
- Quantitative assays and reference standards
- Environmental monitoring
- Mutation detection
- Clinical diagnosis of viral loads (such as SARS-CoV-2)
Speak to an expert today
We pride ourselves on being able to provide ddPCR capabilities to customers from all backgrounds with a wide range of requirements from pharma and biotechs to academic groups. If you would like to learn more about how ddPCR could help accelerate your research, speak to one of our experts today.