Digital PCR (dPCR) is an advanced approach for the absolute quantification of specific DNA and RNA sequences by using partitioned amplification reactions. This method is an adaptation of traditional PCR techniques, offering improved precision, sensitivity, and accuracy. Some advantages of dPCR over other quantification methods include independence from standard curves, higher tolerance to PCR inhibitors, and the capability to detect low-abundance targets. This technology has proven useful for applications such as trace DNA detection, minimal residual disease (MRD) testing, and copy number variation analysis.
Copy number variations (CNVs), also referred to as copy number alterations (CNAs), are repeat sequences within the genome that are duplicated or deleted. These variations range in size from 50 bp to several megabases and can encompass one or more genes and non-coding regions.1 The number of repeats varies between individuals, and these changes are often associated with various health conditions, such as cancer, autoimmune diseases, and neurodevelopmental disorders.2,3 The ability to detect even the smallest changes in CNVs is important for identifying genetic risk factors and developing effective treatments. Due to its heightened sensitivity and precision, dPCR is well-suited for CNV/CNA detection, offering advantages in providing reliable copy number estimates and identifying variations as low as 10%.
In comparison to traditional quantitative PCR (qPCR), dPCR offers several advantages, including true quantification, higher precision over a broad dynamic range, and superior sensitivity. This increased sensitivity is particularly beneficial for detecting small changes in copy number. Unlike qPCR, dPCR does not require the use of a standard curve, which increases accuracy and decreases the number of reactions needed. Moreover, dPCR demonstrates robust performance with challenging samples, such as those with low concentrations or degraded nucleic acids.
Detecting CNVs with dPCR enables highly accurate quantification of DNA sequences. The original dPCR platforms use oil emulsification to form droplets and partition the reactions, while the newer, nanowell-plate-based dPCR systems physically separate the mixtures into tiny wells in nanowell plates, further enhancing detection capabilities by providing equal and consistent partitioning.
To measure CNVs of a specific region using dPCR, two targets are measured: the gene of interest (e.g., HER2) and a reference gene with a known, stable copy number (e.g., GAPDH). Once the PCR mixture (containing the target nucleic acid, primers, buffer, and probes) is prepared, the samples are partitioned into thousands of microreactions, allowing each partition to undergo individual endpoint PCR amplification. During the amplification process, each target is tagged with a distinct fluorophore, enabling simultaneous detection. Then, following amplification, the nanowells are imaged in multiple fluorescent channels to measure the presence or absence of amplification products. Using Poisson statistics, the total number of molecules present in the original sample is estimated by analyzing the positive and negative reaction counts for each target. A ratio between the gene of interest and the reference gene copy number is then used to determine the gene's CNV.
Another advantage of dPCR is its ability to simultaneously detect multiple targets within each partition. This capability, combined with the potential to include several reference genes in one reaction, eliminates the need for setting up additional reactions and allows for efficient multiplexing. Multiplexing minimizes the amount of sample needed for testing, which is beneficial when dealing with limited or precious samples. Additionally, the consolidation of multiple tests into a single reaction shortens turnaround times, increases throughput, and decreases technical errors, such as pipetting inaccuracies. This reduction in errors and sample usage also contributes to lower costs.
High-performance multiplexing is especially advantageous for CNV assays. This technique often requires the detection of multiple genomic regions, along with a no-template control. A dPCR system equipped with multiplexing allows for precise measurement of target and reference gene ratios within the same reaction. This simultaneous assessment improves the accuracy and specificity of CNV detection, enabling the identification of gene amplifications, deletions, and other variations essential for disease research.
dPCR is an advanced method for the absolute quantification of DNA and RNA, ideal for detecting low-abundance targets and CNVs. Its heightened sensitivity, precision, and independence from standard curves make it a superior alternative to traditional PCR methods. In addition, the robust multiplexing capabilities of dPCR allow for the simultaneous analysis of multiple targets while reducing errors and costs and increasing throughput. This powerful tool continues to offer key insights into genetic risk factors and advance the development of effective treatments.
The Roche Digital LightCycler® dPCR System is a flexible instrument featuring three nanowell plate configurations (high resolution, high sensitivity, and a universal plate), six advanced optical channels, and 5x concentrated DNA and RNA master mixes. This comprehensive design enhances the sensitivity and precision required for CNV analysis, ensuring reliable detection and quantification.
1. Pös O, Radvanszky J, Buglyó G, et al. DNA copy number variation: Main characteristics, evolutionary significance, and pathological aspects. Biomed J. 2021;44(5):548-559. doi:10.1016/j.bj.2021.02.003
2. Girirajan S, Campbell CD, Eichler EE. Human copy number variation and complex genetic disease. Annu Rev Genet. 2011;45:203-226. doi:10.1146/annurev-genet-102209-163544
3. Steele CD, Abbasi A, Islam SMA, et al. Signatures of copy number alterations in human cancer. Nature. 2022;606(7916):984-991. doi:10.1038/s41586-022-04738-6