What is Copy Number Variation and how can I use digital PCR to measure CNV?

What are Copy Number Variations (CNVs)

Natural variability of the human genome can range from single nucleotide polymorphisms (SNPs) to larger-scale events such as insertions or deletions of genomic regions. Copy number variation (CNV) refers to the structural alteration of the genome compared to a reference and can affect areas anywhere between 1,000 and 5 megabase pairs in length.1 Often, CNVs can involve many genes and can have many implications in human health and disease.

Dose-Response is relevant to many CNVs

One challenge associated with studying CNVs is that they are not binary. In addition to gene deletions, CNVs can occur from duplication of genomic regions – and the total number of duplicated copies can vary. Since the copy number of a gene can impact the expression of downstream genes, the risk factor for certain diseases could be affected by the number of copies present for a gene of interest. To fully understand these impacts, the precise quantification of copy numbers is crucial. 

Spinal muscular atrophy (SMA), for example, is the leading genetic cause of infant mortality. The loss of the SMN1 gene primarily causes the disease. A duplicate gene – SMN2 – has been found to produce partial function. The presence of SMN2 can partially compensate for the loss of SMN1 and reduce disease severity.

What is Digital PCR?

Digital PCR (dPCR) is fundamentally simple. The digital PCR instrument splits a bulk PCR reaction into many thousands of tiny micro reactions or partitions. The template DNA in the PCR reaction is spread across these partitions alongside the primers, probes, enzymes, and buffer required for PCR. 

The “digital” concept behind dPCR is that each partition will contain only one target DNA molecule of interest. After thermal cycling, all partitions are scanned. Those that generate a fluorescent signal are counted, providing an absolute quantity of the starting template material. 

Furthermore, digital PCR systems equipped with multiple optical channels enable researchers to quantify many targets in a single assay. This means that the several ROIs can be interrogated alongside a reference control in a single digital PCR reaction. 

How can Digital PCR be used to measure CNVs

Absolute quantification can be extremely useful for measuring copy number variation. By measuring the exact number of molecules present in a particular region of interest (ROI) and comparing those values to the number of molecules for a reference, researchers can calculate the precise copy number with higher accuracy. 

Digital PCR, which utilizes simple counting of target molecules to achieve absolute quantification, enables higher precision and accuracy to detect and quantify copy number variations – even if the changes are minor. The Combinati Absolute Q provides four optical channels for multiplexing, enabling more genes to be interrogated per reaction – requiring fewer samples when compared to qPCR. Early identification of copy number profiles related to genetic conditions can facilitate timely interventions that could lead to better patient outcomes.

In this publication, dPCR was used to develop a 4-color assay to genotype and quantify CNVs of clinical SMA samples.


Advances in digital PCR technologies are paving the way to simplify the detection of CNVs that cannot be detected using molecular techniques. Digital PCR enables simultaneous measurement of multiple targets from a single sample alongside a simple workflow and rapid turnaround time. With overall higher accuracy and precision, dPCR is poised to become a leading technique in measuring clinically relevant CNVs.

Interested in learning more about more applications of digital PCR? Visit our applications page to explore a variety of research areas and applications which utilize absolute quantification for higher precision and accuracy.

1.  Eichler, E. E. (2008) Copy Number Variation and Human Disease. Nature Education 1(3):1

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