Copy number variations (CNVs) are involved in a large number of complex human diseases including many cancers and genetic diseases. An important measurement challenge in translational and diagnostic research involves identification of small CNV changes with high confidence. Cell free DNA (cfDNA) in blood plasma promises a readily available source of genetic material for tumor or pre-natal diagnosis with only minimally invasive sampling techniques. However, since only a proportion of the cfDNA is derived from the tumor or embryo, quantification of an associated CNV becomes even more challenging because the target DNA is heavily diluted by the background of normal DNA. For example, a tumor-associated 5-fold increase in CNV becomes a 1.2-fold increase if only 5% of the cfDNA sample is derived from the tumor1. This magnitude of CNV would be undetectable by current approaches such as quantitative PCR (qPCR), which is limited to resolving ~1.5-fold changes 2,3.
Early efforts with digital PCR have reported the ability to detect a 1.25-fold difference in copy number4. Additionally, the binary nature of dPCR means that the precision is more independent of variation in assay amplification, making it easier to optimize and standardize between laboratories.
The ability of dPCR to measure small CNVs is directly dependent upon template concentration. This is a crucial factor when using cfDNA as a template as its concentration can range considerably. Whale1 (Figure 1) demonstrates dPCR techniques that are limited to hundreds or even thousands of reaction chambers (wells or droplets) will be unable to discriminate small copy number difference when template concentrations are low. Only droplet dPCR approaches with millions of reactions, like the RainDrop System, will provide the level of precision required and deliver the clinical benefits of measuring smaller CNVs differences in more challenging clinical samples like cfDNA in a cost effective manner.
Figure 1
Figure 1 – Illustrates the number of PCR reaction chambers required to detect a specified CNV (two tailed, 95% power). Higher numbers of chambers are required to detect small changes, particularly as the template concentration decreases.
References
1. Whale, A.S. et al. Nucleic Acids Res. Published online, doi:10.1093/nar/gks203 (28 February 2012).
2. Chiu RW, Murphy MF, Fidler C, Zee BC, Wainscoat JS, Lo YM, Determination of RhD zygosity: comparison of a double amplification refractory mutation system approach and a multiplex real-time quantitative PCR approach. Clin. Chem. 2001;47:667-672.
3. Zimmermann B, Holzgreve W, Wenzel F, Hahn S. Novel real-time quantitative PCR test for trisomy 21. Clin. Chem. 2002;48:362-363.
