Comparison of microfluidic digital PCR and conventional quantitative PCR for measuring copy number variation

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Nucleic Acids Research (Impact Factor: 9.11). 02/2012; 40(11):e82. DOI: 10.1093/nar/gks203
Source: PubMed


One of the benefits of Digital PCR (dPCR) is the potential for unparalleled precision enabling smaller fold change measurements. An example of an assessment that could benefit from such improved precision is the measurement of tumour-associated copy number variation (CNV) in the cell free DNA (cfDNA) fraction of patient blood plasma. To investigate the potential precision of dPCR and compare it with the established technique of quantitative PCR (qPCR), we used breast cancer cell lines to investigate HER2 gene amplification and modelled a range of different CNVs. We showed that, with equal experimental replication, dPCR could measure a smaller CNV than qPCR. As dPCR precision is directly dependent upon both the number of replicate measurements and the template concentration, we also developed a method to assist the design of dPCR experiments for measuring CNV. Using an existing model (based on Poisson and binomial distributions) to derive an expression for the variance inherent in dPCR, we produced a power calculation to define the experimental size required to reliably detect a given fold change at a given template concentration. This work will facilitate any future translation of dPCR to key diagnostic applications, such as cancer diagnostics and analysis of cfDNA.

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    • "Digital PCR has many advantages in the view of its precision, lack of susceptibility to inhibitors, and reaction optimisation (Whale et al., 2012). Digital PCR also extends the use of advanced applications, such as: high-precision copy number variation and rare mutation analysis (Whale et al., 2012); absolute quantification of bacterial and viral loads, especially of low-level pathogens that cause human illness (Rothrock et al., 2013); and reference and standard quantification, and genetically modified organism detection (Morisset et al., 2013). Numerous studies in recent years have led to accurate interpretations of the results obtained by dPCR (Huggett et al., 2013). "
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    • "Cook et al. [2] showed that the soybean cultivar Fayette, that was developed from the Rhg1 resistant source PI 88788 has ten copies of the Rhg1 gene compared to nine copies in PI 88788. Recent medical science reports highlighted how CNV can be enumerated using digital polymerase chain reaction (dPCR) [22] [23]. The determination of CNV with dPCR is costly and time-consuming and is not suitable for plant breeding applications; however, it is feasible to identify a haplotype that represents a particular CNV that can then be utilized for marker development. "

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    • "reliance on rate-based measurements (C T values) and the need for the use of calibration curves (Hindson et al., 2011; Pinheiro et al., 2012). (2) It demonstrates high sensitivity and precision for lowcopy-number target nucleic acids (Diehl and Diaz, 2007; Hayden et al., 2013; Hindson et al., 2011; Sanders et al., 2011; Whale et al., 2012). In recent years, many studies (Jahn et al., 2014; McDermott et al., 2013; Nathan et al., 2014; Pinheiro et al., 2012) involving nucleic acid quantification have been performed using a commercial ddPCR system such as the Bio-rad QX100/200 (Sykes et al., 1992; Vogelstein and Kinzler, 1999). "
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