Article

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

LGC Limited, Queens Road, Teddington, Middlesex TW11 0LY, UK.
Nucleic Acids Research (Impact Factor: 9.11). 02/2012; 40(11):e82. DOI: 10.1093/nar/gks203
Source: PubMed

ABSTRACT

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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    ABSTRACT: The purpose of this study was to develop a PCR-based method for quantification of Listeria monocytogenes adhesion in microtitre plates. We optimized isolation of DNA in the microtitre plates using cell lysis, ultrasound treatment, heating, and centrifugation of the lysate. Digital PCR was applied for quantification of L. monocytogenes DNA that was used for construction of the standard curve, and real-time PCR was used for quantification of the attached L. monocytogenes cells. This PCR-based method was applied to quantify different strains of L. monocytogenes at different times of biofilm formation, and to study the anti-adhesive actions of natural bioactive substances (epigallocatechin gallate, (−)-α-pinene). The results show that the PCR-based method developed here can be widely used as a novel approach for adhesion assays and biofilm research.
    No preview · Article · Nov 2015
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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). "
    [Show abstract] [Hide abstract]
    ABSTRACT: The purpose of this study was to develop a PCR-based method for quantification of Listeria monocytogenes adhesion in microtitre plates. We optimized isolation of DNA in the microtitre plates using cell lysis, ultrasound treatment, heating, and centrifugation of the lysate. Digital PCR was applied for quantification of L. monocytogenes DNA that was used for construction of the standard curve, and real-time PCR was used for quantification of the attached L. monocytogenes cells. This PCR-based method was applied to quantify different strains of L. monocytogenes at different times of biofilm formation, and to study the anti-adhesive actions of natural bioactive substances (epigallocatechin gallate, (-)-α-pinene). The results show that the PCR-based method developed here can be widely used as a novel approach for adhesion assays and biofilm research. Copyright © 2015. Published by Elsevier B.V.
    Full-text · Article · Aug 2015 · Journal of microbiological methods
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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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    ABSTRACT: Soybean cyst nematode (SCN, Heterodera glycines Ichinohe) is a serious soybean pest. The use of resistant cultivars is an effective approach for preventing yield loss. In this study, 19,652 publicly available soybean accessions that were previously genotyped with the SoySNP50K iSelect BeadChip were used to evaluate the phylogenetic diversity of SCN resistance genes Rhg1 and Rhg4 in an attempt to identify novel sources of resistance. The sequence information of soybean lines was utilized to develop KASPar (KBioscience Competitive Allele-Specific PCR) assays from single nucleotide polymorphisms (SNPs) of Rhg1, Rhg4, and other novel quantitative trait loci (QTL). These markers were used to genotype a diverse set of 95 soybean germplasm lines and three recombinant inbred line (RIL) populations. SNP markers from the Rhg1 gene were able to differentiate copy number variation (CNV), such as resistant-high copy (PI 88788-type), low copy (Peking-type), and susceptible-single copy (Williams 82) numbers. Similarly, markers for the Rhg4 gene were able to detect Peking-type (resistance) genotypes. The phylogenetic information of SCN resistance loci from a large set of soybean accessions and the gene/QTL specific markers that were developed in this study will accelerate SCN resistance breeding programs.
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