Comparison of Methods for Accurate Quantification of DNA Mass Concentration with Traceability to the International System of Units

Bhat, Somanath; Curach, Natalie C.; Mostyn, Thomas; Bains, G.S.; Griffiths, Kate; Emslie, Kerry R.

doi:10.1021/ac100845m

Cited by 92 publications

(95 citation statements)

References 15 publications

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“…Digital PCR (dPCR) is a relatively new technology, requiring no external calibrators, for measuring the absolute and relative copy numbers of target DNA [14,15]. Digital PCR (dPCR) transforms the exponential, analogue signal of classic PCR into a linear, digital signal, retaining the single-molecule sensitivity of PCR.…”

Section: Introductionmentioning

confidence: 99%

Evaluation of droplet digital PCR for characterizing plasmid reference material used for quantifying ammonia oxidizers and denitrifiers

Meng

Wang

et al. 2014

Anal Bioanal Chem

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DNA reference materials of certified value have a critical function in many analytical processes of DNA measurement. Quantification of amoA genes in ammonia oxidizing bacteria (AOB) and archaea (AOA), and of nirS and nosZ genes in the denitrifiers is very important for determining their distribution and abundance in the natural environment. A plasmid reference material containing nirS, nosZ, amoA-AOB, and amoA-AOA is developed to provide a DNA standard with copy number concentration for ensuring comparability and reliability of quantification of these genes. Droplet digital PCR (ddPCR) was evaluated for characterization of the plasmid reference material. The result revealed that restriction endonuclease digestion of plasmids can improve amplification efficiency and minimize the measurement bias of ddPCR. Compared with the conformation of the plasmid, the size of the DNA fragment containing the target sequence and the location of the restriction site relative to the target sequence are not significant factors affecting plasmid quantification by ddPCR. Liquid chromatography-isotope dilution mass spectrometry (LC-IDMS) was used to provide independent data for quantifying the plasmid reference material. The copy number concentration of the digested plasmid determined by ddPCR agreed well with that determined by LC-IDMS, improving both the accuracy and reliability of the plasmid reference material. The reference value, with its expanded uncertainty (k=2), of the plasmid reference material was determined to be (5.19±0.41)×10 9 copies μL −1 by averaging the results of two independent measurements. Consideration of the factors revealed in this study can improve the reliability and accuracy of ddPCR; thus, this method has the potential to accurately quantify DNA reference materials.

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Section: Introductionmentioning

confidence: 99%

Evaluation of droplet digital PCR for characterizing plasmid reference material used for quantifying ammonia oxidizers and denitrifiers

Meng

Wang

et al. 2014

Anal Bioanal Chem

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“…Furthermore, fragmentation of larger, complex gDNA may also improve associated measurement (40 ). When dPCR is used, there is a unique challenge associated with quantifying sequences that may occur as concatamers (repeats of the same sequence that are present on the same molecule); concatameric sequences cannot be measured individually in different partitions.…”

Section: Components Of Dpcr Dna Targetmentioning

confidence: 99%

The Digital MIQE Guidelines: Minimum Information for Publication of Quantitative Digital PCR Experiments

Huggett¹,

Foy²,

Beneš³

et al. 2013

Clinical Chemistry

762

660

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There is growing interest in digital PCR (dPCR) because technological progress makes it a practical and increasingly affordable technology. dPCR allows the precise quantification of nucleic acids, facilitating the measurement of small percentage differences and quantification of rare variants. dPCR may also be more reproducible and less susceptible to inhibition than quantitative real-time PCR (qPCR). Consequently, dPCR has the potential to have a substantial impact on research as well as diagnostic applications. However, as with qPCR, the ability to perform robust meaningful experiments requires careful design and adequate controls. To assist independent evaluation of experimental data, comprehensive disclosure of all relevant experimental details is required. To facilitate this process we present the Minimum Information for Publication of Quantitative Digital PCR Experiments guidelines. This report addresses known requirements for dPCR that have already been identified during this early stage of its development and commercial implementation. Adoption of these guidelines by the scientific community will help to standardize experimental protocols, maximize efficient utilization of resources, and enhance the impact of this promising new technology.

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“…Comparison of spectrophotometric methods for DNA measurements with dPCR has generally shown good agreement, differing by Ͻ2-fold (23,34,35 ). To begin with this is very promising because when molecular quantification is routinely applied in the clinic, decisions are seldom made on changes of Ͻ10-fold.…”

Section: Absolute Quantificationmentioning

confidence: 99%

Considerations for Digital PCR as an Accurate Molecular Diagnostic Tool

2015

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BACKGROUND Digital PCR (dPCR) is an increasingly popular manifestation of PCR that offers a number of unique advantages when applied to preclinical research, particularly when used to detect rare mutations and in the precise quantification of nucleic acids. As is common with many new research methods, the application of dPCR to potential clinical scenarios is also being increasingly described. CONTENT This review addresses some of the factors that need to be considered in the application of dPCR. Compared to real-time quantitative PCR (qPCR), dPCR clearly has the potential to offer more sensitive and considerably more reproducible clinical methods that could lend themselves to diagnostic, prognostic, and predictive tests. But for this to be realized the technology will need to be further developed to reduce cost and simplify application. Concomitantly the preclinical research will need be reported with a comprehensive understanding of the associated errors. dPCR benefits from a far more predictable variance than qPCR but is as susceptible to upstream errors associated with factors like sampling and extraction. dPCR can also suffer systematic bias, particularly leading to underestimation, and internal positive controls are likely to be as important for dPCR as they are for qPCR, especially when reporting the absence of a sequence. SUMMARY In this review we highlight some of the considerations that may be needed when applying dPCR and discuss sources of error. The factors discussed here aim to assist in the translation of dPCR to diagnostic, predictive, or prognostic applications.

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Comparison of Methods for Accurate Quantification of DNA Mass Concentration with Traceability to the International System of Units

Cited by 92 publications

References 15 publications

Evaluation of droplet digital PCR for characterizing plasmid reference material used for quantifying ammonia oxidizers and denitrifiers

Evaluation of droplet digital PCR for characterizing plasmid reference material used for quantifying ammonia oxidizers and denitrifiers

The Digital MIQE Guidelines: Minimum Information for Publication of Quantitative Digital PCR Experiments

Considerations for Digital PCR as an Accurate Molecular Diagnostic Tool

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