Abstract
Digital PCR (dPCR) as an enumeration-based quantification method is capable of quantifying the DNA copy number without the help of standards. However, it can generate false results when the PCR conditions are not optimized. A recent international comparison (CCQM P154) showed that most laboratories significantly underestimated the concentration of supercoiled plasmid DNA by dPCR. Mostly, supercoiled DNAs are linearized before dPCR to avoid such underestimations. The present study was conducted to overcome this problem. In the bilateral comparison, the National Institute of Metrology, China (NIM) optimized and applied dPCR for supercoiled DNA determination, whereas Korea Research Institute of Standards and Science (KRISS) prepared the unknown samples and quantified them by flow cytometry. In this study, several factors like selection of the PCR master mix, the fluorescent label, and the position of the primers were evaluated for quantifying supercoiled DNA by dPCR. This work confirmed that a 16S PCR master mix avoided poor amplification of the supercoiled DNA, whereas HEX labels on dPCR probe resulted in robust amplification curves. Optimizing the dPCR assay based on these two observations resulted in accurate quantification of supercoiled DNA without preanalytical linearization. This result was validated in close agreement (101~113%) with the result from flow cytometry.
Highlights
Accurate DNA concentration measurement that is traceable to the International System of Units (SI) is very important for producing reproducible measurements in many applications involving DNA analysis
There was no significant difference in the Cq values between supercoiled and linearized plasmid when using DNA Free master mix (DF) (Fig. 1c)
This study demonstrates that a substantial improvement in the commonly observed underestimation of the supercoiled DNA concentration can be obtained by digital PCR (dPCR), which obviates the need for preanalytical linearization of supercoiled DNA
Summary
Accurate DNA concentration measurement that is traceable to the International System of Units (SI) is very important for producing reproducible measurements in many applications involving DNA analysis. Like many other analytical measurements, accuracy of DNA quantification requires application of high quality reference materials that are certified for its DNA concentration. The flow cytometric counting technique is unable to distinguish similar size DNA molecules with different sequences, and is only suitable for quantifying an extremely purified DNA sample or a mixture of substantially different sizes Another approach for enumeration-based quantification is digital PCR (dPCR), which can provide absolute DNA copy number concentration[10,11]. The US Food and Drug Administration (FDA) and China food and Drug Administration (CFDA) recommends that the proportion of supercoiled plasmid in the vaccines should be at least 80% and 90%, respectively[23,24] This requirement is based on an understanding that the content of supercoiled DNA is a key indicator of plasmid quality, and that the supercoiled plasmid has superior biological activity as compared to other plasmid forms[25]. Most of the laboratories significantly underestimated the concentration of the supercoiled plasmid DNA by dPCR, which suggests that some factors need to be considered when using dPCR for quantifying supercoiled DNA
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