title: Accurate quantification of supercoiled DNA by digital PCR.
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.
authors.affiliation: National Institute of Metrology, Beijing, 100013, P.R. China.
authors.forename: Lianhua
authors.initials: L
authors.lastname: Dong
authors.affiliation: Korea Research Institute of Standards and Science, Daejeon, Korea.|University of Science and Technology, Daejeon, Korea.
authors.forename: Hee-Bong
authors.initials: HB
authors.lastname: Yoo
authors.affiliation: National Institute of Metrology, Beijing, 100013, P.R. China.
authors.forename: Jing
authors.initials: J
authors.lastname: Wang
authors.affiliation: Korea Research Institute of Standards and Science, Daejeon, Korea.|University of Science and Technology, Daejeon, Korea.
authors.forename: Sang-Ryoul
authors.initials: SR
authors.lastname: Park
mesh_terms: D017931:DNA Primers / Q000737:chemistry / Q000378:metabolism; D004278:DNA, Superhelical / Q000032:analysis*; D005456:Fluorescent Dyes / Q000737:chemistry; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction
chemical_list: D017931:DNA Primers; D004278:DNA, Superhelical; D005456:Fluorescent Dyes