A Candidate Reference Analytical Method for Quantification of Low Concentrations of Plasmid DNA by Exhaustive Counting of Single DNA Molecules in a Flow Stream

Published

August 4, 2014

Author(s)

Hee-Bong Yoo, Dong-Geun Oh, Jae Yong Song, Mamoru Kawaharasaki, Jeeseong C. Hwang, In Chul Yang, Sang-Ryoul Park

Abstract

This work demonstrates accurate measurement of the concentration of low-level plasmid DNA by counting individual DNA molecules using a high-sensitivity flow cytometric setup. Plasmid DNA is a widely used form of DNA, and its quantity often needs to be accurately determined. This work establishes a reference analytical method for direct quantification of low-level plasmid DNA molecules prepared as reference standards for polymerase chain reaction-based DNA quantification. The model plasmid DNA pBR322 (4.58 kbp) was stained with a fluorescent dye and was detected on a flow stream in a micro-fluidic channel with laser-induced fluorescence detection, for which the DNA flow was electro-hydrodynamically focused at the center of the channel. 200-8,000 DNA particles in a 1 μL sample volume were counted within 2 minutes in an 'exhaustive counting' manner, which facilitated quantitation without calibration. The sample volume was measured and validated from the close agreement of the results of two independent measurement methods. Within the given concentration range, an excellent measurement linearity (R = 0.999) was achieved with appropriate data processing for simultaneous exposures of DNA to the detector. The validity of the proposed method was confirmed from the close agreement with the results of quantitation of enzymatically released nucleotides using capillary electrophoresis.

Citation

Metrologia

Volume

51

Pub Type

Journals

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DOI Link

Keywords

single molecule, flow cytometry, plasmid DNA, fluorescence detection

Citation

Yoo, H. , Oh, D. , Song, J. , Kawaharasaki, M. , Hwang, J. , Yang, I. and Park, S. (2014), A Candidate Reference Analytical Method for Quantification of Low Concentrations of Plasmid DNA by Exhaustive Counting of Single DNA Molecules in a Flow Stream, Metrologia, [online], https://doi.org/10.1088/0026-1394/51/5/491 (Accessed October 8, 2025)