Accurate quantification of supercoiled DNA by digital PCR.

Area of Science:

• Molecular Biology
• Metrology

Background:

• Digital PCR (dPCR) quantifies DNA copy number without standards but can yield false results if PCR conditions are suboptimal.
• International comparisons reveal significant underestimation of supercoiled plasmid DNA concentration by dPCR, often necessitating linearization before analysis.

Purpose of the Study:

• To develop an optimized digital PCR (dPCR) assay for accurate quantification of supercoiled DNA without preanalytical linearization.
• To address the underestimation issue observed in previous international dPCR comparisons.

Main Methods:

• Bilateral comparison between the National Institute of Metrology (NIM) and Korea Research Institute of Standards and Science (KRISS).
• NIM optimized dPCR conditions, evaluating PCR master mix, fluorescent labels (HEX), and primer positions for supercoiled DNA.
• KRISS prepared unknown samples and performed quantification using flow cytometry.

Main Results:

• A 16S PCR master mix was identified to prevent poor amplification of supercoiled DNA.
• HEX labels on dPCR probes demonstrated robust amplification curves.
• Optimized dPCR assay accurately quantified supercoiled DNA without linearization, achieving results within 101-113% agreement with flow cytometry.

Conclusions:

• Optimized dPCR protocols, utilizing specific master mixes and fluorescent labels, enable accurate quantification of supercoiled DNA.
• This advancement eliminates the need for preanalytical linearization, simplifying the dPCR workflow for supercoiled DNA analysis.
• The validated dPCR method provides reliable results comparable to established techniques like flow cytometry.