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International Comparison of Enumeration-Based Quantification of DNA Copy-Concentration Using Flow Cytometric Counting
Hee-Bong Yoo1,2, Sang-Ryoul Park1,2, Lianhua Dong3
1Korea Research Institute of Standards and Science , Daejeon 34113, Republic of Korea.
Analytical Chemistry
|February 15, 2017
Summary
Enumeration methods like digital PCR (dPCR) offer absolute DNA quantification without calibration standards. International comparisons show dPCR and flow cytometry align well with chemical methods, validating these absolute quantification techniques.
Area of Science:
- Metrology
- Molecular Biology
- Analytical Chemistry
Background:
- Absolute quantification of DNA copy-concentration is crucial for reliable reference material assignment and SI traceability.
- Enumeration-based methods, such as digital PCR (dPCR) and flow cytometry (FCM), offer a path to absolute quantification without reliance on calibration standards.
- Accurate DNA copy counting enables traceability to the International System of Units (SI) at the single-copy level.
Purpose of the Study:
- To assess enumeration-based DNA copy-concentration determination through an international comparison.
- To compare digital PCR (dPCR) and flow cytometric (FCM) counting against chemical analysis methods for DNA quantification.
- To evaluate the validity and measurement uncertainty of enumeration approaches in DNA quantification.
Main Methods:
- Employed two enumeration-based methods: digital polymerase chain reaction (dPCR) and flow cytometric (FCM) counting.
- Utilized two orthogonal chemical-analysis methods: isotope-dilution mass spectrometry (IDMS) and capillary electrophoresis (CE) for nucleotide quantification.
- Quantified pBR322 plasmid solutions at different concentrations using these comparative methods.
Main Results:
- Digital PCR (dPCR) results from multiple laboratories showed some dispersion (RSD = 11.8%) but aligned closely with FCM and chemical analysis methods.
- When using the mean of dPCR results, the relative standard deviation (RSD) across all four methods decreased significantly to 1.8%.
- This strong agreement supports the validity of recent enumeration approaches for absolute DNA quantification.
Conclusions:
- Enumeration-based methods, particularly dPCR, demonstrate strong potential for absolute DNA quantification and SI traceability.
- While overall agreement between methods is good, individual dPCR results often exceeded reported measurement uncertainties.
- Further investigation is warranted to ensure all contributing factors to dPCR measurement uncertainty are adequately considered by laboratories.
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