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Development of gene-in-plasmid DNA reference materials certified by single-molecule counting
Da-Hye Lee1,2, Hee-Bong Yoo1, Kee-Suk Hong3
1Bio-Metrology Group, Korea Research Institute of Standards and Science, Daejeon, Republic of Korea.
Analytical and Bioanalytical Chemistry
|December 8, 2024
Summary
Gene reference materials were developed using single-molecule counting for accurate quantification. This method ensures mole-traceable measurements for DNA and other biological entities, improving bioanalytical applications.
Area of Science:
- Biotechnology
- Metrology
- Molecular Biology
Background:
- The mole, the SI unit for amount of substance, is now defined by a fixed number of entities, enabling direct counting.
- Quantifying large biological molecules like DNA and proteins is challenging with traditional methods.
- Single-molecule counting offers a precise method for quantifying discrete biological entities.
Purpose of the Study:
- To develop gene reference materials certified by single-molecule counting for mole-traceable measurements.
- To establish methods for quantifying gene-based DNA constructs and their impurities.
- To provide higher-order standards for DNA quantification in bioanalytical applications.
Main Methods:
- Single-molecule counting was used to quantify three plasmid DNA constructs.
- Digital PCR and LC-MS were employed for cross-validation of quantified values.
- Single-molecule real-time sequencing and two-color digital PCR were used to quantify sequence and fragment impurities, respectively.
Main Results:
- Gene reference materials were successfully developed and certified using single-molecule counting.
- Mole-traceable measurements were achieved for DNA constructs.
- Methods for impurity quantification and uncertainty estimation were implemented.
Conclusions:
- Practical gene-based DNA reference materials with metrological traceability were created.
- The developed methods enable precise quantification of DNA and impurities.
- These reference materials will serve as valuable standards for various bioanalytical applications.
