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Related Concept Videos

DNA Isolation01:24

DNA Isolation

DNA isolation protocols can be fast and straightforward or complex and time-consuming depending on the type and quality of DNA required for further processing. For example, plasmid DNA extraction is a bit more complicated than genomic DNA extraction because of the need for an appropriate lysis method to separate plasmid DNA from gDNA during isolation. However, for specific applications, such as long-range DNA sequencing that require a good yield of high- quality DNA samples, we need to follow...

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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
PubMed
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.

Keywords:
DNA CRMDNA quantificationMoleSingle-molecule counting

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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.