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An equation for DNA electrophoretic mobility in agarose gels
C E Willis1, D G Willis, G P Holmquist
1Technology Life Sciences Division, KRUG International, Houston, Texas 77258.
Applied and Theoretical Electrophoresis : the Official Journal of the International Electrophoresis Society
|January 1, 1988
Summary
A new logit transformation method accurately predicts DNA fragment size using agarose gel electrophoresis mobility. This simplifies molecular weight assignment for DNA fragments across a wide range.
Area of Science:
- Molecular Biology
- Biochemistry
- Bioinformatics
Background:
- Accurate molecular weight assignment of DNA fragments is crucial for molecular biology applications.
- The relationship between electrophoretic mobility in agarose gels and DNA molecular weight is non-linear, complicating direct assignments.
- Existing graphical methods often require normalization and may not cover a wide range of DNA sizes.
Purpose of the Study:
- To develop a simplified and accurate method for assigning molecular weight to DNA fragments based on electrophoretic mobility.
- To linearize the sigmoidal relationship between DNA mobility and molecular weight for improved analysis.
- To provide a predictive model for DNA fragment length, migration, resolution, and gel performance.
Main Methods:
- Electrophoresis of linear duplex DNA molecules (118 to 169,200 base pairs) in agarose gels (0.2% to 1.6%) at varying voltage gradients (1 to 6 V/cm).
- Application of a logit transformation to mobility data, graphed against the logarithm of molecular weight.
- Comparison of the logistic representation with conventional graphical methods (log MW vs. mobility, MW vs. reciprocal mobility) and Probit analysis.
Main Results:
- A logit transformation of normalized mobility data versus the logarithm of molecular weight yields a linear relationship.
- This linear relationship is consistent across a wide range of DNA molecular weights and is not affected by changes in voltage gradient or gel composition (only position shifts).
- A single equation was determined that accurately describes DNA mobility as a function of molecular weight, voltage gradient, and gel concentration.
Conclusions:
- The logit transformation provides a robust and simplified method for molecular weight assignment of DNA fragments in agarose gel electrophoresis.
- This approach overcomes the limitations of non-linear mobility-molecular weight relationships, enabling more accurate fragment size determination.
- The developed model and presented curves are valuable tools for predicting and optimizing DNA gel electrophoresis experiments.