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Comprehensive interpretation of gel electrophoresis data
Chongli Yuan1, Elizabeth Rhoades, Daniel M Heuer
1School of Chemical and Biomolecular Engineering, and Department of Applied and Engineering Physics, Cornell University, Ithaca, NY 14853, USA.
Analytical Chemistry
|September 2, 2006
Summary
An entropic force, caused by restricted orientation of DNA in gel pores, explains discrepancies in electrophoresis models. A new model incorporating this force accurately predicts DNA behavior, improving gel electrophoresis data interpretation.
Area of Science:
- Biophysics
- Polymer Science
- Analytical Chemistry
Background:
- Polyacrylamide gel electrophoresis (PAGE) is widely used for separating DNA molecules.
- Existing models (Ogston-Morris-Rodbard-Chrambach) show discrepancies with experimental data for rigid-rod DNA.
- The source of these discrepancies, particularly for anisotropic analytes, requires further investigation.
Purpose of the Study:
- To identify the cause of discrepancies between theoretical models and experimental results in PAGE of rigid-rod DNA.
- To develop a new, comprehensive model for PAGE that accounts for analyte orientation effects.
- To propose a novel length scale for characterizing the impact of analyte topology on electrophoretic mobility.
Main Methods:
- Analysis of temperature-dependent polyacrylamide gel electrophoresis data.
- Time-dependent fluorescence anisotropy decay measurements to assess analyte orientation.
- Development and validation of a new comprehensive electrophoretic model.
- Testing the model with linear and branched rigid-rod DNA analytes.
Main Results:
- An entropic force, arising from reduced orientational freedom of anisotropic analytes in gel pores, is identified as the cause of model discrepancies.
- Fluorescence anisotropy measurements confirm restricted orientation of analytes within polyacrylamide gels, even without external fields.
- The new comprehensive model shows favorable agreement with experimental data across various DNA topologies and sizes.
- A new topology-dependent length scale is proposed, reducing to the radius of gyration for simple geometries.
Conclusions:
- The proposed entropic force and comprehensive model significantly improve the interpretation of PAGE data for rigid-rod DNA.
- The new length scale provides a unified approach to describe the influence of analyte topology on electrophoretic mobility.
- This work offers a general framework for analyzing gel electrophoresis data of charged analytes with diverse topologies.
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