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Quantitative analysis of DNA-sequencing electrophoresis
P D Grossman1, S Menchen, D Hershey
1Applied Biosystems, Inc., Foster City, California 94404.
Genetic Analysis, Techniques and Applications
|February 1, 1992
Summary
DNA sequencing electrophoresis separates oligonucleotide fragments by size. Diffusion limits resolution, while electric field strength is constrained by reptation for long sequences and Joule heating for short sequences, favoring tube formats.
Area of Science:
- Molecular Biology
- Biophysics
- Analytical Chemistry
Background:
- DNA sequencing relies on precise separation of nucleotide fragments.
- Electrophoresis is a key technique for this separation, but its efficiency is limited by various factors.
Purpose of the Study:
- To quantitatively analyze the selectivity and efficiency of DNA sequencing electrophoresis.
- To identify the primary mechanisms and limitations affecting fragment separation.
Main Methods:
- Quantitative analysis of electrophoretic separation.
- Investigation of Ogston sieving and reptation migration mechanisms.
- Assessment of Joule heating and diffusion effects on band broadening.
Main Results:
- Both Ogston sieving and reptation migration are operative in DNA sequencing electrophoresis.
- Diffusion, not Joule heating, is the main contributor to plate height under traditional conditions.
- Analytic expression derived for peak width relative to molecular size.
- Electrical field strength is limited by biased reptation for long sequences and Joule heat dissipation for short sequences.
Conclusions:
- Understanding separation mechanisms and limitations is crucial for optimizing DNA sequencing.
- Tube electrophoresis formats offer a significant speed advantage over slab formats for short sequences due to efficient heat dissipation.