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Three DNA sequencing methods using capillary gel electrophoresis and laser-induced fluorescence
H Swerdlow1, J Z Zhang, D Y Chen
1Department of Chemistry, University of Alberta, Edmonton, Canada.
Analytical Chemistry
|December 15, 1991
Summary
Capillary gel electrophoresis achieves high sequencing rates and improved resolution compared to traditional methods. This technique offers sensitive detection limits for DNA sequencing applications.
Area of Science:
- Biotechnology
- Analytical Chemistry
- Molecular Biology
Background:
- DNA sequencing is crucial for genetic analysis and disease research.
- Traditional slab gel electrophoresis faces limitations in resolution and throughput.
- Capillary electrophoresis (CE) offers potential advantages for high-throughput DNA analysis.
Purpose of the Study:
- To demonstrate the application of capillary gel electrophoresis for different DNA sequencing techniques.
- To evaluate the sequencing rates and resolution achieved by CE.
- To determine the detection limits of CE with various detection methods.
Main Methods:
- Capillary gel electrophoresis was applied to three spectral-channel sequencing techniques (Smith, Prober, Richardson and Tabor).
- Sequencing rates were measured at electric field strengths up to 465 V/cm.
- Detection limits were assessed using on-column and post-column detectors.
Main Results:
- Sequencing rates of up to 1000 bases/hour were achieved.
- Capillary electrophoresis provided 2x better resolution than slab gel electrophoresis for fragments >550 nucleotides.
- Detection limits as low as 2 zmol were obtained with a post-column detector.
Conclusions:
- Capillary gel electrophoresis is a viable and efficient technique for DNA sequencing.
- CE offers significant improvements in resolution and sensitivity over traditional methods.
- The choice of detector impacts the achievable detection limits in CE-based sequencing.