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DNA sequencing by capillary electrophoresis with replaceable linear polyacrylamide and laser-induced fluorescence
M C Ruiz-Martinez1, J Berka, A Belenkii
1Barnett Institute, Northeastern University, Boston, Massachusetts 02115.
Analytical Chemistry
|October 15, 1993
Summary
Replaceable linear polyacrylamide (LPA) enables routine DNA sequencing via capillary electrophoresis (CE). This method achieves high-resolution sequencing of M13mp18 phage DNA up to 350 nucleotides in under 30 minutes.
Area of Science:
- Biochemistry
- Molecular Biology
- Analytical Chemistry
Background:
- Cross-linked polyacrylamide gels present stability issues for routine DNA sequencing.
- Capillary electrophoresis (CE) offers a high-resolution separation technique for DNA analysis.
- Laser-induced fluorescence (LIF) detection is a sensitive method for analyzing fluorescently labeled molecules.
Purpose of the Study:
- To develop a stable and reproducible method for DNA sequencing using replaceable linear polyacrylamide (LPA) as a sieving matrix in CE.
- To optimize a simple LIF detection system for efficient DNA sequencing.
- To achieve high-resolution sequencing of M13mp18 phage DNA, including difficult compressed regions.
Main Methods:
- Utilized replaceable linear polyacrylamide (LPA) as a sieving matrix in capillary electrophoresis (CE).
- Employed a simplified laser-induced fluorescence (LIF) detection system with a single laser and two photomultiplier tubes (PMTs).
- Used FAM- and JOE-labeled primers for excitation with an argon ion laser and optical separation.
- Optimized electrophoresis buffer composition (1x TBE, 3.5 M urea, 30% formamide) and column temperature (32°C).
Main Results:
- Achieved reproducible DNA sequencing of M13mp18 phage up to 350 nucleotides in approximately 30 minutes.
- Successfully resolved challenging compressed sequence sites using optimized LPA and CE conditions.
- Demonstrated effective sequencing by analyzing two fluorescent dyes and two peak height ratios across two optical channels.
Conclusions:
- Replaceable linear polyacrylamide (LPA) overcomes the stability limitations of cross-linked gels for routine DNA sequencing by CE.
- The developed LIF detection system and optimized CE conditions provide a robust and efficient method for high-resolution DNA sequencing.
- This methodology enables rapid and accurate sequencing of DNA fragments, including complex regions.