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Click chemistry to construct fluorescent oligonucleotides for DNA sequencing.
Tae Seok Seo1, Zengmin Li, Hameer Ruparel
1Columbia Genome Center, Columbia University College of Physicians and Surgeons, New York, New York 10032, USA.
The Journal of Organic Chemistry
|January 18, 2003
Summary
Click chemistry enabled efficient labeling of single-stranded DNA (ssDNA) with 6-carboxyfluorescein (FAM). This FAM-labeled ssDNA served as a primer for DNA sequencing, achieving single-base resolution in capillary electrophoresis.
Area of Science:
- Bioconjugation Chemistry
- Molecular Biology
- Analytical Chemistry
Background:
- Fluorescent labeling of nucleic acids is crucial for molecular diagnostics and research.
- Traditional labeling methods can be inefficient or require harsh conditions.
- Click chemistry offers a mild and efficient alternative for bioconjugation.
Purpose of the Study:
- To develop a robust method for fluorescently labeling single-stranded DNA (ssDNA) using click chemistry.
- To evaluate the utility of the labeled ssDNA in downstream applications like DNA sequencing.
Main Methods:
- Utilized "click chemistry", specifically a 1,3-dipolar cycloaddition reaction.
- Conjugated alkynyl 6-carboxyfluorescein (FAM) with azido-labeled ssDNA under aqueous conditions.
- Employed capillary electrophoresis with laser-induced fluorescence detection for analysis.
Main Results:
- Achieved quantitative yields of FAM-labeled ssDNA.
- Demonstrated successful use of FAM-labeled ssDNA as a primer in DNA sequencing.
- Obtained single-base resolution in DNA sequencing products.
Conclusions:
- Click chemistry provides an efficient and reliable method for FAM labeling of ssDNA in aqueous solution.
- FAM-labeled ssDNA is suitable for high-resolution DNA sequencing applications.
- This approach enhances fluorescent detection capabilities in molecular biology.