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Updated: Jul 18, 2026
07:44
Synthesis of Wavelength-shifting DNA Hybridization Probes by Using Photostable Cyanine Dyes
Published on: July 6, 2016
Four-color DNA sequencing by synthesis using cleavable fluorescent nucleotide reversible terminators
Jingyue Ju1, Dae Hyun Kim, Lanrong Bi
1Columbia Genome Center, Columbia University College of Physicians and Surgeons, New York, NY 10032, USA. ju@c2b2.columbia.edu
Summary
Researchers developed novel DNA sequencing technology using chemically modified nucleotides. This method enables efficient DNA sequencing by synthesis (SBS) with improved accuracy, particularly in challenging homopolymer regions.
Area of Science:
- Molecular Biology
- Biochemistry
- Genomics
Background:
- DNA sequencing by synthesis (SBS) on solid surfaces is a key technology for deciphering genetic information.
- Existing SBS methods face challenges, particularly with homopolymer regions, impacting accuracy and efficiency.
- Development of efficient and accurate reversible terminators is crucial for advancing SBS technology.
Purpose of the Study:
- To engineer a novel DNA sequencing system utilizing molecular engineering approaches for enhanced SBS.
- To develop chemically cleavable fluorescent nucleotide reversible terminators for improved polymerase recognition and reaction initiation.
- To assess the efficiency and accuracy of the developed system, especially in sequencing DNA templates with homopolymer regions.
Main Methods:
- Modification of four nucleotides (A, C, G, T) as reversible terminators with cleavable fluorophores and 3'-OH capping.
- Utilizing an allyl moiety as a linker to tether fluorophores to 3'-O-allyl-modified nucleotides (3'-O-allyl-dNTPs-allyl-fluorophore).
- Employing Palladium-catalyzed deallylation in aqueous buffer for simultaneous removal of fluorophore and 3'-O-allyl group.
Main Results:
- Successful synthesis of chemically cleavable fluorescent nucleotide reversible terminators (3'-O-allyl-dNTPs-allyl-fluorophore).
- Demonstration of a one-step dual-deallylation reaction, removing both the fluorophore and the 3'-O-allyl group in 30 seconds.
- Accurate sequencing of DNA templates, including homopolymer regions, using the developed nucleotide analogues on a DNA chip with a four-color scanner.
Conclusions:
- The developed 3'-O-allyl-dNTPs-allyl-fluorophore system represents a significant advancement in SBS technology.
- Simultaneous deprotection enables efficient reinitiation of the polymerase reaction, increasing overall SBS efficiency.
- This novel approach offers high accuracy for DNA sequencing, particularly beneficial for resolving complex genomic regions like homopolymers.
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