ArticleProceedings of the National Academy of Sciences of the United States of America2003
A photocleavable fluorescent nucleotide for DNA sequencing and analysis.
Article in Proceedings of the National Academy of Sciences of the United States of America, 2003. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 18 papers.
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Who cites it
18 citing papers in PubMed, 70 citations in OpenAlex.
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- Decoding the microbial universe with metagenomics: a brief insight.Frontiers in genetics · 2023Review
- Molecular Microbial Community Analysis as an Analysis Tool for Optimal Biogas Production.Microorganisms · 2021Review
- Switchable DNA-origami nanostructures that respond to their environment and their applications.Biophysical reviews · 2018Review
- Synthesis of BODIPY derivatives substituted with various bioconjugatable linker groups: a construction kit for fluorescent labeling of receptor ligands.Journal of fluorescence · 2014Article
- DNA sequencing by synthesis using 3'-RSC advances · 2014Article
- The history and advances of reversible terminators used in new generations of sequencing technology.Genomics, proteomics & bioinformatics · 2013Review
- Manipulation of gene expression in zebrafish using caged circular morpholino oligomers.Nucleic acids research · 2012Article
- Four-color DNA sequencing with 3'-O-modified nucleotide reversible terminators and chemically cleavable fluorescent dideoxynucleotides.Proceedings of the National Academy of Sciences of the United States of America · 2008Article
- A new class of cleavable fluorescent nucleotides: synthesis and optimization as reversible terminators for DNA sequencing by synthesis.Nucleic acids research · 2008Article
- 3'-O-modified nucleotides as reversible terminators for pyrosequencing.Proceedings of the National Academy of Sciences of the United States of America · 2007Article
- Molecular approaches in the diagnosis of primary immunodeficiency diseases.Human mutation · 2006Review
- Design and synthesis of a 3'-O-allyl photocleavable fluorescent nucleotide as a reversible terminator for DNA sequencing by synthesis.Proceedings of the National Academy of Sciences of the United States of America · 2005Article
- Photocleavable fluorescent nucleotides for DNA sequencing on a chip constructed by site-specific coupling chemistry.Proceedings of the National Academy of Sciences of the United States of America · 2004Article
- Design and synthesis of a photocleavable biotinylated nucleotide for DNA analysis by mass spectrometry.Nucleic acids research · 2004Article
- Microfluidic device reads up to four consecutive base pairs in DNA sequencing-by-synthesis.Nucleic acids research · 2004Article
- Photocleavage of a 2-nitrobenzyl linker bridging a fluorophore to the 5' end of DNA.Proceedings of the National Academy of Sciences of the United States of America · 2003Article
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Authors and funding
6 authors at 1 institution in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
DNA sequencing by synthesis during a polymerase reaction using laser-induced fluorescence detection is an approach that has a great potential to increase the throughput and data quality of DNA sequencing. We report the design and synthesis of a photocleavable fluorescent nucleoside triphosphate, one of the essential molecules required for the sequencing-by-synthesis approach. We synthesized this nucleoside triphosphate by attaching a fluorophore, 4,4-difluoro-5,7-dimethyl-4-bora-3alpha,4alpha-diaza-s-indacene propionic acid (BODIPY), to the 5 position of 2'-deoxyuridine triphosphate via a photocleavable 2-nitrobenzyl linker. We demonstrate that the nucleotide analogue can be faithfully incorporated by a DNA polymerase Thermo Sequenase into the growing DNA strand in a DNA-sequencing reaction and that its incorporation does not hinder the addition of the subsequent nucleotide. These results indicate that the nucleotide analogue is an excellent substrate for Thermo Sequenase. We also systematically studied the photocleavage of the fluorescent dye from a DNA molecule that contained the nucleotide analogue. UV irradiation at 340 nm of the DNA molecule led to the efficient release of the fluorescent dye, ensuring that a previous fluorescence signal did not leave any residue that could interfere with the detection of the next nucleotide. Thus, our results indicate that it should be feasible to use four different fluorescent dyes with distinct fluorescence emissions as unique tags to label the four nucleotides (A, C, G, and T) through the photocleavable 2-nitrobenzyl linker. These fluorescent tags can be removed easily by photocleavage after the identification of each nucleotide in the DNA sequencing-by-synthesis approach.
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