Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 100, Issue 2, Pages 414-419Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.242729199
Keywords
nucleoside triphosphate; 2-nitrobenzyl linker; photocleavage; fluorescence; DNA sequencing by synthesis
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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 nucleoticle 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 nucleoticle. 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 nucleoticle in the DNA sequencing-by-synthesis approach.
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