Design and synthesis of a photocleavable biotinylated nucleotide for DNA analysis by mass spectrometry

Bai, Xiaopeng; Kim, Sobin; Li, Zengmin; Turro, Nicholas J.; Ju, Jingyue

We report here the design, synthesis and evaluation of a novel photocleavable (PC) biotinylated nucleotide analog, dUTP‐PC‐Biotin, for DNA polymerase extension reaction to isolate DNA products for mass spectrometry (MS) analysis. This nucleotide analog has a biotin moiety attached to the 5‐position of 2′‐deoxyribouridine 5′‐triphosphate via a photocleavable 2‐nitrobenzyl linker. We have demonstrated that dUTP‐PC‐Biotin can be faithfully incorporated by the DNA polymerase Thermo Sequenase into the growing DNA strand in a DNA polymerase extension reaction and that its incorporation does not hinder the addition of the subsequent nucleotide. Therefore, the DNA extension fragments generated by using the dUTP‐PC‐Biotin can be efficiently isolated by a streptavidin‐coated surface and recovered by near‐UV light irradiation at room temperature in mild condition for further analysis without using any chemicals or heat. Single and multiple primer extension reactions were performed using the dUTP‐PC‐Biotin to generate DNA products for MALDI‐TOF MS analysis. Such nucleotide analogs that carry a biotin and a photocleavable linker will allow the isolation and purification of DNA products under mild conditions for MS‐based genetic analysis by DNA sequencing or multiplex single nucleotide polymorphism (SNP) detection. Furthermore, these nucleotide analogs should also be useful in isolating DNA–protein complexes under non‐denaturing conditions.


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Nucleic Acids Research

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July 20, 2010