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

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

Title:
Design and synthesis of a photocleavable biotinylated nucleotide for DNA analysis by mass spectrometry
Author(s):
Bai, Xiaopeng
Kim, Sobin
Li, Zengmin
Turro, Nicholas J.
Ju, Jingyue
Date:
Type:
Articles
Department:
Chemistry
Volume:
32
Permanent URL:
Book/Journal Title:
Nucleic Acids Research
Abstract:
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.
Subject(s):
Chemistry
Chemical engineering
Publisher DOI:
http://dx.doi.org/10.1093/nar/gkh198
Item views:
349
Metadata:
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