Published online 26 January 2004
Nucleic Acids Research, 2004, Vol. 32, No. 2 535-541
© 2004 Oxford University Press
Design and synthesis of a photocleavable biotinylated nucleotide for DNA analysis by mass spectrometry
1 Laboratory of DNA Sequencing and Chemical Biology, Columbia Genome Center, Columbia University College of Physicians and Surgeons, New York, NY 10032, USA and Departments of 2 Chemical Engineering and 3 Chemistry, Columbia University, New York, NY 10027, USA
*To whom correspondence should be addressed at Room 405A, Russ Berrie Medical Science Pavilion, Columbia University College of Physicians and Surgeons, 1150 St Nicholas Avenue, New York, NY 10032, USA. Tel: +1 212 851 5172; Fax: +1 212 851 5215; Email: dj222{at}columbia.edu
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.