Design and synthesis of a photocleavable biotinylated nucleotide for DNA analysis by mass spectrometry.
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.
Bai
Xiaopeng
X
Laboratory of DNA Sequencing and Chemical Biology, Columbia Genome Center, Columbia University College of Physicians and Surgeons, New York, NY 10032, USA.
Kim
Sobin
S
Li
Zengmin
Z
Turro
Nicholas J
NJ
Ju
Jingyue
J
D001710:Biotin / Q000031:analogs & derivatives / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism; D019921:Biotinylation; D004247:DNA / Q000032:analysis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D003856:Deoxyuracil Nucleotides / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism; D010777:Photochemistry; D017422:Sequence Analysis, DNA / Q000379:methods; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D019809:Streptavidin / Q000378:metabolism
C483775:5-(3-(1-(5-(5-(biotinylamino)pentanamidomethyl)-2-nitrophenyl)ethoxycarbonylamino)propenyl)-2'-deoxyuracil 5'-triphosphate; D003856:Deoxyuracil Nucleotides; D001710:Biotin; D004247:DNA; D019809:Streptavidin; C034970:bacteriophage T7 induced DNA polymerase; D004259:DNA-Directed DNA Polymerase