Articles:
Pyrene binary probes for unambiguous detection of mRNA using time-resolved fluorescence spectroscopy
Angel A. MartÃ; Xiaoxu Li; Steffen Jockusch; Zengmin Li; Bindu Raveendra; Sergey M. Kalachikov; James J. Russo; Irina Morozova; Sathyanarayanan V. Puthanveettil; Jingyue Ju; Nicholas J. Turro
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- Title:
- Pyrene binary probes for unambiguous detection of mRNA using time-resolved fluorescence spectroscopy
- Author(s):
-
MartÃ, Angel A.
Li, Xiaoxu
Jockusch, Steffen
Li, Zengmin
Raveendra, Bindu
Kalachikov, Sergey M.
Russo, James J.
Morozova, Irina
Puthanveettil, Sathyanarayanan V.
Ju, Jingyue
Turro, Nicholas J. - Date:
- 2006
- Type:
- Articles
- Department:
- Chemistry
- Volume:
- 34
- Permanent URL:
- http://hdl.handle.net/10022/AC:P:9344
- 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
- DOI:
- http://dx.doi.org/10.1093/nar/gkl406
- Item views:
- 111