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© Research
Publication : Journal of visualized experiments : JoVE

Nucleoside triphosphates–from synthesis to biochemical characterization

Scientific Fields
Diseases
Organisms
Applications
Technique

Published in Journal of visualized experiments : JoVE - 03 Apr 2014

Hollenstein M, Smith CC, Räz M

Link to Pubmed [PMID] – 24747811

J Vis Exp 2014;(86)

The traditional strategy for the introduction of chemical functionalities is the use of solid-phase synthesis by appending suitably modified phosphoramidite precursors to the nascent chain. However, the conditions used during the synthesis and the restriction to rather short sequences hamper the applicability of this methodology. On the other hand, modified nucleoside triphosphates are activated building blocks that have been employed for the mild introduction of numerous functional groups into nucleic acids, a strategy that paves the way for the use of modified nucleic acids in a wide-ranging palette of practical applications such as functional tagging and generation of ribozymes and DNAzymes. One of the major challenges resides in the intricacy of the methodology leading to the isolation and characterization of these nucleoside analogues. In this video article, we present a detailed protocol for the synthesis of these modified analogues using phosphorous(III)-based reagents. In addition, the procedure for their biochemical characterization is divulged, with a special emphasis on primer extension reactions and TdT tailing polymerization. This detailed protocol will be of use for the crafting of modified dNTPs and their further use in chemical biology.

http://www.ncbi.nlm.nih.gov/pubmed/24747811