UNIT 13.3 Synthesis and Polymerase Incorporation of 5′-Amino-2′,5′-Dideoxy-5′-N-Triphosphate Nucleotides
Published Online: 1 OCT 2004
Copyright © 2003 by John Wiley and Sons, Inc.
Lab Protocol Title
Current Protocols in Nucleic Acid Chemistry
How to Cite
Wolfe, J. L. and Kawate, T. 2004. Synthesis and Polymerase Incorporation of 5′-Amino-2′,5′-Dideoxy-5′-N-Triphosphate Nucleotides. Current Protocols in Nucleic Acid Chemistry. 18:13.3:13.3.1–13.3.17.
- Published Online: 1 OCT 2004
- Published Print: SEP 2004
This unit presents synthetic procedures for the preparation of 5′-amino-2′,5′-dideoxy analogs of adenosine, cytidine, guanosine, and thymidine, as well as corresponding 5′-N-triphosphate nucleotides, using commercially available reagents. The modified nucleosides are prepared in high yields from naturally occurring 2′-deoxynucleosides using robust chemical reactions including tosylation, azide exchange, and the Staudinger reaction. Efficient conversion of these 5′-amino nucleosides to corresponding 5′-N-triphosphate nucleotides is achieved through a one-step reaction with trimetaphosphate in Tris-buffered aqueous solution. The 5′-amino modification renders these nucleoside and nucleotide analogs markedly increased reactivity, which is useful for a variety of biochemical, pharmaceutical, and genomic applications. Also included in this unit are protocols for polymerase incorporation of the 5′-amino nucleotides, either partially or completely replacing their naturally occurring counterparts, and subsequent sequence-specific cleavage at the modified nucleotides under mildly acidic conditions.
- modified nucleosides;
- modified nucleotide triphosphates;
- chemical synthesis;
- polymerase incorporation;
- sequence specific DNA cleavage