Unit

UNIT 13.3 Synthesis and Polymerase Incorporation of 5′-Amino-2′,5′-Dideoxy-5′-N-Triphosphate Nucleotides

  1. Jia Liu Wolfe,
  2. Tomohiko Kawate

Published Online: 1 OCT 2004

DOI: 10.1002/0471142700.nc1303s18

Current Protocols in Nucleic Acid Chemistry

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.

Author Information

  1. Massachusetts General Hospital, Cambridge, Massachusetts

Publication History

  1. Published Online: 1 OCT 2004
  2. Published Print: SEP 2004

Abstract

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.

Keywords:

  • amine;
  • azide;
  • modified nucleosides;
  • modified nucleotide triphosphates;
  • chemical synthesis;
  • polymerase incorporation;
  • sequence specific DNA cleavage