title: Synthesis and polymerase incorporation of 5'-amino-2',5'-dideoxy-5'-N-triphosphate nucleotides.
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.
authors.affiliation: Massachusetts General Hospital, Cambridge, Massachusetts, USA.
authors.forename: Jia Liu
authors.initials: JL
authors.lastname: Wolfe
authors.forename: Tomohiko
authors.initials: T
authors.lastname: Kawate
mesh_terms: D000588:Amines / Q000138:chemical synthesis / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D054306:Dideoxynucleotides / Q000138:chemical synthesis* / Q000378:metabolism*; D008954:Models, Biological
chemical_list: D000588:Amines; D054306:Dideoxynucleotides; D004259:DNA-Directed DNA Polymerase