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