Polymerase directed incorporation studies of LNA-G nucleoside 5′-triphosphate and primer extension involving all four LNA nucleotides

Abstract

For the first time, the enzymatic incorporation of LNA-G nucleotides into DNA strands has been investigated. KOD DNA polymerase accepted LNA-GTP as a substrate and provided a full-length dsDNA product modified with LNA-G nucleotides. Most importantly, an extension experiment was conducted using a reaction mixture containing LNA-A, LNA-T, LNA-G and LNA-mC nucleotides as the only NTP sources and this showed that KOD DNA polymerase can even incorporate 21 LNA nucleotides successively to a full-length extension product. Also, the experiments revealed that LNA-containing extension products were much more resistant towards the 3′→5′ exonucleolytic cleavage activity of the enzyme than DNA extension products.