Enzymatic incorporation of chemically-modified nucleotides into DNAs

Modified 2'-deoxyuridine triphosphates bearing proteinous amino acids at a C5 position were prepared, and their substrate properties were investigated for KOD Dash DNA polymerase during PCR. The modified dUTPs bearing histidyl, lysyl, glutaminyl or seryl group produced the aimed 108 nt PCR products in good yields. In contrast, the analog bearing glutamyl group did not work as a substrate for KOD Dash while the analog bearing aspartyl group gave the product in a low yield. Moreover, not only KOD Dash but also three other thermostable DNA polymerases were tested as catalysts by use of C5 modified dUTPs with two different types of linker arms. Both Pfu and Vent(exo-) were relatively tolerant for the modification at the C5 position as well as KOD Dash.