Simultaneous incorporation of three different modified nucleotides during PCR.

Modified analogs of 2'-deoxycytidine triphosphates bearing (6-aminohexyl)carbamoylmethyl or 7-amino-2,5-dioxaheptyl linker at a C5 position were designed and synthesized. Both analogs were found to be good substrates for Vent(exo-) DNA polymerase during PCR, resulting in the corresponding full-length modified DNAs, respectively. Moreover, we have demonstrated simultaneous incorporation of three different modified nucleotides into a DNA strand by PCR using triphosphates of 5-(3-aminopropynyl)dUTP, 5-[(6-aminohexyl)carbamoylmethyl]dCTP and 2-amino-dATP (dDTP) or N6-methyl-dATP in place of the natural nucleoside triphosphates TTP, dCTP and dATP.