5-[3-(E)-(4-Azido-2,3,5,6-tetrafluorobenzamido)propenyl-1]-2 '-deoxy-uridine-5 '-triphosphate substitutes for thymidine-5 '-triphosphate in the polymerase chain reaction

The DNA targets may be labeled and simultaneously amplified in the polymera se chain reaction (PCR) using a pair of respective primers after elongation with nucleoside-5'-triphosphates carrying photoreactive groups. The amplif ied DNA may be subsequently photoactivated by irradiation above 300 nm, res ulting in photo-cross-linking of the strands. For this goal 5-[3-(E)-(4-azi do-2,3,5,6-tetrafluorobenzamido)propenyl-1]-, 5-{N-[N'-(4-azido-2,3,5,6-tet rafluorobenzoyl)-3-aminopropionyl]aminomethyl}- and 5-{N-[N'-(2-nitro-5-azi dobenzoyl)-3-amimopropionyl]aminomethyl}-2'-deoxyuridine-5'-triphosphate (V II, VIa, and VIb) derivatives have been synthesized. It was found that VII is capable of efficiently elongating DNA primers with both Klenow fragment DNA polymerase I and Thermus aquaticus DNA polymerase. Thereto, it turned o ut to provide quantitative incorporation in DNA as revealed by the formatio n of the full-length amplificate by PCR in the presence of this photoreacti ve analogue without any dilution with natural dTTP. On the contrary, it was found, that incorporation of VIa and VIb do not permit further DNA replica tion.