A comparative study of the modification efficiency of DNA polymerases and DNA template by the DNA primers with various photoreactive groups at their 3′-termini

The dependence of the modification efficiency of DNA polymerases and DNA template on the nature of photoreactive group and the length of the linker that joins the group with the heterocyclic base of the primer 3'-terminal nucleotide was studied. The primers that contained the photoreactive groups at their 3'-termini were obtained using the rat DNA polymerase beta or the DNA polymerase from Thermus thermophilus in the presence of one of the dTTP analogues carrying the photoreactive group in position 5 of thymidine residue. After irradiating the reaction mixture with LTV light and separating the modification products, the level of covat lent attachment of the [5'-(32)p]primer to DNA polyrnerases and template was determined. The primers containin- 4-azido-2,5-difluoro-3-chloropyridyl group were shown to be the most effective in the modification of DNA polymerases.