Chemical primer extension at submillimolar concentration of deoxynucleotides

Template-directed primer extension usually requires a polymerase, nucleoside triphosphates, and magnesium ions as cofactors. Enzyme-free, chemical primer extensions are known for preactivated nucleotides at millimolar concentrations. Based on a screen of carbodiimides, heterocyclic catalysts, and reactions conditions, we now show that near-quantitative primer conversion can be achieved at submillimolar concentration of any of the four deoxynucleotides (dAMP, dCMP, dGMP and dTMP). The new protocol relies on in situ activation with EDC and 1-methylimidazole and a magnesium-free buffer that was tested successfully for different sequence motifs. The method greatly simplifies chemical primer extension assays, further reduces the cost of such assays, and demonstrates the potential of the in situ activation approach.