Effects of pH and cations on E.coli Endo IV activity was determined using the probe, enhancer and target ( Figure 3A) . It was shown that Endo IV cleavage of the probe has a relatively flat pH optimum in the range between pH 8.5 and 9.5. For compatibility with post-PCR Endo IV amplification the assay buffer with pH 8.6 was chosen, similar to that of the PCR master mixture. Inclusion of the divalent magnesium cation in the buffer ranging from 0 to 20 mM, resulted in more than 2-fold increase in cleavage rate, the optimum range was $5-8 mM. A concentration of 5 mM was chosen for the Endo IV assay. It was determined that the monovalent cations, such as Li, Na, K and Rb, inhibit Endo IV cleavage activity for the concentrations studied in the range from 0 to 100 mM. An inhibition of $30% was observed at 20 mM monovalent cation concentration, which increased to $60-70% at 100 mM concentration. The optimum probe concentration was determined to be $600 nM, please see Supplementary Figures 1-3 . Figure 3A shows a model Endo IV assay that requires a target complementary to an enhancer and a probe labeled at the 5 0 end with an Eclipse Dark Quencher and a fluorescent dye at the 3 0 end. As shown, the probe (calculated T m ¼ 46.7 C) and enhancer (calculated T m ¼ 50.2 C) are separated by one base to mimic a natural abasic site. Four probes with different fluorescent dyes and linkers ( Figure 3B ) were evaluated for their performance in the new assay. Fluorophores FL1, FL2 and FL3 of the probes I, II and III (with similar quantum yields) contained a hydroxyprolinol linker between the 3 0 -phosphate of the probe sequence and the core of the dyes. Fluorophore FL4 of probe IV was analogous to FL3 of probe III, with an additional aminocaproic spacer introduced between the hydroxylprolinol moiety and the dye; the purpose of this spacer was to distance the fluorophore from the cleavage site.