It appears that both the structure of the sterically constrained hydroxyprolinol linker and proximity of the bulky dye moiety to the cleavage site moderate cleavability of the 3 0 -terminal phosphodiester bond and practically eliminate non-template-dependent cleavage. Figure 4A shows the target, probe and the enhancer aligned with the target to indicate different gaps. The T m of the enhancer is typically chosen to be at least 5 C higher than that of the probe. The cleavage rates in the presence of enhancer are shown with 1-5 base gaps ( Figure 4B ). As expected, based on the natural abasic substrate requirement shown in Figure 1B , a one base gap gave the highest cleavage rate. The probe in the absence of enhancer showed a cleavage Table 1 . Effects of dyes and linkers on the rates and ratios of specific and non-specific cleavage Probe nos Dye Substitution on Figure 3B Cleavage rate Specific/non-specific rate ratios Target-specific %/min Target-non-specific %/min rate nearly equivalent to that of a 4 base gap in the presence of enhancer. As shown in Figure 4C , the maximum cleavage rate is achieved at a concentration of enhancer greater than $80 nM. In practice, the concentration of the enhancer is typically at least equivalent or larger than that of the probe. Some cleavage is observed with no enhancer present.