Another interesting observation was the relative difference in NNSA for conditions C1 and C2 for substrates 1C, 4C, and 11C (Figure , Table ). The precursors of the cDNAs in this study contained a nick (for 1C) or a recessive 3-termini (for 4C and 11C) both of which are preferred substrates for Exo III digestion over Exo I digestion. However, the precursors of 4C and 11C differed from each other by the presence of a protruding 5-termini in the latter. Interestingly, both 1C and 4C had higher NNSA for Exo I digestion (condition C1) than Exo III digestion (condition C2). This indicated poor digestion by Exo I under condition C1 and relatively efficient digestion by Exo III under condition C2 for both 1C and 4C. In comparison, the NNSA for conditions C1 and C2 did not vary significantly from each other for substrate 11C, with a slightly higher magnitude for C2. This may imply a yet unknown (to the best of our knowledge) substrate selectivity for Exo III, reflecting relatively poor substrate binding for protruding single-stranded 5-cohesive end compared to nicks and 3recessive end. This aspect of poor substrate selectivity for cohesive end precursors was later revisited while investigating the NSA associated with varied lengths of cohesive ends. Overall, the investigation of these literature reported conditions demonstrated that either single exonuclease digestion or absence of enzymatic treatment insufficiently mitigated NSA, while dual digestion (as in C3) was relatively more effective in reducing it.