Interestingly, both substrates, like the cDNA 11C derived from 14 nt overhang containing precursor, demonstrated low NSA under LRCA conditions (Figure and 4B, Figure ). However, the NNSA under LRCA conditions for cDNA 13C from either 5 nt overhang precursor (16.3 and 12.7% for C6 and C7 conditions, respectively) or 12C from 9 nt overhang precursor (6.1 and 8.5% for C6 and C7 conditions, respectively) remained higher than that of cDNA 11C from 14 nt precursor (3.3 and 0.4% for C6 and C7 conditions, respectively). This trend was also present for the NNSA quantified for HRCA conditions, where the cDNA from shorter overhangs again reflected higher NNSA than the cDNA from 14 nt precursor. Overall, this indicated that contrary to our postulate, precursor cohesive end oligonucleotide containing shorter 5-protruded (i.e., 3-recessed) cohesive end probably are relatively more difficult to be digested by Exo III or dual digestion.