Cohesive end ligated cDNA with 14 nt long overhangs also showed the least NSA for LRCA conditions under dual exonuclease digestion with 1X Exo III buffer (supplemented with 33 mM Mg 2+ ) as well as with 3X Exo III buffer (having 33 mM Mg 2+ ). Perplexingly and unlike the other two substrates, its NSA under HRCA conditions increased substantially compared to its LRCA counterpart assays. Furthermore, it seemingly demonstrated a length-dependent NSA trend which increased with reducing cohesive end length. This observation, in addition to another observation concerning relative degrees of single enzyme Exo I vs Exo III digestion (in Figure ) for the three substrates may indicate a poor affinity of Exo III towards cohesive end ligating precursors. The cohesive length end might also play a role in controlling the enzyme affinity, digestion efficiency, or both. The experiments in our study could not comprehensively explain the origin of NSA in cohesive end ligation and we suspect that a possible strand-invasion mechanism between the precursors and cDNA could be responsible.