Next, we investigated the NSA for cohesive end ligation with 5 or 9 nt long overhangs for LRCA and HRCA conditions. In experiments quantifying NSA in cohesive end ligated cDNA substrates, low NSA for LRCA was observed that increased substantially under HRCA conditions (above). We postulated that this could be due to the inability of Exo III, a 3-to 5-digesting exonuclease, to digest 14 nt long recessive 3-termini and could improve the NSA for shorter overhang lengths. To investigate, precursor oligonucleotides containing 9 nt (12a and 12b for cDNA 12C) and 5 nt (13a and 13b for cDNA 13C) long 5-overhangs were designed. These oligonucleotides, when circularized, would utilize the same LRCA primer and HRCA primer sequences as that of cDNA 11C (see Figures and for primer and HRCA primer binding sites). After ligation, these substrates were treated with dual exonuclease treatments with C6 or C7 conditions (due to their higher Mg 2+ concentration) and subjected to LRCA (for quantifying NSA in absence of HRCA primer) and HRCA (for quantifying NSA in presence of HRCA primer) amplification conditions. A summary of these experiments has been provided in Table .