The ligated cDNAs 1C, 4C, and 11C were analyzed by denaturing PAGE to have a qualitative insight into the ligation efficiency (Figure ). The denaturing PAGE analysis indicated that all three ligations, despite using desalted (i.e., non-HPLC purified) precursor, were able to utilize nearly 100% of the precursor oligonucleotide. The formation of the cDNA in the ligation reaction was indicated by the presence of a slower-moving band in denaturing PAGE. Furthermore, when the band persisted after exonuclease digestion (C5 condition), it was confirmed as the desired circular product. Interestingly, we noticed that there are more than one prominent slower-moving bands (compared to the precursors) in the lanes containing the ligated DNA for splint-padlock ligation (lane 2 in Figure ). The slowest moving band disappeared after exonuclease digestion and therefore revealed to be not the intended circular product. The presence of more than one prominent slow-moving band in ligation-probing denaturing PAGE was a persistent observation for even smaller splint-assisted splint-padlock ligations explored later in this work (below). We suspect that the slowest band could be due to the formation of a long linear oligomeric splint-padlock complex as seen in the hybridization chain reaction , one that is susceptible to exonuclease digestion. Another interesting observation for splint-padlock ligation was the change in migration of cDNA 4C after exonuclease digestion (lane 2 vs 3, Figure ). This observation probably indicated continued annealing of the splint with cDNA despite the denaturing nature of the PAGE. The depreciated migration of cDNA due to continued splint-padlock complexation was later independently verified for shorter splint-mediated splint-padlock ligated substrates as well (below). Such an association would contribute towards a high degree of NSA as encountered with splint-padlock ligated cDNA 4C later in this study. Finally, the comparison of the gel images with that of a linear oligonucleotide of known concentration in the same gel was utilized to assess the final concentrations of the cDNAs (not shown).