In summary, this novel investigative study, with the objective of finding the best condition for mitigating non-specific amplification, subjected three cDNA substrates from three different ligations to nine digestion treatments having a plethora of combinations of enzymatic, buffer, and salts under LRCA conditions. Some of these were further investigated for NSA under HRCA conditions. We found that cDNA preparations using underutilized (sometimes unutilized) methodologies such as self-annealing ligation or splint-padlock ligation using 16 nt splint, or dual Exo I and III enzymatic digestion using Exo III buffer, when applied in tandem, led to minimal NSA. In separate work, we have demonstrated an application of the optimized self-annealing cDNA preparation followed by C5 digestion described here . Utilizing HRCA-mediated signal amplification and an in-house fabricated bi-electrode device, it detected the dengue virus sequence with fluorescence and end-point electrochemistry readouts .