Ratio of the Primers Used in Polymerase Chain Reaction-Stop Analysis Impacts the Resultant Banding Pattern

G-quadruplex (G4), as a dynamic nucleic acid secondary structure, widely exists in organism genomes and plays regulatory roles in a variety of cellular functions. Polymerase chain reaction stop assay (PCR-Stop) is a simple, quick, and low-cost widely used method for detection of the binding between G4 and its binding compounds. Different from the common PCR approach, no double-stranded DNA template is needed in the PCR-Stop assay, in which the forward and reverse primers extend against each Other in the presence of DNA Polymerase to produce a single DNA product. However, unexpected results, such as two or more PCR products, are often generated, and the mechanism is unclear. We found that the ratio of pair primers significantly impacts the generation and components of PCR-Stop products, which is crucial for the interpretation of the experiment results.