Wang, J.; Wang, J.; Wang, Z.; Wang, P.; Sun, S.; Li, X.; Tian, Z.; Xu, R.; Shi, Y.; Wang, Y.

Protein-DNA interactions are crucial for cellular processes, but current quantification methods lack sensitivity. We developed the Real-time PCR-based DNA Binding Assay (RP-DBA) to detect and quantify these interactions. The target protein, expressed as a Strep-tag II fusion, is purified and incubated with double-stranded DNA probes containing 4 bp 3\' overhangs. Protein-DNA complexes are immobilized on Strep-Tactin beads, washed, and eluted. A complementary single-stranded DNA amplification arm is added, extended by Taq polymerase, and quantified via qPCR with SYBR Green. RP-DBA enables real-time kinetic analysis and is 4- to 10-fold more sensitive than EMSA, depending on amplification arm length (30-90 bp). Its simplicity, speed, accuracy, and high-throughput potential make it a valuable tool for advancing molecular biology research.