Quantitative DNA Sequencing to Determine the Relative Protein-DNA Binding Constants to Multiple DNA Sequences

Abstract

DNA sequencing technology was modified into a quantitative assay, which for multiple DNA sequences allowed the simultaneous determination of relative protein-DNA binding constants. The band mobility shift of the protein-DNA binding reactions partitions the mixture of DNA sequences into bound and unbound fractions. The quantitation of that partitioning gives directly the relative binding constants, usually with accuracies of better than ±20%. The protein of interest was the Mnt repressor of Salmonella bacteriophage P22, and the synthetic DNA contained Mnt′s natural operator with a randomized position.