Binding site selection analysis of protein-DNA interactions via solid phase sequencing of oligonucleotide mixtures

摘要

By combining the concept of degenerate oligonucleotide mutagenesis (1,2,3,4) and the convenience of solid phase chemical DNA sequencing (5), we have developed a rapid procedure for determining the specificity of DNA-binding proteins in vitro. Starting with a degenerate oligonucleotide mixture, the technique assays for alternative nucleotides in fractions that are bound or non-bound to the protein of interest. In contrast to previous approaches using degenerate oligonucleotides, it does not involve cloning but rather employs direct sequencing of the oligonucleotide mixtures after attachment to a solid support. Solid state processing obviates the need for both DNA extractions from polyacrylamide gels and time-consuming ethanol precipitations. Because of its convenience and sensitivity, this binding site selection analysis is well suited to determining rapidly the sequence preference of DNA-binding proteins that are available in small amounts, and complements well established approaches like methylation interference or missing contact assays. The solid phase reaction protocol we propose can also improve these latter approaches.