Mapping transcription factors on extended dna: A single molecule approach

The ability to determine the precise loci and distribution of nucleic acid binding proteins is instrumental to our detailed understanding of cellular processes such as transcription, replication, and chromatin reorganization. Traditional molecular biology approaches and above all Chromatin immunoprecipitation (ChIP) based methods have provided a wealth of information regarding protein-DNA interactions. Nevertheless, existing techniques can only provide average properties of these interactions, since they are based on the accumulation of data from numerous protein-DNA complexes analyzed at the ensemble level. We propose a single molecule approach for direct visualization of DNA binding proteins bound specifically to their recognition sites along a long stretch of DNA such as genomic DNA. Fluorescent Quantum dots are used to tag proteins bound to DNA, and the complex is deposited on a glass substrate by extending the DNA to a linear form. The sample is then imaged optically to determine the precise location of the protein binding site. The method is demonstrated by detecting individual, Quantum dot tagged T7-RNA polymerase enzymes on the bacteriophage T7 genomic DNA and assessing the relative occupancy of the different promoters.