Footprinting with an Automated Capillary DNA Sequencer

Abstract

Footprinting is a valuable tool for studying DNA-protein contacts. However, it usually involves expensive, tedious and hazardous steps such as radioactive labeling and analyses on polyacrylamide sequencing gels. We have developed an easy four-step footprinting method involving (i) the generation and purification of a PCR fragment that is fluorescently labeled at one end with 6-carboxyfluorescein; (ii) brief exposure of the fragment to a DNA-binding protein and then DNase I; (iii) spin-column purification; and (iv) analysis of partial digestion products on the ABI PRISM™ 310 capillary DNA sequencer/genetic analyzer. Very detailed and sensitive footprints of large (> 400 bp) DNA fragments can be easily obtained, as illustrated by our use of this method to characterize binding of PhcA, a LysR-type activator, to two sites greater than 100 bp apart in the 5′ untranslated region of xpsR, one of its regulated target genes. The advantages of this new method are that it (i) uses long-lived, safe and easyto-make fluorescently labeled target fragments; (ii) uses sensitive, robust and highly reproducible fragment analysis using an automated DNA sequencer, instead of gel electrophoresis and autoradiography; and (iii) is cost effective.