IN-VIVO FOOTPRINTING OF THE INTERACTION OF PROTEINS WITH DNA AND RNA

zAnalysis of the interaction of proteins with either DNA or RNA sequen ces by in vivo footprinting involves two steps: (i) the in situ modifi cation of nucleic acids by the footprinting reagent and (ii) the visua lization of the footprints. Ligation-mediated PCR (LM-PCR) procedures provide a level of sensitivity and specificity that is suitable for vi sualization of footprints of single-copy genes or low-abundance mRNAs in higher eukaryotes. In this article, we discuss several of the techn ical aspects of these multistep procedures that contribute to the qual ity of the results, particularly the parameters that affect the specif icity and fidelity of the reactions: (i) the design of the primers, wh ich is important to achieve optimal specificity; (ii) the choice of po lymerases so that the amplified material represents faithfully the ini tial nucleic acid population; and (iii) the impact of the plateau effe ct within the PCR on the interpretation of the data. We then discuss a spects of in vivo nucleic acid manipulation that may affect the qualit y of the footprinting image, in particular the choice of the footprint ing reagent and its condition of use (e.g., on intact or permeabilized cells or prepared nuclei) and the extent of nucleic acid modification . Finally, we provide detailed experimental procedures corresponding t o the techniques we have developed or modified: LM-PCR, reverse ligati on-mediated PCR, and nuclease treatment of RNAs in vivo. (C) 1997 Acad emic Press.