In vivo footprinting of the interaction of proteins with DNA and RNA.
Analysis of the interaction of proteins with either DNA or RNA sequences by in vivo footprinting involves two steps: (i) the in situ modification of nucleic acids by the footprinting reagent and (ii) the visualization of the footprints. Ligation-mediated PCR (LM-PCR) procedures provide a level of sensitivity and specificity that is suitable for visualization of footprints of single-copy genes or low-abundance mRNAs in higher eukaryotes. In this article, we discuss several of the technical aspects of these multistep procedures that contribute to the quality of the results, particularly the parameters that affect the specificity and fidelity of the reactions: (i) the design of the primers, which is important to achieve optimal specificity; (ii) the choice of polymerases so that the amplified material represents faithfully the initial nucleic acid population; and (iii) the impact of the plateau effect within the PCR on the interpretation of the data. We then discuss aspects of in vivo nucleic acid manipulation that may affect the quality of the footprinting image, in particular the choice of the footprinting 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 to the techniques we have developed or modified: LM-PCR, reverse ligation-mediated PCR, and nuclease treatment of RNAs in vivo.
Grange
T
T
Institut Jacques Monod du CNRS, Université Paris 7, France.
Bertrand
E
E
Espinás
M L
ML
Fromont-Racine
M
M
Rigaud
G
G
Roux
J
J
Pictet
R
R
D001483:Base Sequence; D006528:Carcinoma, Hepatocellular; D002460:Cell Line; D004247:DNA / Q000378:metabolism*; D018983:DNA Footprinting / Q000379:methods*; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase; D003850:Deoxyribonuclease I; D006801:Humans; D008113:Liver Neoplasms; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D016133:Polymerase Chain Reaction / Q000379:methods*; D011401:Promoter Regions, Genetic; D012313:RNA / Q000378:metabolism*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000737:chemistry; D016601:RNA-Binding Proteins / Q000378:metabolism*; D011990:Receptors, Transferrin / Q000096:biosynthesis / Q000737:chemistry; D012680:Sensitivity and Specificity; D019914:Taq Polymerase
D017931:DNA Primers; D004268:DNA-Binding Proteins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D011990:Receptors, Transferrin; D012313:RNA; D004247:DNA; D019914:Taq Polymerase; D004259:DNA-Directed DNA Polymerase; D003850:Deoxyribonuclease I