Nonradiochemical DNase I footprinting by capillary electrophoresis.
A new application for DNase I footprinting using capillary electrophoresis (CE) has been developed in order to decrease analysis time and to eliminate the use of radiochemicals. An additional advantage of the new method over the traditional radioactive methods is that the DNA probe can be labeled on both ends with different fluorescein dyes. This provides an internal check of the identification of protein-binding sites on DNA, because the binding region can be observed from both DNA strands. The initial parameters for the CE method were developed using the Promega Core Footprinting Kit for analysis of AP-2 binding sites in the SV40 enhancer sequence. After optimization of the method, the protocol was found to be effective for footprint analysis of the immediate upstream region (bases -1 to -370) of the rat glutathione peroxidase (GPX) and it permitted identification of a previously unknown binding site in the upstream sequence of the GPX gene.
Wilson
D O
DO
Department of Chemistry and Biochemistry, Ohio University, Athens 45701, USA.
Johnson
P
P
McCord
B R
BR
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000032:analysis / Q000235:genetics; D018983:DNA Footprinting / Q000379:methods*; D017931:DNA Primers / Q000235:genetics; D015342:DNA Probes; D004279:DNA, Viral / Q000032:analysis / Q000235:genetics; D004268:DNA-Binding Proteins / Q000378:metabolism; D003850:Deoxyribonuclease I; D019075:Electrophoresis, Capillary / Q000379:methods*; D004742:Enhancer Elements, Genetic; D019793:Fluorescein; D005979:Glutathione Peroxidase / Q000235:genetics; D066298:In Vitro Techniques; D016133:Polymerase Chain Reaction; D051381:Rats; D013539:Simian virus 40 / Q000235:genetics; D050656:Transcription Factor AP-2; D014157:Transcription Factors / Q000378:metabolism
D017931:DNA Primers; D015342:DNA Probes; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D050656:Transcription Factor AP-2; D014157:Transcription Factors; D004247:DNA; D005979:Glutathione Peroxidase; D003850:Deoxyribonuclease I; D019793:Fluorescein