DNA annealing and DNA-protein interactions by capillary electrophoresis.
This work deals with annealing of single-stranded DNA and the binding of a serum respond factor to a DNA probe containing specific binding site. Capillary electrophoresis (CE) method is explored and compared with the mobility-shift gel electrophoresis (GE) procedure. The results indicate the CE method offers direct and rapid annealing of the DNA strands. It requires no prior incubation with additives (polynucleotides, proteins) to reduce nonspecific DNA-protein interactions. Unwanted nonspecific interactions are not observed in the CE method. The presence of a fluorescein tag to the DNA probe yields identical results to those with the radioactive label. A fluorescein tag in the CE work can be used without any adverse effects. The dissociation constant (Kd) of this protein-DNA complex by the CE method was similar to those determined by the GE method (approximately 10(-6) M). The proposed method is extremely powerful, highly sensitive, quantitative, and fast. It can determine even very small conformational differences of the DNA probe.
Singhal
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Department of Chemistry, Wichita State University, Kansas 67266-0051, USA. Singhal@twsu.edu
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D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D004247:DNA / Q000302:isolation & purification* / Q000378:metabolism*; D015342:DNA Probes / Q000737:chemistry; D004277:DNA, Single-Stranded / Q000302:isolation & purification / Q000378:metabolism; D004268:DNA-Binding Proteins; D019075:Electrophoresis, Capillary / Q000379:methods*; D005452:Fluoresceins; D006801:Humans; D008823:Micelles; D009687:Nuclear Proteins; D011089:Polydeoxyribonucleotides; D011485:Protein Binding; D026362:Serum Response Factor
D015342:DNA Probes; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D005452:Fluoresceins; D008823:Micelles; D009687:Nuclear Proteins; D011089:Polydeoxyribonucleotides; D026362:Serum Response Factor; C031156:poly d(I-C); D004247:DNA