Detection of A + T-rich DNA in gels by differential fluorescence.
The fluorochrome Hoechst 33258 preferentially forms complexes with A + T-rich duplex DNA, whereas ethidium bromide binds nucleic acids independent of base composition. Both compounds can be conveniently used to visualize DNA fractionated by gel electrophoresis. Determination of fluorescence emission from Hoechst 33258-stained restriction fragments normalized to fluorescence derived from the same sample after ethidium bromide staining provides a measure of emission due to A + T content, and allows easy identification of A + T-rich restriction fragments. To demonstrate the utility of this procedure, an A + T map of bacteriophage lambda DNA was constructed and found to be comparable to similar maps derived by alternate techniques. Analysis of recombinant plasmid DNAs with established nucleotide sequences demonstrated that the A + T content of individual restriction fragments could be estimated to within an accuracy of 5%.
D001482:Base Composition; D004247:DNA / Q000032:analysis*; D004262:DNA Restriction Enzymes; D004587:Electrophoresis, Agar Gel; D010957:Plasmids
D004247:DNA; D004262:DNA Restriction Enzymes