A new procedure for determining thymine residues in DNA sequencing. Photoinduced cleavage of DNA fragments in the presence of spermine.
A new procedure for T specific cleavage of DNA fragments utilizing photoreaction with spermine has been described. Irradiation of 3'-[32P]-end-labeled DNA fragments for 10-20 min with a germicidal lamp emitting mainly 254-nm light in the presence of 1 M spermine in distilled water resulted in a T specific cleavage of the DNA chains. This method does not require piperidine treatment. By contrast, when the DNA fragments were irradiated in the presence of methylamine under similar conditions, both G and T bands with the intensity of G greater than T have appeared. A similar but less selective T cleavage has also been observed in the irradiation of 5'-[32P]-end-labeled DNA fragments in the presence of spermine followed by brief heating of the photolysate in a loading buffer for gel electrophoresis. The T specific photoreaction with spermine and the G greater than T reaction with methylamine described here may be conveniently used in combination with the standard Maxam-Gilbert's reactions to provide independent confirmatory readings.
D001345:Autoradiography; D010584:Bacteriophage phi X 174; D001483:Base Sequence; D055598:Chemical Phenomena; D002621:Chemistry; D004262:DNA Restriction Enzymes; D004279:DNA, Viral / Q000528:radiation effects*; D010761:Phosphorus Radioisotopes; D010782:Photolysis; D013096:Spermine; D013941:Thymine / Q000032:analysis*; D014466:Ultraviolet Rays
D004279:DNA, Viral; D010761:Phosphorus Radioisotopes; D013096:Spermine; D004262:DNA Restriction Enzymes; D013941:Thymine