DNA sequencing by the chemical method.
This procedure is written for a novice with no chemical sequencing experience. It is assumed, however, that the investigator has experience with DNA fragment isolation and DNA labeling with [32P]dNTPs. A DNA fragment labeled at only one end with 32P or 35S is divided into four aliquots and subjected to base-specific modification reactions. Piperidine is then added to catalyze strand scission at the modified bases. Finally, the four reactions are subjected to electrophoresis on adjacent lanes of a high-resolution denaturing polyacrylamide gel. The gel is then autoradiographed and the sequence is read from the film.
Eckert
R L
RL
Case Western Reserve University, Cleveland, Ohio, USA.
D001331:Automation; D001483:Base Sequence; D004247:DNA / Q000737:chemistry*; D053938:DNA Fragmentation; D007202:Indicators and Reagents; D007553:Isotope Labeling / Q000379:methods; D010761:Phosphorus Radioisotopes; D012680:Sensitivity and Specificity; D013462:Sulfur Radioisotopes
D007202:Indicators and Reagents; D010761:Phosphorus Radioisotopes; D013462:Sulfur Radioisotopes; D004247:DNA