An enzymatic procedure for the purification of DNA restriction fragments without gel electrophoresis and ethidium bromide staining

A rapid and simple enzymatic method for the purification of a DNA fragment from a restriction digest was developed. The method is based on the two fea tures of exonuclease III activity: digestion of DNA from a 3'-OH at blunt o r recessed ends and failure to initiate digestion at DNA ends with four-bas e 3' overhangs. Herein, we establish a method for purification of a DNA res triction fragment without any physical separation via gel electrophoresis. The elimination of the ethidium bromide staining and ultraviolet irradiatio n steps should increase the quality and the safety of the purified DNA, a m atter of major concern in the perspective of human gene therapy. In additio n, since the method described does not use the visualization of the restric tion fragments or their difference in size it can be used to purify a DNA f ragment from a pool of DNA fragments with the same size even when microquan tities of material are available. (C) 2000 Academie des sciences/Editions s cientifiques et medicales Elsevier SAS.