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 features of exonuclease III activity: digestion of DNA from a 3'-OH at blunt or recessed ends and failure to initiate digestion at DNA ends with four-base 3' overhangs. Herein, we establish a method for purification of a DNA restriction fragment without any physical separation via gel electrophoresis. The elimination of the ethidium bromide staining and ultraviolet irradiation steps should increase the quality and the safety of the purified DNA, a matter of major concern in the perspective of human gene therapy. In addition, since the method described does not use the visualization of the restriction fragments or their difference in size it can be used to purify a DNA fragment from a pool of DNA fragments with the same size even when microquantities of material are available.
Ratel
D
D
Inserm U318, CHU Michallon, Grenoble, France.
Dupré
I
I
Allamane
S
S
Berger
F
F
Verna
J M
JM
Benabid
A L
AL
Wion
D
D
D004247:DNA / Q000737:chemistry / Q000235:genetics*; D004262:DNA Restriction Enzymes; D004587:Electrophoresis, Agar Gel / Q000379:methods; D004996:Ethidium; D005090:Exodeoxyribonucleases; D006801:Humans; D008156:Luciferases / Q000235:genetics; D020932:Nerve Growth Factor / Q000235:genetics; D009838:Oligodeoxyribonucleotides / Q000302:isolation & purification*; D010957:Plasmids / Q000737:chemistry*; D015183:Restriction Mapping / Q000379:methods*
D009838:Oligodeoxyribonucleotides; D004247:DNA; D020932:Nerve Growth Factor; D008156:Luciferases; D005090:Exodeoxyribonucleases; C020133:exodeoxyribonuclease III; D004262:DNA Restriction Enzymes; D004996:Ethidium