Escherichia coli exonuclease III enhances long PCR amplification of damaged DNA templates.
Recent development of the long PCR technology has provided an invaluable tool in many areas of molecular biology. However, long PCR amplification fails whenever the DNA template is imperfectly preserved. We report that Escherichia coli exonuclease III, a major repair enzyme in bacteria, strikingly improves the long PCR amplification of damaged DNA templates. Escherichia coli exonuclease III permitted or improved long PCR amplification with DNA samples submitted to different in vitro treatments known to induce DNA strand breaks and/or apurinic/apyrimidinic (AP) sites, including high temperature (99 degrees C), depurination at low pH and near-UV radiation. Exonuclease III also permitted or improved amplification with DNA samples that had been isolated several years ago by the phenol/chloroform method. Amelioration of long PCR amplification was achieved for PCR products ranging in size from 5 to 15.4 kb and with DNA target sequences located either within mitochondrial DNA or the nuclear genome. Exonuclease III increased the amplification of damaged templates using either rTth DNA polymerase alone or rTth plus Vent DNA polymerases or TAQ: plus PWO: DNA polymerases. However, exonuclease III could not improve PCR amplification from extensively damaged DNA samples. In conclusion, supplementation of long PCR mixes with E.COLI: exonuclease III may represent a major technical advance whenever DNA samples have been partly damaged during isolation or subsequent storage.
Fromenty
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INSERM Unité 481 and Centre de Recherches sur les Hépatites Virales de l'Association Claude Bernard, Hôpital Beaujon, 92118 Clichy, France. fromenty@bichat.inserm.fr
Demeilliers
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C
Mansouri
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A
Pessayre
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D
D000818:Animals; D004247:DNA / Q000235:genetics / Q000302:isolation & purification / Q000528:radiation effects; D004249:DNA Damage; D004272:DNA, Mitochondrial / Q000235:genetics / Q000378:metabolism / Q000528:radiation effects; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D005090:Exodeoxyribonucleases / Q000378:metabolism*; D006358:Hot Temperature; D006801:Humans; D051379:Mice; D009691:Nucleic Acid Denaturation; D016133:Polymerase Chain Reaction / Q000379:methods*; D051381:Rats; D013698:Templates, Genetic; D014466:Ultraviolet Rays
D004272:DNA, Mitochondrial; D004247:DNA; C093010:DNA polymerase, Thermus thermophilus; D004259:DNA-Directed DNA Polymerase; D005090:Exodeoxyribonucleases; C020133:exodeoxyribonuclease III