Removal of contaminating DNA from polymerase chain reaction using ethidium monoazide.
The presence of exogenous DNA in PCR reagents and DNA polymerase is a common occurrence. In particular, the amplification of 16S rRNA genes with universal primers for non-culture-based study is often hampered by the formation of false positives. Here, we describe the use of ethidium monoazide (EMA) to eliminate contaminating DNA in a polymerase chain reaction. The advantage of the proposed methodology is the retention of the highly sensitive nature of PCR with the ability to amplify template DNA at concentrations lower than those of contaminating DNA. The treatment of PCR master mix with EMA concentrations that exceeded those required to remove contaminating DNA can interfere with the amplification of low-template concentrations. The methodology presented is straightforward and can be accomplished within 10 min.
Rueckert
Andreas
A
Thermophile Research Unit, Department of Biological Sciences, University of Waikato, Private Bag 3105, Hamilton, New Zealand. Andreas.Rueckert@alumni.TU-Berlin.de
Morgan
Hugh W
HW
D000345:Affinity Labels / Q000737:chemistry; D001386:Azides / Q000737:chemistry; D001406:Bacillaceae / Q000737:chemistry / Q000235:genetics; D004269:DNA, Bacterial / Q000737:chemistry*; D003666:Decontamination / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D019914:Taq Polymerase / Q000737:chemistry*
D000345:Affinity Labels; D001386:Azides; D004269:DNA, Bacterial; C014535:8-azidoethidium; D019914:Taq Polymerase