Use of uracil DNA glycosylase to control carry-over contamination in polymerase chain reactions.
Polymerase chain reactions (PCRs) synthesize abundant amplification products. Contamination of new PCRs with trace amounts of these products, called carry-over contamination, yields false positive results. Carry-over contamination from some previous PCR can be a significant problem, due both to the abundance of PCR products, and to the ideal structure of the contaminant material for re-amplification. We report that carry-over contamination can be controlled by the following two steps: (i) incorporating dUTP in all PCR products (by substituting dUPT for dTTP, or by incorporating uracil during synthesis of the oligodeoxyribonucleotide primers; and (ii) treating all subsequent fully preassembled starting reactions with uracil DNA glycosylase (UDG), followed by thermal inactivation of UDG. UDG cleaves the uracil base from the phosphodiester backbone of uracil-containing DNA, but has no effect on natural (i.e., thymine-containing) DNA. The resulting apyrimidinic sites block replication by DNA polymerases, and are very labile to acid/base hydrolysis. Because UDG does not react with dUTP, and is also inactivated by heat denaturation prior to the actual PCR, carry-over contamination of PCRs can be controlled effectively if the contaminants contain uracils in place of thymines.
Longo
M C
MC
Life Technologies, Inc., Gaithersburg, MD 20877.
Berninger
M S
MS
Hartley
J L
JL
D045647:DNA Glycosylases; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D003856:Deoxyuracil Nucleotides / Q000378:metabolism; D066298:In Vitro Techniques; D009699:N-Glycosyl Hydrolases / Q000378:metabolism*; D009841:Oligonucleotides; D016133:Polymerase Chain Reaction / Q000379:methods*; D051981:Uracil-DNA Glycosidase
D003856:Deoxyuracil Nucleotides; D009841:Oligonucleotides; D004259:DNA-Directed DNA Polymerase; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D051981:Uracil-DNA Glycosidase