Reliability of PCR decontamination systems.
A major problem in the application of PCR is contamination with material amplified previously. Repeated PCRs result in the accumulation of intact and degraded amplicons and primer artifacts that can contaminate following amplification reactions. Post-PCR UV treatment and pre-PCR uracil DNA glycosylase (UDG) digestion have been recognized to efficiently inactivate or decompose intact amplification fragments. We show here that degraded amplification products and primer artifacts account for decreased sensitivity and may cause false-negative results. Our experiments indicate that partly degraded PCR products and primer artifacts containing sequences homologous to the primer oligonucleotides in the succeeding PCR reaction compete efficiently with sample DNA for the primers. The experiments done in this study may explain unexpectedly low PCR sensitivities reported in an increasing number of publications. In an attempt to solve this problem, we evaluated three post-PCR treatment methods to completely eliminate sequences competing for the amplification primers, namely, 8-methoxypsoralen (MOPS) or hydroxylamine treatment of amplified DNA and use of oligonucleotides containing 5'-ChemiClamps. However, all three methods did not sufficiently inhibit artificially produced carryover contaminations. In conclusion, false-positive results can be eliminated with UDG or UV treatment, but physical barriers are indispensable to avoid the occurrence of false-negative results.
Niederhauser
C
C
Laboratory of Food Chemistry, Institute of Biochemistry, University of Bern, Switzerland.
Höfelein
C
C
Wegmüller
B
B
Lüthy
J
J
Candrian
U
U
D016477:Artifacts; D001483:Base Sequence; D045647:DNA Glycosylases; D017931:DNA Primers; D003666:Decontamination / Q000295:instrumentation / Q000592:standards*; D005188:False Negative Reactions; D005189:False Positive Reactions; D008969:Molecular Sequence Data; D009699:N-Glycosyl Hydrolases; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D014466:Ultraviolet Rays; D051981:Uracil-DNA Glycosidase
D017931:DNA Primers; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D051981:Uracil-DNA Glycosidase