Reliability of PCR decontamination systems.

Détails

ID Serval
serval:BIB_7D0B633C49E8
Type
Article: article d'un périodique ou d'un magazine.
Collection
Publications
Titre
Reliability of PCR decontamination systems.
Périodique
Pcr Methods and Applications
Auteur(s)
Niederhauser C., Höfelein C., Wegmüller B., Lüthy J., Candrian U.
ISSN
1054-9803 (Print)
ISSN-L
1054-9803
Statut éditorial
Publié
Date de publication
1994
Volume
4
Numéro
2
Pages
117-123
Langue
anglais
Notes
Publication types: Journal Article Publication Status: ppublish
Résumé
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.
Mots-clé
Artifacts, Base Sequence, DNA Glycosylases, DNA Primers, Decontamination/instrumentation, Decontamination/standards, False Negative Reactions, False Positive Reactions, Molecular Sequence Data, N-Glycosyl Hydrolases, Polymerase Chain Reaction/methods, Reproducibility of Results, Ultraviolet Rays, Uracil-DNA Glycosidase
Pubmed
Création de la notice
09/11/2014 15:45
Dernière modification de la notice
20/08/2019 14:38
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