Eliminating PCR contamination: is UV irradiation the answer?
The sensitivity of the polymerase chain reaction (PCR) can mean that even very low levels of contamination with the target DNA will result in a positive signal. At present this aspect is a major limitation in the use of PCR as a routine diagnostic method. By exposing PCR reagents to UV light, contaminating DNA can be inactivated, thus providing an opportunity to eradicate false positive reactions. UV irradiation was applied to PCR systems used for the detection of human cytomegalovirus (CMV) and human immunodeficiency virus (HIV) and shown to be effective in eradicating both laboratory encountered contamination and plasmid DNA (below 100 pg) added to PCR systems prior to UV exposure. The sensitivity of a PCR system to amplify the long terminal repeat (LTR) sequence of HIV-1 was not affected by the irradiation procedure; however, the ultimate sensitivity of a PCR system for the amplification of an early gene promotor sequence of the CMV genome was reduced 1000-fold. UV irradiation did not affect the size of the PCR product as determined by strand separating polyacrylamide gel electrophoresis of a 32P-labelled amplimer. Thus, a simple pre-exposure to UV light would seem a worthwhile step to incorporate into PCR protocols provided that the effects on sensitivity have been determined empirically for each PCR system.
Fox
J C
JC
Division of Communicable Diseases, Royal Free Hospital School of Medicine, London, U.K.
Ait-Khaled
M
M
Webster
A
A
Emery
V C
VC
D001483:Base Sequence; D003587:Cytomegalovirus / Q000235:genetics / Q000302:isolation & purification / Q000528:radiation effects; D004279:DNA, Viral / Q000032:analysis* / Q000528:radiation effects; D006678:HIV / Q000235:genetics / Q000302:isolation & purification / Q000528:radiation effects; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014466:Ultraviolet Rays
D004279:DNA, Viral