Endogenous and Exogenous Contamination and Other Factors

Contamination and other factors intimate to the reaction, even apparently inert components, may cause inhibition at any of the points of molecular interaction to be discussed. Their modes of action are not yet understood, but there may be chemical or physical interference with the availability or activity of an essential reaction component. Contamination may be endogenous to the reaction components (e.g., sample, enzyme, tubes) or exogenous (e.g., bacteria, dust, pollen).

The most obvious origin of PCR inhibitors in endogenous contamination is compounds present in insufficiently purified target DNA. Inhibition can also arise from other endogenous sources, including reaction components. Commercial preparations of Taq polymerase, including a low-DNA product, have been shown to be contaminated with eubacterial DNA that originates in neither E. coli nor Taf (23). In particular, this may reduce the effective application of PCR using broad-range eubacterial primers because of the production of false-positive reactions. For more specifically targeted reactions, such contamination generally is of little importance. This study demonstrated that enzyme-contaminating sequences can be destroyed by UV irradiation. It also showed that microcentrifuge tubes from different manufacturers gave greatly different results. Strong inhibition has also been identified in some brands and batches of PCR reaction tubes by other workers, but the cause could not be discovered (73). Considerable variability in the performance of Taq polymerase within batch, by concentration, and between suppliers has been documented in a commentary that details many parameters affecting multiple arbitrary amplicon profiling (39). In addition to reaction failure, enzyme contamination may be manifested as spurious background bands during amplification-based DNA fingerprinting using methods such as random amplified polymorphic DNA (RAPD). By nature of their low stringency, such typing techniques rely on both specific and nonspecific priming and combine the detection of artifac-tual variation with true polymorphism. Contamination, concentration-dependent effects, spurious bands, and inhibition may complicate the interpretation of banding patterns and give rise to misleading results.

Like reaction tubes, other apparently inert components may be inhibitory. Cellulose and nitrocellulose filters were found to inhibit PCR (74). In this study, polycarbonate filters proved not to be inhibitory, perhaps because of differences in binding properties for DNA or its contaminants relative to those of cellulose-based filters. Mineral oil has been shown to have an inhibitory effect on PCR when irradiated with UV light (75). The inhibition is dependent on the UV dose. Unirradiated mineral oil has been reported as a facilitator in "oil-free" reactions containing high concentrations of nonionic detergents (76). This author suggested that components of detergent preparations (monomers, micelles, or impurities) could be responsible for adverse effects on the specificity of annealing. It is likely that detergents allow the greater solubilization of inhibitors that might otherwise aggregate and precipitate during preparation or in the reaction tube. Oil overlays may facilitate amplification by segregating inhibitors at the oil-water interface and remain an option in thermal cyclers designed for "oil-free" reactions.

Reaction failure may also be caused by exogenous contamination. This differs from the other sources of inhibition discussed earlier that result from compounds present in insufficiently purified DNA or from contaminants in reaction components. However it may involve the same mechanisms of inhibition. Inhibition may the result of even <10 grains of pollen that may enzymically digest an essential reaction component (46), glove powder (45,77), which may nonspecifically bind DNA, or other factors that enter reaction tubes when hygienic conditions are not sufficiently controlled.

Contamination can be prevented by Good Laboratory Practice (GLP) and scrupulous attention to aseptic technique that also serves to protect from cross-contamination of target sequences, although these can be dealt with using UV irradiation or uracil n-glycosylase (UNG) (78). Restriction enzymes have been shown to be inhibited, or their specificity altered, by multiple uracil substitutions in restriction sites (79). This may also have implications for the fidelity of PCR typing methods, as discussed later. Laminar-flow cabinets may be of use in preventing airborne contamination and cabinets equipped with a UV light source are available specifically to minimize contamination during PCR work. Ironically, the good practice of changing gloves may in some cases actually predispose reactions to failure. Powders from gloves were shown to have variable inhibitory effects on PCR, depending on manufacturer (45). If it is suspected that this is the source of problems, washing gloves and the selection of nonpowdered brands may be helpful.

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