Protocol for ultraviolet irradiation of surfaces to reduce PCR contamination.

Abstract

1Department of Laboratory Medicine, University of Washington, Seattle, Washington 98105; 2Department of Pathology, Wayne State University School of Medicine, Detroit, Michigan 48201 False-positive PCRs arise from contamination with exogenous genomes, plasmids, or PCR products. (1~ Contaminated laboratory surfaces represent one of the many potential sources of exogenous DNA. UV irradiation of dry DNA provides just one tool in the arsenal necessary to prevent PCR contamination. Although this type of decontamination was recommended previously as a way to quickly damage any DNA left on exposed surfaces, ''~ further work has revealed a slow time course and sequence dependence. (2-4~ UV irradiation has also been proposed for decontaminating DNA in reagent solutions, (s-7) a procedure that has met with mixed reviews.(4,8,9) Most UV-induced DNA damage occurs via the formation of cyclobutane rings between neighboring pyrimidine bases, thymidine or cytidine. The cyclobutane rings form intrastrand pyrimidine dimers that inhibit polymerasemediated chain elongation. Dimer formation is reversible, establishing a steady-state equilibrium that favors monomers over dimers. As such, ~10% of the possible pyrimidine dimers actually exist in irradiated DNA at one time.(~0) UV irradiation of laboratory surfaces has some important limitations. First, the surface must be perpendicular to the light source to achieve optimal light intensity. Skewed surfaces dilute the intensity, and three-dimensional objects, such as pipettors, cannot be effectively decontaminated by UV light because only a fraction of the surface actually faces the light source. This drawback is compounded by the fact that almost all laboratory surfaces, such as pipettors, centrifuges, door handles, test tube racks, and so forth, present potential sources of contamination. ~ Second, other materials dried with the target DNA, such as irrelevant DNA and nucleotides, can shield the target, making inactivation less efficient. (9~ Third, very short PCR products may not contain adequate numbers of neighboring pyrimidines to make them susceptible targets. The UV sensitivity of an amplified region can be estimated by counting the number of dimerizable sites (neighboring pyrimidines: CT, TT, TC, CC) in each single strand of the sequence. Based on theoretical considerations (1~ and limited experimental data, (2'7) sequences with ~10 dimerizable sites will be relatively UV resistant. Meticulous technique remains the most important way to prevent contamination. However, UV irradiation can provide an additional margin of safety for keeping the PCR setup laboratory contamination free. This procedure describes a method for reducing DNA contamination on laboratory surfaces by using UV light to inactivate dried DNA. (2) Different procedures have been proposed for UV inactivation of DNA in solutions. (s-7~