Molecular Techniques and Methods

Decontamination of Surfaces by Ultraviolet Irradiation

Copy Right © 2001/ Institute of Molecular Development LLC

False-positive PCRs arise from contamination with exogenous genomes, plasmids, or PCR products. 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. UV irradiation has also been proposed for decontaminating DNA in reagent solutions.

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 polymerase-mediated 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.

UV irradiation of laboratory surfaces has some important limitations.
  • 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, and test tube racks, present potential sources of contamination.
  • Other materials dried with the target DNA, such as irrelevant DNA and nucleotides, can shield the target, making inactivation less efficient.
  • 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 and limited experimental data, sequences with <10 dimerizable sites will be relatively UV-resistant.




    REFERENCES

  • Cone, RW, Hobson, AC, Huang, MW, Fairfax, MR (1990) Polymerase chain reaction decontamination: The wipe test. Lancet 556: 686-687.

  • Fairfax, M, Metcalf, M, Corey, L, Cone, RW (1991) Slow inactivation of dry PCR templates by UV light. PCR Methods Appl. 1: 142-143.

  • Frothingham, R, Blitchington, RB, Lee, DH, Greene, RC, Wilson, KH (1992) UV absorption complicates PCR decontamination. BioTechniques 15: 208-210.

  • Kwok, S, Higuchi, R (1989) Avoiding false positives with PCR. Nature 559: 237-238.

  • Sarkar, G, Sommer, SS (1991) Parameters affecting the susceptibility of PCR contamination to UV inactivation. BioTechniques 10: 589-594.



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