Racemisation of Air Transported DNA Aerosols with the Help of a UVC Work Chamber

Inactivation of Air Transported DNA Aerosols with the Help of a UVC Work Chamber

Jochen Schulz, Andreas Bermpohl, Brigitte Dreiseikelmann

Introduction

Air transported DNA molecules can lead to misleadingly positive results for certain PCR applications. Particularly in PCR laboratories, where the same diagnostic systems are routinely employed, this can sooner or later lead to an accumulation of the target molecules to be verified. DNA particles can be contaminated by the adsorption on surfaces through the distribution of particles by the air, appliances, reagents, samples, controls, water etc. By the use of strong UVC spotlights (253.7 nm) typical photoproducts such as cyclobutane pyrimidine dimers and 6-4 photoproducts [1] are formed in the DNA. These photoproducts lead to a blockage of the polymerisation enzymes [2], such as the taq polymerase [3], which is frequently used in PCR technology. Air transported DNA molecules should thus be inactivated by UVC radiation when neighbouring pyrimidines can be found in the DNA region to be amplified. This results in a simple possibility to avoid air transported contamination in order to improve the consistency of results in the routine diagnosis. The technical implementation of a PCR chamber involves the integration of a 55-watt UVC spotlight in the upper part of a work chamber that is coated with polished stainless steel. (Ill. 1). The spotlight is located in a burner chamber, the chamber air is supplied via a ventilator. The problem of the “open UV radiation” is avoided by the encapsulation of the UVC spotlight. The radiation dose is optimised so that the air current is made to pass close by the UV burner. The decontaminated air is subsequently channelled back to the workroom via an air vent system so that the system functions as an air circulation system, thus generating an additive effect. Due to their construction from a special glass, the UVC spotlights used are not ozone- inducing. In addition to the closed burner chamber for the decontamination of air transported DNA aerosols, there is a separately switchable 55 watt radiator integrated in the workroom, which enables the disinfection of the chamber’s surface and contained materials when the face-plate is closed.

Material and Methods

The results check was carried out using a 500 bp fragment of the DNA of the bacteriophage lambda. 1.05 ml of the purified fragment with a concentration of 0.894 µg/ml was atomised in the chamber with a closed face-plate. A PARI LC PLUS Turbo atomiser and a PARI BOY compressor were used for the atomisation. The DNA aerosols were isolated by the adsorption of the aerosols in H2O (10µl) filled wells of a chilled microtitre plate. This microtitre plate remained protected from contamination 60 minutes after atomisation. Then it was exposed for 30 minutes to the air current in the chamber. The water from 18 wells was subsequently used as a test in a Finnzymes PCR (polymerase Kit F-551L without template, 35 cycles), which amplifies the atomised fragment. The DNA fragment from the atomised suspension was used as a positive test and water was used as a negative test. The amplificates were split in four 1.5% agarose minigels. A mix of cut λ-DNA Hind III and cut ΦX174- DNA Ilae III was used as length standard. After dying with SYBR Gold, the gels were photographed with a GelCam system and Polaroid 667 black-and-white films. The experiment was carried out once without the UVC radiation and once with UVC radiation of the circulating air. The L traces show the length standard, the P traces show the positive tests and the N traces the negative tests. In traces 1 to 36, 10µl amplificate + 3µl blue marker was applied on each trace. The results without UVC radiation show that 17 out of 18 wells (traces 1 to 18) of the microtitre plate with aerosols containing the 500 bp fragment were contaminated. On repeating the experiment with the UVC radiation of the chamber’s circulating air, no contamination by aerosols could be demonstrated in any of the 18 wells (traces 19 to 36).

Conclusion

The results show that DNA aerosols could be inactivated by the UVC radiation of the circulating air in the chamber. An effective radiation dose which penetrates the burner chamber is vital for this, as the development of UVC-induced radiation damage of the DNA increases with the dose (1, 4, 5). The sensitivity of the PCR as regards the atomised fragment is ≥ 8 molecules [6]. Due to the DNA concentration of the atomised solution, an aerosol with a diameter of 2.1µm may already be contaminous. Compared with the data of the atomiser manufacturer (PARI, 1997), which relates to the atomisation of a solution of comparable density, several billion potentially contaminous particles developed in this experiment. The number of DNA aerosols placed in the chamber’s air space greatly exceeds the contamination to be expected in a PCR laboratory and thus emphasises the contamination protection that can be achieved by using this chamber. Moreover, the chamber can not only offer protection from DNA molecules that are already in the air, but also from unintentional release of template DNA during the manufacture of the PCR extensions. The work was backed by the Carl Severing Berufskolleg Bielefeld, department for Biological Technical Assistance, as well as by the WEGEmbH Wirtschaftsentwicklungsgesellschaft [Economic Development Association] Bielefeld.

Literature

[1] Friedberg, E.C., Walker, G.C. and Siede, W. (1995) DNA Repair and Mutagenesis, ASM Press, Washington, D.C. [2] Chan, G.I., Doetsch, P.W. and Haseltine, W.A. (1985) Biochemistry, 24, 5723- 5728 [3] Wellinger, R.E. and Thoma, F. (1996) Nucleic Acids Research, 24, No. 8, 1578- 1579 [4] Ciarrocchi, G. and Pedrini, A.M. (1982) J. Mol. Biol., 155, 177-183 [5] Govan, H.I., Valies-Ayoub, Y., and Braun J. (1990) Nucleic Acid Research, 18, No. 13. 3823-3830 [6] Schulz, J. (2001) Dissertation at the Chair for Microbiology and Genetic Engineering of the University of Bielefeld [7] PARI (1997) Verneblercharakteristik [Atomiser Characteristics] PARI BOY + PARI I.C. PLUS Turbo

More detailed information on the PCR chamber: Biotec-GmbH, Elbrachtsweg 76, 33332 Gütersloh, Germany Tel: +49 (0)5241/904123, Fax: +49 (0)5241/904124 Captions: Working surface: B = 920 mm, T = 520 mm, H = 540 mm Results without UVC radiation: Results with UVC radiation: