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New Insights Into DNA Decontamination

Dr. Karl-Heinz Esser, multiBIND biotec GmbH,
Dr. Wolfram H. Marx, AppliChem GmbH and
Prof. Dr. Thomas Lisowsky, multiBIND biotec GmbH

Advanced experiments in gene technology demonstrate that even small amounts of free DNA molecules are sufficient to cause infections, recombinations or biological transformations [1,2]. Additionally, the complete decontamination of equipment and surfaces from any DNA molecules is important for biological containment, safety and prevention of artifacts in PCR amplification experiments. Using new methods available to detect extremely low levels of DNA molecules, we investigated the molecular mechanism of action of various commercially available DNA decontamination reagents. When tests were performed under extreme conditions, i.e. high concentrations of DNA and short incubations times, two major problems were apparent: First, none of the conventional reagents destroyed DNA molecules efficiently and second, all reagents contained components with corrosive or even toxic properties. Consequently, we saw the need to develop new solutions for effective DNA decontamination. Now, we present the latest developments and assays comparing the new AppliChem DNA decontamination reagent, DNA-ExitusPlus, with other conventional products. DNA-ExitusPlus guarantees fast and efficient destruction of nucleic acids without corrosive or toxic properties.

DNA decontamination reagents use three different molecular principles for destruction or inactivation of genetic material: modification, denaturation and degradation. Depending on the composition of the reagents, the different mechanisms may be combined. Safe DNA decontamination depends on the degradation of DNA into very small fragments. We developed a DNA degradation test to compare conventional deconta mination reagents with the novel DNA-ExitusPlus. This test allows sensitive quantification of the fragmentation process (see Figure 1 and 2).

One surprising result was the finding that some of the conventional reagents only use the principle of modification or denaturation for the inactivation of DNA molecules. This was determined in the test by the complete absence of any degraded DNA molecule. The genetic information encoded in these DNA strands is only masked but not destroyed. By chemical demasking reactions the genetic information of these molecules would be available again and could also be amplified by enzymatic reactions. Based on our knowledge about gene technology and the principles of recombination, we concluded that these reagents are no longer sufficient.

But even reagents that show degradation of DNA cause only limited and partial destructions. Hence, very large DNA fragments which contain the complete genetic information, still survive treatment (Fig. 1). Only our novel DNA-ExitusPlus achieves rapid and efficient degradation. By spraying DNA-ExitusPlus on surfaces in the lab complete decontamination will be achieved, when the whole surface, especially the critical points, are wetted. To remove heavy contaminations, we recommend to repeat the treament with DNAExitusPlus.

The strand breaking activity of DNA-ExitusPlus is independent of the size and sequence of the DNA fragments since the destruction is based on a chemical action and not an enzymatic activity. Large plasmids require a longer incubation time than small ones (e.g. a primer). Assuming that a theoretical nicking activity of 100,000 nicks per minute is present, all DNA fragments will be destroyed, independent of their size. Smaller fragments will disappear before the larger ones. Applying this theory to a test molecule (ccc form, 6 kb plasmid) onl y a small fraction of fragments with 200 to 500 bp in size will remain after 5 minutes. The nicks will be introduced statistically at any site, leaving not a single class of fragments. Therefore, PCR analysis will be negative.

Efficient degradation of DNA molecules by DNA-ExitusPlus was monitored by PCR analysis (see Figure 3) proving that no amplifiable DNA templates are present. Defined DNA samples where dried on the inner surface of reaction tubes, followed by treatment with DNA-ExitusPlus.

Today, only very different non-standardized PCR tests are used as controls for successful DNA decontamination. In case of large DNA control templates, low DNA concentrations, and high dilutions in the washing steps, evidence for a successful DNA decontamination is very limited. Therefore one has to be very cautious about using a single PCR test as evidence for complete decontamination since such a PCR test would also be negative in cases where the DNA is only modified or masked. For complete evaluation of the potential of a DNA decontamination reagent one has to use PCR analysis in combination with a sensitive DNA degradation test.

Another severe disadvantage of conventional reagents is revealed in a test for their corrosive potential. For this purpose different metal plates were incubated with aliquots of the reagents (see Figure 4). This test demonstrates that all conventional products contain aggressive chemicals with corrosive, harmful or even toxic effects. Known ingredients of conventional reagents are azides, mineralic acids like phosphoric acid or hydrochloric acid, aggressive peroxides or strong alkaline substances like sodium hydroxide. Therefore, even after only 20 minutes of incubation irreversible damages of metal surfaces are observed (see Figure 4). The newly developed and patented DNA-ExitusPlus exhibits its unique characteristics especially in this test. For all metal surfaces tested no corrosion is observed. DNAExitusPlus was also tested on many different plastic surfaces without any indication of damages (data not shown). DNA-ExitusPlus offers an efficient, gentle and environmentally safe alternative and proves its superiority towards all other commercially available decontamination reagents. DNA-ExitusPlus not only degrades and removes all DNA molecules with high efficiency but in addition is neither toxic nor corrosive.

Currently, the most effective method for DNA decontamination was believed to be autoclaving. Under the standard conditions for autoclaving, DNA molecules are degraded into fragments of 20 to 30 base pairs. In reality, PCR analysis demonstrate that even after autoclaving, larger DNA fragments can be identified [1]. This will be observed, especially when nucleic acids are protected by protein envelopes (e. g. viruses) or within microorganism (e. g. bacteria). Furthermore, autoclaving can only be used with heat-resistant materials and equipment that fit into the autoclave. Decontamination of laboratory benches or larger equipment is impossible.

The reaction time of DNA-ExitusPlus corresponds to the incubation time. The reagent dries within 10 to 20 minutes after spraying on a surface. Due to its chemical composition DNA-ExitusPlus is not heat-sensitive and does not contain volatile and harmful ingredients it could be tested at elevated temperatures for its effects on bacterial cultures and the bacterial nucleic acids (Fig. 5 and 6). Only the addition of DNA-ExitusPlus leads to an efficient degradation of bacterial DNA, while under the standard conditions (aqueous solutions, medium) the controls are always positive in terms of undegraded / partially degraded DNA.

In consi deration of the new findings, performing a PCR test alone to prove the complete removal of DNA molecules is not sufficient. Such a PCR test will be negative for decontamination reagents that modify or mask DNA, while the DNA is neither removed nor destroyed. The true determination of the decontamination potential of a reagent requires PCR analysis in combination with a DNA degradation test. Also, the standard method of destroying nucleic acids, namely autoclaving, has to be re-evaluated, since the latest data show that DNA from viruses and microorganisms are not inactivated properly.

These are the outstanding and unique characteristics of DNA-Exitus- Plus:
I. catalytic and cooperative effects of the components cause a very rapid non-enzymatic, non-sequence-specific degradation of DNA and RNA molecules.

II. all components of DNA-ExitusPlus are readily bio-degradable and not harmful or toxic for humans.

III. no aggressive mineralic acids or alkaline substances are used. Equipment and materials are not damaged or corroded even after prolonged incubation times.

IV. no harmful aerosols when spraying on surfaces.

[1] Elhafi, G. et al. (2004) Microwave or autoclave treatments destroy the infectivity of infectious bronchitis virus and avian pneumovirus but allow detection by reverse transcriptase-polymerase chain reaction. Avian Pathology 33, 3003-306.

[2] Burns, P.A. et al.(1991) Transformation of mouse skin endothelial cells in vivo by direct application of plasmid DNA encoding the human T24 H-ras oncogene. Oncogene 6(11), 1973-1978.



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