Contaminations. Nucleic Acids. Problems & Practical Solutions. Take the Pink Link! www. .com

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1 Contaminations Problems & Practical Solutions ucleic Acids by Take the Pink Link!

2 a b o u t u s s o m e t h i n g Vision We have to start developing products today that you may need tomorrow. This, of course, is a quite demanding task. A task we can successfully tackle thanks to the highly educated people employed at AppliChem. All of them also strive to extend and to maintain an international network. Only by staying in close contact with colleagues in the various fields in many countries all over the world will it be possible to manage these current tasks. Today, AppliChem has subsidiaries in Scandinavia, Asia and the US; all of them play an essential role in the further development of our product portfolio. We intensively and continuously strive to become better and better as partners of our customers, as chemists and biologists supporting chemists, biologists and other scientists. We are convinced that your work will benefit from this. Quality Management We will provide nothing but the best to our customers this is the motto of our quality management. Every single AppliChem employee knows: a product features the extraordinary and uniform quality demanded by us, once it meets the exacting requirements of our customers. This excellent quality of each individual product is above all the result of an optimum collaboration of all members in the overall process. Every employee bears a maximum of responsibility. We are well aware of the fact that our customer requirements and the market are subject to a continuous process of change. Our quality management works daily on the improvement and further development of all process flows. The certification in accordance with DI E ISO 9001 : 2000 guarantees the highest possible quality standard in all process phases. Quality control The high demands on the quality of all our products are a challenge that we are ready to face on a daily basis. Each and every employee gets involved at every stage of the order processing with his knowledge and experience from the receipt of goods to the production laboratory to storage. Our quality assurance is applied in the production of our AppliChem products and, of course, in the custom synthesis at an identical level. AppliChem stands for a continuous control of the product quality during production, always in view of the customer requirements. We permanently test in accordance with the legal requirements and the high standards of our own AppliChem test specifications. Of course, we realize additional, customer-specific tests at any time. Part of this process is the permanent supervision of the testing equipment, among other things.

3 c o n t e n t s Introduction ucleic acids are omnipresent 2 Chapter 1 Structure and chemistry of nucleic acids 3 Chapter 2 Risk potential of free nucleic acids 6 ature & Artificiality: atural and artificial nucleic acids 7 atural recombination and genetically modified nucleic acids: Biological safety under scrutiny 18 Chapter 3 Ancient DA: Preventing contamination in DA clean room laboratories 28 Chapter 4 Sources of contamination and decontamination products 30 Chapter 5 ew solutions for practical application: Products and applications 34 ucleic acid surface decontamination I 35 ucleic acid surface decontamination II 42 Regeneration of DA binding columns with silica matrix 44 Regeneration of silica-based anion exchanger 55 ucleic acid decontamination during autoclaving 61 Literature AppliChem ucleic acid decontamination 1

4 introduction ucleic acids are omnipresent ucleic acid molecules, DA and RA carry the genetic information of living cells (Alberts et al. 2002). By the spread of living organisms on earth, nucleic acids today are omnipresent in our environment, caused by living organisms, as well as by the release of nucleic acids from dead cells (Pääbo et al. 2004, Mulligan 2005). By desiccation and mineralization, encapsulated nucleic acid molecules of dead organisms were conserved in the environment for millennia or even millions of years (Green et al. 2006, oonan et al. 2006). Researching this fossil genetic information ( ancient DA ) opens a completely new view at evolution, but also at archeology (Bollongion et al. 2006, aak et al. 2004, oonan et al. 2006). Such new fields of research are based on new methods for the treatment of nucleic acids. Good examples are methods for the purification, analysis and amplification of nucleic acids (Sambrook & Russel 2001). A fast and reproducible isolation and cleaning of nucleic acids was not possible until the development of the silica matrix. This also established the automation of these processes for high throughput. This again, was a prerequisite for the optimization of DA sequencing to such a level, as to permit the sequencing of complete genomes to become standard. One milestone of this work is the decoding of the entire human genome (Collins et al. 2003). Finally, for a quick amplification of DA and RA molecules, the Polymerase Chain Reaction (PCR) technology proved to be decisive. Today, this method has been refined to detect even individual molecules (Innis et al. 1990). All these methods have been the driving force for the fast development in genetic engineering over the last 30 years (Demain 2001). Recombinant techniques in genetic engineering laboratories now produce more and more artificial nucleic acid molecules (Bensasson et al. 2004). These recombinant nucleic acid molecules are important tools for research and development while making completely new demands to biological safety, since an uncontrolled release or widespread distribution has to be prevented (Kaiser 2005a, 2005b). Keeping the distribution of nucleic acids in check by employing efficient decontamination products is therefore a current topic. On the one hand, an efficient decontamination is necessary to appropriately use highly sensitive processes for analysis, as incorrect results due to contaminating nucleic acid molecules can be observed with increasing frequency. Such incorrectly positive test results can have serious consequences for medical diagnostics, for criminology or for scientific analyses. On the other hand, the unrestricted distribution of problematic nucleic acid molecules, such as multi-resistance cassettes, oncogenes, recombinant infectious, viral genomes, etc. must be prevented (Bensasson et al. 2004, Burns et al. 1991, Davison 1999, Dzidic & Bedekovic 2003, Guyot et al. 1999, o et al. 2001, Kaiser 2005a, Lorenz & Wackernagel 1994). To our knowledge, no technical literature is available covering the current problem of nucleic acid decontamination. It is the objective of this brochure to collect the most important data and facts. 2 ucleic acid decontamination AppliChem 2008

5 chapter 1 Structure and chemistry of nucleic acids Basic elements of the nucleic acids are the nucleotides. They are composed of 3 important components: the base, sugar and a phosphate residue. The phosphate groups release + ions in the water; thus, these molecules act as weak acids, which eventually led to the nomenclature of nucleic acid (acid inside the nucleus ). The sugar types are ribose and deoxyribose. The former can be found in the ribonucleic acids (RA), the latter in the deoxyribonucleic acids (DA). Sugar and phosphate are called the sugarphosphate backbone, the supporting structure for bases. They don t contribute to the real genetic information, as they are always identical. Both types of nucleic acids get by with different nucleotides each that are polymerized via the phosphate residues. The bases that are, in fact, the carriers of the information are eponyms of the respective nucleotides and can be subdivided into purines (adenine, guanine), or pyrimidines (cytosine, uracil, thymine), respectively. The order of the bases provide for the sequence. Today, it is assumed that the first biological genetic information was stored in the shape of single-stranded RA molecules (Vlassov et al. 2005). These, however, are less stable than in the form of DA molecules, but additionally offer certain catalytic characteristics. Therefore, especially at the beginning, they particularly accelerated the evolution of biological molecules in their capacity as multifunctional molecules. Currently, functional RA molecules can still be found for example as Transfer-RAs for the binding of amino acids, as ribosomal RA inside the ribosomes for the 2 2 Purines 2 Adenine 2 O O 2 C C C C O O C C 2 Pyrimidines C C 2 Guanine O O O 3 C C C C C O C O Cytosine Thymine Uracil O O C C O O C C 2008 AppliChem ucleic acid decontamination

6 OC 2 O O 2 C C O Deoxyribose O C C O OC 2 O O O O O P O O C C C O O C C Ribose A C AT CG CG AT AT CG CG AT A C C A C G A A T G G T GC CG CG TA GC CG CG TA G C G A G C C G C T base pairing double strand double strand with breakages single strand with breakages Double strand with breakages after denaturation protein synthesis and as catalytic RAs in ribozymes or telomerases (Chen et al. 2007, Isaacs et al. 2006). For a better conservation of the genetic information, evolution provided a pairing of the single strand with a complementary strand to ensure that in case of a damaged strand a second backup copy would be available. This is possible since the bases can be matched in a precisely defined way by hydrogen bonds and a steric fit. Only one adenine (A) can match up with one thymine (T) or uracil (U) and one guanine (G) with one cytosine (C). In the shape of a double strand, nucleic acid molecules are much more stable than in that of a single strand. By base pairing, reactive chemical groups are protected from the outside. Thus, undesired reactions with other foreign molecules, as well as chemical modifications aren't possible as easily as they are with single strands. Further, even individual breakages in the sugar phosphate structure have serious consequences for the single strand, since the molecule immediately breaks apart completely. In contrast to that, a double strand can suffer many such single strand breakages ( nicks ) without breaking apart, since the paired strands support the coherence of the overall molecule as long as the single strand breakages occur in different areas of the molecule. It is after denaturation of the molecules only that strand breakages in double stranded DA leads to a decomposition into fragments. As an increased stability has many advantages, a gradual transition from RA molecules to DA molecules for primary genetic information storage occurred. As a consequence, virtually all organisms living today make use of the double stranded DA for primary genetic information storage. owadays, the far less stable RA molecules mainly serve for the short-term relay of information in the shape of messenger RA for the protein ucleic acid decontamination AppliChem 2008

7 TCTCACAGTG TACGGACCTA AAGTTCCCCC ATAGGGGGTA CCTAAAGCCC 51 AGCCAATCAC CTAAAGTCAA CCTTCGGTTG ACCTTGAGGG TTCCCTAAGG 101 GTTGGGGATG ACCCTTGGGT TTGTCTTTGG GTGTTACCTT GAGTGTCTCT 151 CTGTGTCCCT ATCTGTTACA GTCTCCTAAA GTATCCTCCT AAAGTCACCT 201 CCTAACGTCC ATCCTAAAGC CAACACCTAA AGCCTACACC TAAAGACCCA O C TCAAGTCAAC GCCTATCTTA AAGTTTAAAC ATAAAGACCA GACCTAAAGA Section from the sequence of the phage T7 DA A O T O C 3 O A O T G O C (1) AT base pair (2) GC base pair O G O C biosynthesis. ere, the faster degradation and the reduced half-life of these molecules even show regulatory advantages for obtaining a timely and quantitydependent exploitation of information. Today, single stranded or double stranded RA can only be found in viruses or viroids (Becker 1999) AppliChem ucleic acid decontamination

8 chapter 2 Risk potential of free nucleic acids For several years, we have made free nucleic acids a topic for our studies and we have come to the conclusion that the significance of this topic should not be underestimated, but given more attention instead. We define as free nucleic acids those nucleic acids that are no longer enveloped by a cell or nuclear membrane. The first important distinction, of course, concerns the natural incidence of nucleic acids on the one hand and artificial molecules created through genetic engineering and molecularbiological methods on the other hand. The naturally prevalent nucleic acids are a source of contaminations in a variety of areas. Forensic tests, for instance, microbiological, medical analyses and analyses of ancient DA samples require a working environment that is free from nucleic acids (Balogh et al. 2003, aak et al. 2004). Similarly, there are natural multi-resistant plasmids among clinically relevant bacteria that increasingly pose problems in the area of hospital hygiene (Cohen 2000, Croft et al. 2007, Knobler 2003, Tillotson & Watson 2001). Since the early days of genetic engineering some 30 years ago, more and more recombinant DA and RA molecules are created, the controlled decontamination of which is essential for biological safety (Bensasson et al. 2004, Brower 1998, DeVries & Wackernagel 1998, o et al. 2001, Bush 2004). The resistance genes for antibiotics used in genetic engineering concern the same antibiotics that are being employed in therapeutic treatment (Amyes 2001, Guy et al. 1999, Levy & Marshall 2004, White et al. 2001). At the same time it becomes evident that the transformation of living cells and bacteria also occurs under natural conditions, i. e., not by experimental work (Burns et al. 1991, Lorenz & Wackernagel 1994, Maiden 1998, Mercer et al. 1999, Steinmoen et al. 2003) Preventing an uncontrolled release of recombinant nucleic acids must therefore be an integral part of all genetic engineering work. ucleic acid decontamination AppliChem 2008

9 environment ature & Artificiality: atural and artificial nucleic acids Dr. Wolfram Marx, AppliChem Gmb, Germany As we all know, not everything existing in nature was created in a natural way. Man likes to give a helping hand. The release of genetically engineered plants in field-grown tests and in agricultural production, the formation of resistance in microorganisms against antibiotics and antimycotics (hospital germs, livestock farming) or gene therapy (viral vectors) have become part of the discussion in public. Special aspects of this debate are the risks and dangers for man and the environment emanating from free nucleic acids also known as naked nucleic acids. ot least due to recent findings from research, this topic is repeatedly discussed in a controversial manner. These discussions carry the entire spectrum of opinions from the probably intentional distortion of the truth (fear-mongering) to trivialization. I recently came across these topics, when AppliChem launched DA-ExitusPlus, their new product for DA decontamination, which had been developed in collaboration with multibid Biotec in Cologne. The special feature of this product is that nucleic acids and proteins are destroyed and not only modified or denatured as is the case with most other products. What happens with bacterial or viral DA/RA that is, in fact, released by the treatment with conventional decontamination products? Free nucleic acid is the term for DA and RA that is not bound by proteins or protected by a protein envelope. What really happens with recombinant nucleic acids created in laboratories and what happens with antibiotic resistance genes or other nucleic acids, released from genetically engineered, manipulated (micro-)organisms (GMOs) after they have died? At present time, studies are under way to research the absorption of such nucleic acids by human, animal and plant cells and, in particular, by microorganisms in the laboratory and in nature. ere, again, the spontaneous absorption is of high interest, as this way represents the real danger for the environment as man has really no control over it. In a first step, we need to find out what occurs naturally in the environment and what, by contrast, is produced by man in an artificial way. atural nature: atural exchange of nucleic acids and free, natural nucleic acids Science has often taken nature as a paradigm in the development of new technologies (e. g., PCR) or simply used natural molecules and mechanisms as tools (e. g., restriction enzymes, plasmids). This way, the protracted breeding process could be cut short. Man recombines the way he wants to and decides to the large extent about selection. In other words, man creates a revolutionary evolution in the sense of an accelerated, imagined progress quick and dirty, with all its unknown consequences. Is it by accident that evolution and revolution are distinguished by a single letter only? Reproduction and GT: The naturally intended exchange of nucleic acids for one is effected by natural reproduction within one species ( vertical genetic transfer ). During the procreation of offspring (reproduction), the genes of the parents are recombined during the fusion of the ovule and the sperm. e who gains advantages in his habitat by this process will prevail; he who does not has to look for a niche (specialization) or gets the short end of the stick. A mating of direct relatives was not envisioned by nature, since 2008 AppliChem ucleic acid decontamination

10 nature always looks for the highest possible degree of genetic recombination. The consequences of inbreeding if viable cannot be overlooked (e. g., overbred dogs). Alternatively, in contrast to natural reproduction, there is a nucleic acid exchange between species termed horizontal gene transfer (GT). By principle, a GT can be obtained by different mechanisms: 1.) Zygosis a direct exchange of DA between cells by physical contact; 2.) Transduction DA transfer by viruses. Some infectious viruses are capable to move between the DA of host organisms; 3.) Transformation direct absorption of DA from the environment, originating from the soil, water or, for instance, digestive tracts. The GT is a central point in the GMO discussion. The absorption or the exchange of genetic information by or between microorganisms in nature has been a known fact for a long time. The most recent example was the development of highly infectious influenza viruses from avian flu pathogens (type 51) and human flu viruses. Through the exchange of plasmid DA, bacteria develop resistances against antibiotics. Particularly dreaded are the resistant hospital germs, cause for the failure of many therapies. Without the existence of the selection pressure by antibiotics, the resistance would not offer the bacteria an advantage for survival. Their progeny would even take longer, as they also have to reproduce the genetic information of resistance. For more than thirty years, viroids have been known, infectious envelope-free ribonucleic acid molecules (circular closed and single-stranded), identified as causative organisms of diseases in plants. Their genetic information consists of only 200 to 400 nucleotides, making them about 20 times smaller than the smallest known viruses or bacteriophages. On the one hand, they show similarities to transposons, direct, as well as inverted repeats. On the other hand, sequence homologies exist with small nuclear RAs, which play an important role in the splicing of animal genes. This would also explain the pathogenicity, namely interaction with natural splicing. If this is as it indicates to be an effect comparable to RAi, nature was again faster in its ingenuity than man. Since the viroid genome does not encode for proteins, the effect triggering the symptoms has to stem from the sequence and the structure of the viroids (ribozymes; interaction with introns inside the host DA, etc.). Most probably, injured cells are required for infection to take place. Transport within the plant is realized by cell-cell connections (plasmodesms) and the nutrient transport system (phloem = stele). Viroid RA is highly resistant against enzymatic digestion, since it does not have any free ends. It should also be remarked that the genome of the human pathogenic hepatitis D virus (DV) shows high similarities to the viroids. 8 ucleic acid decontamination AppliChem 2008

11 or how dangerous is man? I am going into this much detail, since these smallest pathogens show natural mechanisms of evolution in an excellent way and a further development can be expected. Let s bear in mind that evolution has not stopped. As already mentioned, viroids show similarities to transposons an important tool in biotechnology. A transmission of the viroids from plant to plant by insects is possible. Up to now, infections caused by viroids normally occur in the tropics and subtropics where they also affect important agricultural crops (potato, lemon, cucumber, avocado, etc.). Today, the worldwide trade and exchange of goods provides for a fast spreading of diseases, animal and plant species that would normally have difficulties in overcoming continental separations. Among man, free nucleic acids can also be observed, for instance i) fetal, cell-free nucleic acids in the blood of pregnant women, ii) plasma nucleic acids and nucleic acids in the urine, used to diagnose diseases (tumors) or iii) autoimmune mediated diseases that can lead to anti-da antibodies (systemic lupus erythematosus). These types of free nucleic acids have not yet been studied in every detail and may be present in apoptotic bodies enveloped by proteins. The so-called naked viruses (e. g., parvovirus, adenoviruses, enteroviruses, rhinoviruses) are not really naked either. Their nucleic acids are associated with proteins; however, they are lacking the envelope itself, which is typical for viruses. Apoptotic bodies: One consequence of the programmed cell death (apoptosis) is the dissection of genetic material by nucleases into bigger fragments. These are released as apoptotic bodies upon the disintegration of the cell membrane. The horizontal transfer of DA by the absorption of apoptotic bodies by phagocyting cells has been shown in vitro (phagocytosis = absorption of solid particles not from the cells). Cells carrying the Epstein- Barr-Virus-DA integrated in the genetic information were co-cultivated with other cells. The absorption of the DA in the shape of apoptotic bodies and their expression inside the cell nucleus of the co-cultivated cells could be proven (olmgren et al. 1999). This way, cells that do not possess a receptor for the virus on the surface can be infected as well. Free DA inside the plasma: the origin of the free DA (and RA!) and the shape of the nucleic acids could not yet be determined. In the case of tumor DA (e. g., K-ras, Epstein-Barr-virus DA) it may stem from dead tumor cells or circulating tumor cells. either is it known, whether the release of nucleic acids is an active or a passive process. Studies have shown, however, that the tumor DA can be absorbed by other cells (transfections) and that the genetic information inside the transfected cells is also expressed (Garcia-Olmo et al. 1999, Garcia-Olmo et al. 2000). The authors proposed the term genometastasis. Free DA inside the cytoplasm: Viral infections or damage to tissue can trigger autoimmune reactions. In such a case, an abnormal expression of the major histocompatibility complex (MC) genes of classes I and II and of other genes for antigen processing or presentation occurs inside the cells. The same phenomenon can be observed in non-immune cells when admitting doublestranded nucleic acids (sequence independent). 25 base-pair sized DA pieces inside the cytoplasm are sufficient to lead to an increased gene expression (Suzuki et al. 1999). Since in tissue damages doublestranded genomic nucleic acids are released as well, the artificial addition (transfections) of nucleic acids might reflect a natural mechanism. Authors Suzuki et al. speculated on the possibility of genetic therapeutic treatments with respective plasmids or other plasmid DA vaccines being able to trigger such reactions. Transposons ( jumping genes, transponable = mobile genetic elements) are rare, but they occur in all types or organisms. These are short DA sequences that can be replicated (multiplied) and change their position inside the genome or on the plasmids, i. e., they jump to different positions. The locations, where transposons are integrated into the genome are usually random. At its ends, a transposon contains nearly identical sequences and reverse, repeated sequences going into the opposite direction ( direct and indirect repeats ). The transposon encodes for the transposase enzyme, which, in turn, catalyses the insertion into the chromosome. Thus, the insertion process is independent from the recombination system of the host cell. Prokaryotic transposons can carry genes that provide the host with new phenotypical characteristics, such as, for instance, a resistance to antibiotics. Depending on their mode of actions, transposons can be divided into two different groups: i) Class I transposons proliferate and move inside the genome 2008 AppliChem ucleic acid decontamination 9

12 by creating RA copies ( reverse ) transcribed back into DA. The new copies can again be introduced into the genome. In their behavior, these transposons correspond to that of retroviruses (e. g., IV) and are thus termed retrotransposons. ii) Class II transposons carry in their sequence the information of the transposase enzyme, cutting it out from the DA with the possibility to introduce it at another location. The majority of natural mutations is caused by transposons. From a human point of view, viruses, and retroviruses in particular, have the big disadvantage of not being particularly orderly, or to put it another way: they are quite variable. Errors and changes are thus tolerated in the own genome or even used for fast modification. Those integrating into the host genome tend to take a few neighboring nucleic acids or complete genes of the host along once they leave it again. Under the influence of the, at least in part, very strong promoters and mutations oncogenes evolved (e. g., v-src, v-ras, v-myc, v-fos). The knowledge of the behavior of viruses is important, when speaking about the utilization of those in gene therapy or another biotechnological exploitation and molecular-biological treatment. They are, as already mentioned previously, not really free from proteins; however they are handled in protein-free form (see below). Dead organisms and the stability of free nucleic acids: Considering that to human cells divide daily, and that a similar number of cells have to die to maintain the tissue homeostasis, some 1 10 g of DA waste is produced daily. In nature, there is a continual coming and going: old people die; newborns see the light of day. Those dying leave a huge quantity of DA behind entering the soil. To a great extent, the soil consists of silicates, quartz sand or clay materials showing similar characteristics to those of artificial DA purification matrices. These materials can bind free nucleic acids and thus even stabilize them. Consequently, DA remains a part of nature much longer than originally anticipated. Even 60 days after free nucleic acids were introduced into the soil, bacteria could be transformed with intact DA molecules (Chamier et al. 1993; Romanowski et al. 1993). This finding is particularly interesting, as it was previously assumed that free nucleic acids were unable to survive for longer periods of time once outside the protection of a cell, where nucleases are kept under control. The extended availability of these nucleic acids increase the probability or risk, depending which way you look at it of (micro) organisms to absorb such nucleic acids: released nucleic acids of animals, plants and microorganisms, or from contaminations in laboratories. It has to be mentioned, though, that the uptake of plasmid DA by soil bacteria is much less than linear, chromosomal DA. The reason for this may be the reduced availability of the small plasmid molecules by binding to the soil (Chamier et al. 1993; ielsen et al. 1997). In the publications it is also stated that the transformation of the bacteria requires an exponential growth of the subject of the case study (A. calcoaceticus). atural soil of this type being very poor in nutrient content and permitting no growth, the transformation efficiency is extremely low. ielsen et al. come to the conclusion that a transformation of A. calcoaceticus probably does not occur, unless the nucleic acids were recently released and the bacteria are in their growth phase. Artificial, yet present in nature and the laboratory Man has added a number of sources of free nucleic acids: viral vectors in gene therapy, sera (vaccines), cloning vectors in molecular and cell biology especially those to study oncogenes, complete viral genomes, and transposons, to name but the most problematic ones. Transposons: In laboratories, for example, they are employed in the genome-wide transposon mutagenesis in yeast, plants, mice or fruit flies (P-elements). This technology permits the creation of a high number of mutations that are then studied based on the phenotype and the specific gene expression pattern. Transposons can be used to insert reporter genes and regulatory elements among others into the host genome. For details on the yeast technology, we recommend Current Protocols in Molecular Biology (2000) Supplement 51. On mouse transposons and techniques, I would recommend Roberg-Perez et al Viruses: In the purification process of nucleic acids from human-pathogenic microorganisms, and viral nucleic acids in particular, the bigger part of infectiousness is lost. The degree of infectiousness of free nucleic acids depends on the virus type. A virus possesses respective enzymes and structures on its surface that enable or expedite the binding to and integration into the host cell, respectively. Free nucleic acids lack same; however, they may be able to resort to the cell's own enzymes, for instance for replication. The RA extracted from flaviviruses or alphaviruses is infectious, if inoculated intracerebrally in newborn mice. Therefore, this RA is classified as the same safety hazard as the complete virus particle. The risk of infections of cells in standard media by free viral nucleic acids is lower by a factor of 10 6 to The variety of hosts subject to infections, however, is much wider, as the special 10 ucleic acid decontamination AppliChem 2008

13 receptors on the cell surface for binding the virus with protein envelope are not required (see above). Due to the fact that nucleic acids are more stable than proteins, infectious nucleic acids can be isolated from viruses inactivated by heat. DA copies of certain RA viruses are infectious (e. g., poliovirus). Also, free nucleic acids sidestep the immune defense by antibodies formed against viral proteins. aked linear RA is extremely unstable, because ribonucleases (Rases) can be found just about anywhere. Therefore, the theoretical risk in this case is minimal. This and additional information is offered at Cloning vectors: To a great extent, the activity of a gene is controlled by promoters, typically found immediately adjecent to the gene in the genome. In order to sustain a big or at least a sufficient quantity of the genetic product, in most cases strong or constantly active promoters are used in the artificial genetic constructs. Among them are, for example, the promoters of the Cytomegalovirus (CMV), the uman Immune Deficiency Virus (IV), and the Simian Virus (SV40) and, in the case of constructs for plants the Cauliflower Mosaic Virus (CaMV) promoter. Task of the viral promoters is the conversion of the cell metabolism to the virusspecific production. Promoters that are also functional in the human cell are problematic. If they are integrated in the genome, they can take over the regulation of the activity of neighboring genes. In addition, most cloning vectors carry an antibiotic resistance gene for selection purposes in most cases the bone of contention in the discussions on released nucleic acids. As already mentioned above, cloning vectors with oncogenes require particular attention. For the handling of nucleic acids with oncogenic potential the ZKBS (Central Commission for Biocontainment) recommends that persons with bigger skin lesions (open eczemas, wounds and infections) or with a pronounced verrucosis (warts) should not conduct any work with such nucleic acids. many years, antibiotics were used as growth stimulant in livestock husbandry rather than for medical reasons. Estimates go as high as 9,000 tons of antibiotics per year within the European Union fed to livestock, a third of which for medical reasons. Today, feeding antibiotics as a growth stimulant is prohibited in the EU. The most recently approved antibiotics were not used in human medicine. Resistant hospital germs result from incorrectly used antibiotics, not from genetic engineering. With increasing frequency, this incorrect use of antibiotics in human medicine is deplored. On the one hand, even in case of minor infections antibiotics are being prescribed. Frequently, incorrect doses are given doses that are too low giving rise to formation of resistant strains. Antibiotics are released undigested into the environment through the digestive tract in the clinical field, as well as in agriculture. ucleic acids in gene therapy: These nucleic acids are employed for the treatment, the healing or the prevention of diseases. Possible targets are either somatic cells (body cells) or germ cells (egg, sperm). Whereas in gene therapeutic treatment of somatic cells only the genome of the recipient is changed, changes can also be transmitted to offspring when treating germ cells. The latter is not taken into consideration, not least for ethical reasons. Gene therapeutic treatment has nothing to do with cloning, since no genetically identical being is created. Ideally, gene therapy has to be conducted only once, if the transgene is integrated into the genome in a stable way (e. g., plasmids capable of transposition = transposon system). ere, the diseasecausing gene is replaced with the therapeutic gene (homologous recombination), or a healthy gene is additionally placed in another position of the genome. Further, a repair or the correct regulation might be feasible as mechanisms. Pathogenic, resistant microbial and fungi strains: Despite the fact that these bacteria and fungi strains are not free nucleic acids, mentioning them at this point is worthwhile. They in particular are in genetic exchange with their conspecifics and are capable of spreading recombinant nucleic acids or transfer acquired resistances. The increasing resistance formation among pathogens should not be inferred from the release of microorganisms from the laboratories modified by genetic engineering, but rather from the hospital sewage and from agricultural production (Kümmerer et al. 2002). Over a period of 2008 AppliChem ucleic acid decontamination 11

14 There are different ways to introduce a new gene into the respective cells i) Viral vectors; viruses have found a way to infiltrate the host cell with their disguised nucleic acids and to express them pathologically. The disease-triggering genes are replaced with the healing genes. The vectors used are modified retroviruses, adenoviruses, adeno-associated viruses and herpes simplex viruses. As virus genomes are usually quite small, the size of therapeutic DA that can be introduced is restricted. ii) Free nucleic acids, requiring huge quantities of DA and can be used for certain tissues only, iii) Liposomes; the nucleic acids are wrapped in a lipid envelope that can fuse with the cell membrane, or iiii) a human artificial chromosome (AC), which is very big and thus difficult to insert into the cell. It is a prerequisite for all of the above that the new gene is accepted and that the correct expression and regulation has to be assured. There must be no triggering of an immune response. Deaths traced back to an immune response to viral vectors have been described (example: Jesse Gelsinger 1999). In addition, there is a theoretical risk of viral vectors in the body reacquiring the capacity to trigger diseases. Until now, an exclusion of toxicity, of an immune or an inflammation response, the gene control, or the control of the insertion into a certain targeted sequence (place of integration) are not possible with viral vectors. It is possible, that other genes are mutated or destroyed (inactivated), that their regulation is changed and that other diseases are triggered. For instance, symptoms similar to leukemia could be observed (refer to articles/2004/01/23/gene_therapy.php; Davé, U.P. et al. (2004) Gene therapy insertional mutagenesis insights. Science 303, 333; acein-bey-abina et al. (2003) LMO2-associated clonal T-cell proliferation in two patients after gene therapy for SCID-X1. Science 302, ). In somatic treatments, gene transfer vectors were found in seminal fluids. Accidental changes of the genome in germ cells cannot be ruled out. Meanwhile, the use of ribozymes, antisense RA, sira, and shra ( small hairpin containing inhibitory RA ) are being tested to downregulate the gene expression. Genetic immunization for antibody production Originally, antigens for immunization are produced with bacterial expression plasmids and the purified protein is dissolved in adjuvants and injected into the laboratory animal. The body then raises antibodies against the respective proteins. A more recent technology circumvents the intermediate step of bacterial expression, which has the additional disadvantage that the antigens are not modified posttranslationally as in mammal cells. In genetic immunization, the research animals are intravenously injected with an expression plasmid, either into a tail vein ( ydrodynamic Tail Vein Delivery = TV) or a limb vein ( ydrodynamic Limb Vein Delivery = LV). In the case of the TV, the protein encoded on the plasmid DA is primarily expressed in hepatocytes, the spleen, the lungs and the myocardium, or the skeletal muscle (LV), respectively. This method is currently used with mice, rats and rabbits. Thus, we are speaking of direct transfections. The cells produce the antigen with all naturally occurring modifications (e. g., glycosylation), and the body reacts by producing antibodies. Parts of the expression plasmids are controlled by the CMV or Ubiquitin promoter (Bates et al. 2006). Transgenic plants and animals Transgenic organisms carry a foreign gene, which is stably integrated in the genome. There are different occasions for the production of transgenic organisms. Some of them are used for the production of foreign proteins: The desired proteins are harvested from sheep and goat milk or from the egg whites of hen s eggs. Others, for instance, are bred for medical basic research: wild type mice cannot be infected with the polio virus, as they are lacking a respective receptor protein on the cell surface. In order to be able to study the disease in the relatively cheap mouse model, transgenic mice are bred that express the human receptor protein. They then show the corresponding symptoms of a polio infection. In plants, frequently a higher crop yield thanks to an improved adaptation to climatic conditions or a resistance to pests plays a role. In the past, selection markers (antibiotic resistance genes) in particular were the target of criticism. 12 ucleic acid decontamination AppliChem 2008

15 atural defense mechanisms In nature, hosts and parasites meet constantly, also in the shape of their free nucleic acids. Both sides are perfectly primed for battle. At this point, our interest focuses on man s options to fend off undesired intruders, especially microorganisms. Today we know that bacteria or viruses cannot penetrate intact skin. Only skin lesions permit their intrusion. The mucous membranes are much more sensitive and are therefore the preferred entry. Several defense mechanisms already exist on the surface of the skin and the mucous membranes: 1. Sweat contains lysozyme, an enzyme employed for the isolation of plasmid DA in the alkaline lysis of bacteria. This enzyme is present on the surface of mucous membranes as well. ere, in addition, secretionary antibodies (IgA) can be found. Last but not least, nucleases are present as well! 2. The acidic p value inside the stomach and the alkaline p value of the intestine supported by various digestion enzymes also serve to create inhospitable conditions. 3. The urogenital tract is protected by the acidic p value of the urine, in females additionally by the colonization with the lactic acid producing bacteria Lactobacillus acidophilus (Doderlein s bacillus). The acidic environment created by the lactic acid also prevents colonization by the potentially pathogenic yeast Candida albicans. 4. By principle, the entire surface of the body (skin and, for instance, intestinal flora) is colonized with microorganisms that are tolerated on the surface. In their own interest, they ensure a growth containment of undesired, mutually competitive germs. 5. All body fluids contain a great variety of antimicrobial substances as well (lysozyme, the enzymatic complement system, peroxidase, fibronectin, interferons, interleukins, lactoferrins and transferrins). Interleukins cause fevers. An increased body temperature is also counted among the important defense mechanisms. Therefore, in many cases it is preferable to weather a fever especially children to permit an effective healing process to take place and not to intervene immediately with antipyretic products. 6. Phagocytes (scavenger cells) are amoeboid-moving cells capable of phagocytosis. They include macrophages, monocytes, as well as neutrophile and eosinophile granulocytes. They are not only moving inside the tissue, they also patrol the surface of mucous membranes. Once they have absorbed intruders, these are digested. 7. Inside the cell, the defense activities continue. Lysosomes contain alkaline proteins perturbing the permeability of the bacterial cell walls. The acidic p value (up to p 4.0) inside the phagolysosomes optimizes the activities of different lysosomal enzymes (lysozyme, glycosylases, phospholipases, and nucleases). 8. The various classes of antibodies bind to the intruded, exogenous substances and microorganisms and contribute to the inactivation. 9. DA methylation: This process was described in bacteria for the first time. Most bacterial strains contain so-called restriction endonucleases. These restriction enzymes recognize certain short DA sequences and digest (cut) the DA at these sites. By modifying the own DA with a strain-specific methylation pattern, the bacterium can distinguish between its own and the intruded foreign DA. Methylation protects the bacterial DA against digestion by its own restriction enzymes. It is assumed that in mammals methylation represents a defense mechanism to protect their own genome against foreign DA, such as viruses. Frequently, viral DA integrated after infection into the host DA is methylated. The methylation of eukaryotic DA does not mark same for purposes of digestion, but methylation can inactivate promoters and thus silence the expression of genes regulated by viral promoters. This assumption is further corroborated by the fact that most methylated cytosines in the mammal genome lie within viral sequences and transposon DA. In addition to turning off ( silencing ) the expression of foreign DA promoters, it could be demonstrated that methylation prevents the movement of transposable elements to other sites inside the genome. This way, methylation can prevent the spreading of infectious viruses from cell to cell or the negative effects of transposon sequences. One has to be aware of the fact that in lysis of bacteria or viruses, their nucleic acids are released. Therefore, nucleases are always part of the defense mechanism! Viral sequences can also be found in the human genome. There are, of course, viruses and bacteria that can cross all barriers (also refer to Lisowsky 2006) AppliChem ucleic acid decontamination 13

16 In conclusion to the topic of natural defense let me remark that frequently microorganisms do not become dangerous until they enter the body by injection or through open wounds. either should one ignore the potential risk to laboratory personnel, if nucleic acids are dissolved in solvents that permeate the skin or have been mixed with membrane fusion reagents. The interdictions to pipette with the mouth and to eat or drink in the laboratory, the recommendation to use plastic instead of glass (danger of breaking) wherever possible and to avoid the generation of aerosols are evident. Apart from that, extracellular nucleases (defense on the skin, on mucous membranes, in tear fluid) are the biggest enemy of the nucleic acid experimenter in the laboratory. The problem created by man is the fact that the frequency of contact has risen exponentially in certain professions for quite some time already, for the world at large quite recently. Absorption of free nucleic acids by cells The absorption of naked nucleic acids by cells seems to be a natural phenomenon. Should that be the case, this has to be based on a mechanism. ow is nucleic acid waste, released by apoptotic cells (cf. Review by Gewirtz et al. 1998), disposed of? Since nucleic acids, thanks to their phosphate backbone, have a high negative charge, a simple absorption by diffusion through the lipophilic cell membrane is hardly possible, yet cannot be completely ruled out. Therefore, a receptor-controlled absorption is favored, all the more, because marked oligo-nucleotides in so-called Clathrin coated pits that have been known from other endocytosis processes could be detected in lysosomal and endosomal compartments (Beltinger et al. 1995). In view of the administration of gene therapeutic nucleic acids, this type of absorption is considered as highly inefficient. Once the nucleic acids can leave the endosomes or lysosomes in the cells, respectively, they collect probably by diffusion inside the cell nucleus (Beltinger et al. 1995). There, they are presumably held by nuclear binding proteins and are possibly no longer available for biological processes. The availability of oligonucleotides inside the human body in the sense of pharmacodynamics was studied as well. Within a time period of 24 hours, 50 % of the originally intravenously infusion-administered oligo were excreted with the urine, in part intact, in part in a degraded state; within 96 hours this figure rises to approx. 70 %. Comparable experiments in mice and monkeys have shown that oligos accumulate most inside the liver and the kidney. (cf. Review by Gewirtz et al. 1998). Free foreign DA ingested with food is not completely degraded in the gastrointestinal tract of the mouse. In experiments, phage DA (M13mp18) was fed to mice and later detected in peripheral leukocytes, the spleen and the liver. A mere 2 to 8 hours after feeding, phage DA already circulated in the blood of the mouse. In the feces, DA fragments of sizes between 100 to approx 1,700 base pairs could be isolated. From the total spleen DA, phage DA fragments of sizes up to 1,300 base pairs could be isolated, covalently linked with mouse DA (Schubbert et al. 1994; Schubbert et al. 1997). These research animals were fed daily with foreign DA M13 for periods of 3 days and one week respectively. After one-time ingestion, no stable phage DA integrated in the mouse s genome could be detected. Although, after feeding chickens with transgenic corn, the DA of the transgenic plants could be detected in crop and stomach, this was not the case in the subsequent sections of the digestive tract (Chambers et al. 2002). atural mitochondrial plant DA does not survive the digestion in the chicken stomach either. The absorption of free DA or DA from foodstuffs after digest by saliva can start in the oral cavity. A great variety of bacteria exist inside the oral cavity, bacteria that are in part naturally competent, i. e., they are capable of absorbing foreign DA. During incubation of free DA with human saliva, DA is partially digested in vitro; however, this happens so slowly as to leave sufficient time to transform the oral cavity bacterium Streptococcus gordonii DL1 in an in vitro experiment with the remaining DA (Mercer et al. 1999). The discussion on the dangers of free nucleic acids was triggered or intensified, respectively, by the experiments of Burns et al. (1991). They were able to prove in vivo the generation of tumors in the skin of mice by applying plasmid DA that encodes the genetic information for the human T24 -ras oncogene. o further agents (tumor promoters) were required to transform endothelial cells of the skin to form lymphangiosarcomas. By comparison, the absorption of DA by epithelial cells is far less effective (multiple treatment with oncogene or tumor promoter) than by endothelial cells in vivo. After all, it was this study that caused the ZKBS in 1991 to make a general statement by recommending precautionary measures when handling nucleic acids with an oncogene potential. It is explicitly pointed out that laboratory surfaces and laboratory equipment having come in contact with those nucleic acids should be cleaned thoroughly upon completion of the work and laboratory waste containing such nucleic acids should be denatured either chemically or by autoclave treatment. 14 ucleic acid decontamination AppliChem 2008

17 another 2 to 6 genetic experiments and we ll have gotten rid of the head as well 2008 AppliChem ucleic acid decontamination 15

18 And now? Man has been sensitized by nuclear power, chemical accidents, foodstuff scandals, pollution of the environment and natural catastrophes the effects of which seem to be compounded by human behavior. Regardless, we continue to build our homes next to the runway, in earthquake zones, on slopes prone to landslides, or in floodplains. Following the catastrophe, political campaigning floods the area like tourism and once this has been weathered and the water has drained away, aid moneys for reconstruction pour in. ow, genetic engineering comes on top. Ripe tomatoes that look it but don t taste it; that stay ripe longer, because they no longer rot. Are the consequences clear to us? Can the advantages justify possible disadvantages? Were enough checks completed before the world at large was confronted with a genetically engineered product? Chemical contaminations are washed out (diluted) with time or chemicals decompose or are degraded. DA contaminations can be transmitted, recombined in nature and multiplied. The sequence analysis of homologous genes of different species has shown that complete genes or partial gene sequences are identical, even between organisms not evolutionarily related. The actual cause can only be a direct genetic transposition. ave we humans learned by now, how to deal with such sensitive topics? The opponents of the spreading of GMOs point out various negative effects and badly calculable risks: 1. Bacillus thuringiensis (Bt) has been used for many years as pesticide (particularly wheat and cotton). Genetically modified plants express the Bt delta endotoxin. There is increasing evidence of farm workers developing allergies from this toxin. 2. Antibiotics resistance as selection marker in the production of transgenic plants. DA released into the environment is more stable than originally anticipated. Bacteria can absorb this DA. Particularly critical is the fact that the Ampicillin-resistance in ovartis Bt grain is under the control of a bacterial promoter instead of a plant promoter. One could counter that the probability of this very DA section being absorbed by bacteria in the soil or during ingestion is extremely low, since it represents but a minute fraction of the overall plant DA. In contrast to multi-resistant germs in clinical areas, the resistance should normally not represent a selection advantage for soil bacteria and disappear again. 3. Posttranslational modification: Depending on different organisms, acetylation and glycosylation of transgenic products can lead to a modified toxicity. 4. on-predictability of the place of integration and the expression of the transgenic inserts: the number of inserts, their localization (chromosome or organelle chloroplast, mitochondria) and their exact position (where on which chromosome) can barely be predicted. 5. Positional effect: The insertion point influences the expression of the transgene. The transgene, in turn, also influences the expression of neighboring genes or silences them, if the insertion takes place in the middle of a gene. Since frequently only a weak expression of the desired gene could be detected, strong promoters are being employed. Therefore, it is desirable to know the sequences before and after the transgene, since strong promoters can have an effect across many thousands of base pairs. 6. orizontal gene transfer (GT) is primarily discussed in connection with microorganisms. The three variants are the direct absorption of naked nucleic acids from the environment, the absorption of DA by viruses (bacteriophages) and by conjugation between different species of bacteria. Once again, the absorption of transgenes (e. g., resistance genes) is the focus of interest. The above explanations make it clear that the probability of such an incident is extremely low, yet theoretically it cannot be ruled out completely. If an absorbed transgene under a respective selection pressure offers an advantage, same can establish itself in a population. 7. Genetic constructs with a corresponding replication unit and promoter can be active in different organisms. 16 ucleic acid decontamination AppliChem 2008

19 People are unsettled, because no information intelligible to all or formulated in a neutral way is available. Even experts have difficulties to be unbiased in verifying and evaluating all existing information. And it is difficult for the experimenter to create conditions that come at least close to the real conditions. obody working in a laboratory and sticking to the rules will swallow DA by the gram or rub it in his skin. Yet is it not necessary that the odd genetically modified plant grows on open land so we can find out what really happens? Something that must not happen under any circumstance is to play irresponsibly with the fear of people. That incorrect information, processed pseudoscientifically, is fed to the public. And who can safely rule out that it is used unlawfully for reasons of greed or other base motives? Bioterrorism, unlabeled, genetically modified ingredients in foodstuffs it is easy to paint a bleak picture of the future. Why does man feed a herbivore with badly processed animal waste? ature fights back. If BSE (TSE) is a pathogen to humans (Creutzfeld-Jacob variant?), if the avian flu recombines with human influenza viruses to form new, highly virulent strains and if IV was transmitted from the ape to man, who is to say whether or not another occurrence takes place with even more serious consequences? The question is not whether or not ; it should read earlier or later. Man increases the chance of a corresponding occurrence in nature happening; without this intervention, recombination and exchange occur naturally and only the sustainable model gets a chance. In their recommendations, the ZKBS does not speak of no risk, but of a very low risk or a low probability. In other words, a (residual) risk and a probability do exist. To my knowledge, the Federal Office for Civil Protection does not explicitly list free nucleic acids under biological agents. Another point to think about. Literature Bates, M.K. et al. (2006) Genetic immunization for antibody generation in research animals by intravenous delivery of plasmid DA, BioTechniques, 40(2), Beltinger, C. et al. (1995) Binding, uptake, and intracellular trafficking of phosphorothioate-modified oligodeoxynucleotides, J. Clin. Invest. 95(4), Burns, P.A. et al. (1991) Transformation of mouse skin endothelial cells in vivo by direct application of plasmid DA encoding the human T24 -ras oncogene, Oncogene 6, Chambers, P.A. et al. (2002) The fate of antibiotic resistance marker genes in transgenic plants feed material fed to chickens, J. Antimicrobiol. Chemother. 49, Chamier, B. et al. (1993) atural Transformation of Acinetobacter calcoaceticus by Plasmid DA Adsorbed on Sand and Groundwater Aquifer Material, Appl. Environ. Microbiol. 59, Garcia-Olmo, D. et al. (1999) Tumor DA circulating in the plasma might play a role in metastasis. The hypothesis of genometastasis, istol. istopathol. 14, Garcia-Olmo, D. et al. (2000) orizontal transfer of DA and the genometastasis hypothesis, Blood 95, Gewirtz, A.M. et al. (1998) Review Article: ucleic Acid Therapeutics: State of the Art and Future Prospects, Blood 92, olmgren, L. et al. (1999) orizontal Transfer of DA by the Uptake of Apoptotic Bodies, Blood 93, Kümmerer, K. et al. (2002) Abschlussbericht Antibiotika-Resistenz und Übertragung in Abwasser, Oberflächenwasser und Trinkwasser Teil 2. Lisowsky, T. (2006) atürliche Rekombination und gentechnischmodifizierte ukleinsäuren: eubewertungen zur biologischen Sicherheit, labor&more 2 (1), 6 9. Mercer, D.K. et al. (1999) Fate of free DA and Transformation of the Oral Bacterium Streptococcus gordonii DL1 by Plasmid DA in uman Saliva, Appl. Environ. Microbiol. 65, ielsen, K.M. et al. (1997) atural Transformation and Availability of Transforming DA to Acinetobacter calcoaceticus in Soil Microcosms, Appl. Environ. Microbiol. 63, Roberg-Perez, K. et al. (2003) MTID: a database of Sleeping Beauty transposon insertions in mice, ucleic Acids Res. 31, Romanowski, G. et al. (1993) Use of Polymerase Chain Reaction and Electroporation of Escherichia coli To Monitor the Persistence of Extracellular Plasmid DA Introduced into atural Soils, Appl. Environ. Microbiol. 59, Schubbert, R. et al. (1994) Ingested foreign (phage M13) DA survives transiently in the gastrointestinal tract and enters the bloodstream of mice, Mol. Gen. Genet. 242, Schubbert, R. et al. (1997) Foreign (M13) DA ingested by mice reaches peripheral leukocytes, spleen, and liver via the intestinal wall mucosa and can be covalently linked to mouse DA, Proc. atl. Acad. Sci. USA 94, Suzuki, K. et al. (1999) Activation of target-tissue immune-recognition molecules by double-stranded polynucleotides, Proc. atl. Acad. Sci. USA 96, AppliChem ucleic acid decontamination 17

20 genetic engineering atural recombination and genetically modified nucleic acids Biological safety under scrutiny Dr. Karl-einz Esser and Prof. Dr. Thomas Lisowsky, multibid biotec Gmb, Germany With the new synthesis of the genome of the highly dangerous 1918 influenza pandemic virus proving that ancient, normally extinct infectious virus particles can resurge in eukaryote cells, a new milestone has been reached in genetic engineering. At the same time, this triggers controversial discussions on the topic of biological safety. This also requires a re-evaluation of the risk potential inherent in free genetically modified DA or RA molecules. By now, recombinant nucleic acid constructs are produced in growing numbers worldwide. The original assessments of genetic engineering were based on the assumption that free DA or RA molecules are not dangerous. Consequently, only the controlled disposal of genetically modified organisms is mandated by the applicable laws on genetic engineering with regards to biological safety. Latest studies show, however, that in certain cases free nucleic acid molecules are sufficient to cause biological transformations, functional expressions or new genetic recombinations. In the short run, these occurrences are still infrequent, in the long run, however, and with constantly rising numbers of recombinant nucleic acid molecules, this could lead to grave consequences. Therefore, anybody employing genetic engineering methods should be interested in minimizing the risk potential of recombinant nucleic acid molecules as a preventive step as well as in a sustainable way. In the interest of a safe exploitation of genetic engineering as a future key technology, an environmentally safe disposal of recom binant DA and RA molecules must be ensured. The latest data derived from the current standard method number one for the professional and correct disposal by autoclave treatment have shown that new technologies or solutions have to be developed for this very purpose. By employing the sensitive method of PCR analysis it could be established that after the autoclave treatment of, for instance, infectious microorganisms, big sections or even complete molecules of the DA remain intact. The current studies highlight a possible gap in biological safety that can be closed by sustainable, long-term safety measures only. The most recent data on the current developments in this area are summarized in this article and practical consequences are proposed based on the example of the endosymbiontic hypothesis of the evolution. Developments for the evaluation of the risk potential of free DA molecules At the beginning of the systematic use of genetic engineering methods since approximately 1980, nobody considered free DA molecules to be potentially dangerous. General opinion was that free DA molecules would not be able to last long in the environment and that their efficient absorption by living organisms was hardly possible. As a consequence, only genetically modified organisms were subjected to statutory regulations regarding their safe disposal. To this day, no legal requirements exist anywhere in the world to professionally dispose of free nucleic acid molecules whether natural or artificially engineered. 18 ucleic acid decontamination AppliChem 2008