EN 12683-1998 en Biotechnology - Modified Organisms for Application in the Environment - Guidance for the Characterization of the Genetically Modified Organism by Analysis of the M.pdf

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1、STD-BSI BS EN L2b83-ENGL 1998 lb24bb9 073955b 842 = BRITISH STANDARD BS EN 12683: 1998 Biotechnology - Modified organisms for application in the environment - Guidance for the characterization of the genetically modified organism by analysis of the molecular stability of the genomic modification “he

2、 European Standard EN 12683:1998 has the status of a British Standard ICs 07.080; NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAW BS EN 12683:1998 been prepared under the direction of the Sector Committee for Materials and Chemicals, was published under the authority of the St

3、andards Committee and comes into effect on 16 December 1998 Amd. No. Q BSI 1998 ISBN O O80 30177 X National foreword Date Text affected This British Standard is the English language version of EN 12683:1995. The UK participation in its preparation was entrusted to Technical Committee CIV58, Biotechn

4、ology, which has the responsibility to: - aid enquirers to understand the text; - present to the responsible European committee any enquiries on the - monitor reM intemaiional and European developments and promulgate inkqretation, or proposals for change, and keep the K interests informed; them in t

5、he UK. A list of organizations represented on this committee can be obtained on request to its secretary. Cross-references The Brish $tandar - the functional expression of the genomic modification (see EN 12682); - the moleculas stability of the genomic modification. This European Standard deals wit

6、h the analysis of the molecular stabiity of the genomic modification of GMOs. In principle, this European Standard refers to the analysis of the molecular stability of GMOs during their prerelease evaluation and in monitoring of experimental releases. If specific questions concerning molecular stabi

7、lity occur during or after the release, especially if the release is scheduled for more than one generation, it is this standard that could apply (see The analysis of the moleculas stability can be based on: nnex 131, 41). - the physical analysis of the genetic modification of interest as it exists

8、in the GMO (genomic modification) (see EN 12687); and/or - the analysis of the functional expression of the genetic modification of interest (genomic modification) (see EN 12682). 1 scope This European Standaxd provides guidance for factors and criteria considered by the experimenter for the valid d

9、esign, execution and evaluation of an analysis of the molecular stability of the genomic modification with respect to life cycle, heritability and extemal factors. It describes the steps in the characterization of a GMO that should be followed to ensure the validity of the analysis of the molecular

10、stability of the genomic modification. The type of molecular stability analysis is dependent on the objectives of the experiment. 2 Normative references This European Standard incorporates by dated or undated reference, provisions from other publications. These normative references are cited at the

11、appropriate places in the text and the publications are listed hereafter. For dated references, subsequent amendments to or r - in relative tem by ordering the data signal strength with respect to (a) defined control(s); - quantitatively by giving their output strength in absolute terms; - by positi

12、on or movement; - qualitatively by describing parameters not addressed by strength or position. 3.3 detection recognition of the presence of an organism or of a molecular structure within a sample 3.4 gene probe specific nucleic acid sequence used to identify c - the transcriptional or RNA-level; -

13、the level of funconal expression; - phenotype such as morphology, resistance, host 4.3 Factors influencing the molecular stability Molecular stability of a genomic modification at the structural or genomic level can depend on factors which directly influence the genomic modification such specific -

14、recombination and/or transposition; - gene transfer, - copy number of accessory genetic elements like plasmidq - methylation. These factors influence mainly the genomic modification, the type of integration into, and/or the localization within the genome. Molecular st. - tissue and/or organ specific

15、ity, - dependence upon metabolic activiw, - developmental stage (e.g. germination, senescence); - exterml factors (e.g. wounding, UV- and visible light, temperature, habitat). Factors which influence the genomic modification can act either in Examples for factors acting in or in m. are: - integratio

16、n site; - chromatin structure; - copy number, - methylation. - life cycle; - heritihilisr; - external factors. - DNA - nucleic acid amplification methods, - sequencing metho - test of enzymatic activities; - immuno-detection; - nuclear-run-on-assays and Northern Blot. 5 Materials If nucleic acids, g

17、ene probes, labelled gene probes are used, they should be prepared in accordance with EN 126871998, clause 6. 6 Considerations for experimental procedures 6.1 General The main steps of the experimental procedures for the analysis of the molecular stability of the genomic modincation are: a) precise

18、statement of the objective for the intended analysis (see 6.2); b) experimental design according to various criteria (see 6.2); c) execution of the analysis according to the experimental protocol (see 6.3); d) appropriate record keeping (see 6.4); e) evaluation of the validity of the results (see f)

19、 documentation of the resulis (see clause 8). clause 7); Page 5 EN 12683:1998 6.2 Experimental design The considerations for experimental procedures to analyse the molecular stability of the genomic modification should start with the definition and statement of the objective for this analysis follow

20、ed by the design of the experiment, which should be written down in a protocol, but keeping the flexibility needed to handle unexpected observations. The reason for choosing a particular method or methods should be stated in the experimental design. The following points should be considered in the d

21、esign of an experiment to test the molecular stability of a genomic modification: - objective of the molecular stability analysis, - type of molecular stability analysis: structural or genomic, functional or phenotypic molecular stability; - choice of the method) to analyse molecular stabili - type

22、of organism (micro-organism, plant, animai); - type of genetic modification (e.g. deletion, insertion, rearrangement); - part of the organism used for analysk, - numbers of samples during one generation; - number of generations tested; - growth and senescence; - population size to be tested, - exter

23、nal factors (e.g. light, temperature, humidity, soil variables, other site factors); - use of appropriate controls. NOTE This list should not be considered as exhaustive. 6.3 Execution of the experimental protocol 6.3.1 General Methods of molecular stabiity testing at the functional or phenotypic an

24、d at the molecular level are described in 6.3.2 and 6.3.3. Not all of the methods are necessarily applicable in one experiment to analyse the molecular stability of the genomic modification. The choice of the method or the combination of methods depends on the experimental design which itself is inf

25、luenced by the factors described in 6.2. 6.3.2 Functional or phenotgpic analysis If appropriate, phenotypic testing can be used to analyse the molecular stbity of the genetic modification of interest. These tests combine the analysis of the performance of the GMO under the experimental conditions wi

26、th the molecular stability analysis. Mostly, the analysis is based on biological tests. Examples for the appropriateness of these kinds of tests are based on phenotypic analysis, such as resistance tests against pests or pesticides. In general, phenotypic analysis is cost-effective and can be perfor

27、med with a statistically relevant number of organisms. Additionally or alternatively to biological tests, biochemical analysis can be suitable (see also 6.3.3.4). O BSI 1998 STD-BSI BS EN L2b83-ENGL 1978 = lb24bb7 07395b3 TA2 9 Page 6 EN 12683:1998 6.3.3 Molecular analysis 6.3.3.1 Geneml Molecular a

28、naiysis of the stabiity of the genetic modification of interest can be performed at various levels, such as the structure of the genome, RNA- transcription, protein-expression. The various methods are described in 6.3.3.2 to 6.3.3.6. 6.3.3.2 Structurai or gencwn.iC analysis At this level, nucleic ac

29、id hybridization and DNA- or RNA-fragment amplincation methods can be applied. In order to avoid misinterpretation of the results appropriate controls should be included in ail experiments. Methods for analysis structure or genomic molecular stability include the foliowing. a) Nucleic acid hybridiza

30、tion methods Sloffdot blot can be applied, if cultivaiion of the GMO is not possible or necem. The advanage of this method is that a higher number of samples can be screened within reasonable time and a more reasonable cd than with Southern hybridization analysis. However, as a disadvantage, false p

31、ositive results can often be obtained from closely-related sequences, if the hybridization stringency is not sufficient. Colony or plaque hybridization can be applied when the molecular stability of a large number of microorganisms has to be analysed. The specificity of the technique depends on the

32、ability of the gene probe to differentiate between the target sequence and cross-reacting sequences. To identify artifacts, it is necessary to use duplicate flters. Sloffdot blot and colony and plaque hybridization give no information about the integrity of the structure. With these methods, only th

33、e presence or absence of a certain genomic modification can be determined. Southern hybridization is characterized by a higher sensitivity and specificity than the previously described methods. Howeveq as it is time- and cosbintensive, generally oniy a smaller number of samples can be handled. There

34、fore, it is usually used for diagnosis or verification rather than for screening. The specificity of the technique depends mainly on the gene probe used and the nature of the target nucleic acid. This method is particularly suitable to detect rearrangements which can give rise to questions concernin

35、g molecular stability or instability of the genomic modification. Data signais from false positive hybridization are recognized if a fragment other than the size predicted from the genetic modification of interest is detected by the gene probe. However, in this case it is also possible that a deleti

36、on or insertion has occurred within the genomic modification. For the case of hybridization analysis, degradation of nucleic acid in the samples can also lead to unexpected hybridization data signals. For ail methods, a positive control should contain the modified sequence, whose molecular stability

37、 is being analysed In the case of Southern hybridization, the positive control should be obtained by digestion of the DNA with a restriction enzyme or combination of enzymes having at least two cum sites in the modified genetic sequence. A negative control should be obtained by using the nucleic aci

38、d of an organism which is identical to the GMO except for the lack of the modified genetic sequence. b) Nucleic acid amplification methods Vcurious methods can be applied, depending on the type of molecular stability question to be answered (e.g. PCR, reverse tsdnscriptaseER, inverse-ER). Nucleic ac

39、id amplification methods are very sensitive techniques. Therefore, special care should be given to the purity of the sample, the optimization of the stringency during the amplification reaction and the inclusion of appropriate controls. The detection should be done in such a way that false positive

40、results are excluded. For example, this could include a Southern analysis or restriction enzyme analysis of the amplified DNA Additionally, crude DNA samples sometimes contain components that inhibit the activity of the polymerase. Control ampication reactions using a second pair of primers specific

41、 for an additionai endogenous DNA sequence should be performed to ensure the purity of the DNA sample. 6.3.3.3 Rute of tmnscription Nuclear-runamwiys, performed according to laboratory manuais (see annexA 7), SI) can be applied to determine the molecular stabiity of the rate of transcription. The fo

42、liowing controls should be used: - a positive control corresponds to a host gene which has no homology with the genetic modifidon of interest and which is tramcm bed in the tissue of inter- - a negative control corresponds to an assay performed in the presence of alpha-amanitin which acts as an inhi

43、bitor of RNA polymerase II. The method is very timeconsuming and laborious. 6.3.3.4 SWg state levels of RNA Methods to determine the steady state levels of the transmibed RNA include RNA slot/dot blot, Northern hybridization, RNase-protection-assays and reversetranscriptase ER. The foliowing control

44、s should be used: - a positive control corresponds to a host gene which has no homology with the genetic modification of interest and which is transrribed in the tissue of inter- - a negative control corresponds to RNA extracted from the non-genetically modified organism. O BSI 1998 STD-BSI BS EN L2

45、b83-ENGL 1998 I lb24bb7 07395b4 919 E 6.3.3.6 Molecular sttg of W expression of a protein Vrious methods to detect and quant the expression level of a protein of inkrest can be applied The method of choice depends on the experimental design and especially on the type of a protein expressed due to th

46、e genomic modification. Examples of applicable methods are Western blot analysis and other immuno-detection techniques. The applicability of these methods depends on the availability of a specific antibody or antiserum. If an expressed protein has an enzymatic activity and a suitable test system is

47、available, the measurement of this enzymatic activity can be a suitable method. Positive and negative controls should be included in order to detect false positive due to antibody cross-reactiviQ or non-specific enzymatic reactions. Positive controls should be protein extracts from organisms which a

48、re known to exprem the protein to be detected or pure preparations of the protein to be deteded Negative controls should be protein ex - person(s) performing the experiment; - date of the experiment; - method chosen for the moleculas stabiity analysis; - origin of samples; - number of samples analys

49、ed; - controls used - deviations from the protocol; - duration of the experiment; Page 7 EN 126831998 h) a Statistically relevant number, as determined by the experimental design (see 6.2), of generations has been andysed; and i) appropriate extemal conditions have been taken into account. 7.2 Specific considerations 7.2.1 Validity of data analysis based on nucleic acid hybridization methods Using sloVdot or colony blot, samples and controls should be applied or transferred to the same filter. Using Southern or Northern hybridization methods, samples and controls should