1、| | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | BRITISH STANDARD BS EN 12683:1998 The Euro
2、pean 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 Biotechnology Modified organisms for application in the environment Guidance for the characterization of the genetically modified organism by analysis o
3、f the molecular stability of the genomic modificationBS EN 12683:1998 This British Standard, having been prepared under the direction of the Sector Committee for Materials and Chemicals, was published under the authority of the Standards Committee and comes into effect on 15 December 1998 BSI 1998 I
4、SBN 0 580 30177 X Amendments issued since publication Amd. No. Date Text affected National foreword This British Standard is the English language version of EN 12683:1998. The UK participation in its preparation was entrusted to Technical Committee CII/58, Biotechnology, which has the responsibility
5、 to: aid enquirers to understand the text; present to the responsible European committee any enquiries on the interpretation, or proposals for change, and keep the UK interests informed; monitor related international and European developments and promulgate them in the UK. A list of organizations re
6、presented on this committee can be obtained on request to its secretary. Cross-references The British Standards which implement international or European publications referred to in this document may be found in the BSI Standards Catalogue under the section entitled “International Standards Correspo
7、ndence Index”, or by using the “Find” facility of the BSI Standards Electronic Catalogue. A British Standard does not purport to include all the necessary provisions of a contract. Users of British Standards are responsible for their correct application. Compliance with a British Standard does not o
8、f itself confer immunity from legal obligations. Summary of pages This document comprises a front cover, an inside front cover, the EN title page, pages 2 to 8, an inside back cover and a back cover.CEN European Committee for Standardization Comite Europe en de Normalisation Europa isches Komitee fu
9、 r Normung Central Secretariat: rue de Stassart 36, B-1050 Brussels 1998 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 12683:1998 E EUROPEAN STANDARD EN 12683 NORME EUROPE ENNE EUROPA ISCHE NORM July 1998 ICS 07.080 Descriptors:
10、biotechnology, genetics, modified organisms, environments, environmental protection, analysis method, bioassay, experimental design English version Biotechnology Modified organisms for application in the environment Guidance for the characterization of the genetically modified organism by analysis o
11、f the molecular stability of the genomic modification Biotechnologie Organismes modifie s disse mine s dans lenvironnement Guide pour la caracte risation de lorganisme ge ne tiquement modifie par lanalyse de la stabilite mole culaire de la modification ge nomique Biotechnik Vera nderte Organismen zu
12、m Einsatz in der Umwelt Leitfaden fu r die Charakterisierung des gentechnisch vera nderten Organismus durch Untersuchung der molekularen Stabilita t der Genomvera nderung This European Standard was approved by CEN on 1 July 1998. CEN members are bound to comply with the CEN/CENELEC Internal Regulati
13、ons which stipulate the conditions for giving this European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such national standards may be obtained on application to the Central Secretariat or to any CEN member. This Europ
14、ean Standard exists in three official versions (English, French, German). A version in any other language made by translation under the responsibility of a CEN member into its own language and notified to the Central Secretariat has the same status as the official versions. CEN members are the natio
15、nal standards bodies of Austria, Belgium, Czech Republic, Denmark, Finland, France, Germany, Greece, Iceland, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and United Kingdom.Page 2 EN 12683:1998 BSI 1998 Foreword This European Standard has been prepared by Te
16、chnical Committee CEN/TC 233, Biotechnology, the Secretariat of which is held by AFNOR. This European Standard shall be given the status of a national standard, either by publication of an identical text or by endorsement, at the latest by January 1999, and conflicting national standards shall be wi
17、thdrawn at the latest by January 1999. This European Standard has been prepared under a mandate given to CEN by the European Commission and the European Free Trade Association. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are boun
18、d to implement this European Standard: Austria, Belgium, Czech Republic, Denmark, Finland, France, Germany, Greece, Iceland, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and the United Kingdom. Contents Page Foreword 2 Introduction 3 1 Scope 3 2 Normative ref
19、erences 3 3 Definitions 3 4 Molecular stability testing 4 5 Materials 5 6 Considerations for experimental procedures 5 7 Validity of data analysis 7 8 Documentation of results 7 Annex A (informative) Bibliography 8Page 3 EN 12683:1998 BSI 1998 Introduction This European Standard relates to part of t
20、he characterization of genetically modified organisms (GMOs). It is designed as a guideline for adaptation of experimental procedures to the requirements of the specific experimental design. The characterization of a GMO can include the analysis of: the genomic modification (see EN 12687); the funct
21、ional expression of the genomic modification (see EN 12682); the molecular stability of the genomic modification. This European Standard deals with the analysis of the molecular stability of the genomic modification of GMOs. In principle, this European Standard refers to the analysis of the molecula
22、r stability of GMOs during their prerelease evaluation and in monitoring of experimental releases. If specific questions concerning molecular stability 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 An
23、nex A 3, 4). The analysis of the molecular stability can be based on: the physical analysis of the genetic modification of interest as it exists in the GMO (genomic modification) (see EN 12687); and/or the analysis of the functional expression of the genetic modification of interest (genomic modific
24、ation) (see EN 12682). 1 Scope This European Standard provides guidance for factors and criteria considered by the experimenter for the valid design, execution and evaluation of an analysis of the molecular stability of the genomic modification with respect to life cycle, heritability and external f
25、actors. It describes the steps in the characterization of a GMO that should be followed to ensure the validity of the analysis of the molecular stability of the genomic modification. The type of molecular stability analysis is dependent on the objectives of the experiment. 2 Normative references Thi
26、s European Standard incorporates by dated or undated reference, provisions from other publications. These normative references are cited at the appropriate places in the text and the publications are listed hereafter. For dated references, subsequent amendments to or revisions of any of these public
27、ations apply to this European Standard only when incorporated in it by amendment or revision. For undated references the latest edition of the publication referred to applies. EN 12687:1998, Biotechnology Modified organisms for application in the environment Guidance for the characterization of gene
28、tically modified organism by analysis of the genomic modification. EN 12682:1998, Biotechnology Modified organisms for application in the environment Guidance for the characterization of genetically modified organism by analysis of the functional expression of the genomic modification. 3 Definitions
29、 For the purposes of this standard, the following definitions apply: 3.1 control preparation of known characteristics used to standardize an analysis 3.2 data signal output of a test system NOTE Data signals can be characterized: by binary decision: presence/absence (+/2); in relative terms by order
30、ing the data signal strength with respect to (a) defined control(s); quantitatively by giving their output strength in absolute terms; by position or movement; qualitatively by describing parameters not addressed by strength or position. 3.3 detection recognition of the presence of an organism or of
31、 a molecular structure within a sample 3.4 gene probe specific nucleic acid sequence used to identify certain DNA or RNA fragments by means of hybridization 3.5 genetic modification of interest conceptual design for altering the genetic material within an organism NOTE 1 The genetic modification of
32、interest can be described at different levels of molecular detail. NOTE 2 The conceptual design can include insertion, substitution or deletion of genetic material. 3.6 genetically modified organism organism in which the genetic material has been altered in a way that does not occur naturally by mat
33、ing and/or natural recombination NOTE Within the terms of this definition, genetic modification occurs at least through the use of the techniques listed in the Directive 90/220/EEC or its appropriate annexes (see annex A 2).Page 4 EN 12683:1998 BSI 1998 3.7 genomic modification actual physical struc
34、ture of the genetic modification of interest as it exists in the genetically modified organism 3.8 identification establishment of identity by comparison with a reference NOTE 1 The reference could be an organism, a molecular structure or the genetic modification of interest. NOTE 2 The certainty of
35、 identification is affected by the types and/or number of characteristics investigated. 3.9 molecular stability maintenance of the integrity of the desired structure and/or the desired function of the genomic modification with respect to time and/or generation 3.10 organism biological entity capable
36、 of replication or of transferring genetic material 3.11 reproducibility precision under reproducibility conditions ISO 3534-1 NOTE 1 Reproducibility conditions are conditions where test results are obtained with the same method on identical test items in different laboratories with different operat
37、ors using different equipment. NOTE 2 Results should be expressed as reproducibility standard deviation or reproducibility coefficient of variation. 4 Molecular stability testing 4.1 General considerations The relative molecular stability of traits introduced by genetic modification, over time and g
38、eneration, can be important in order to ensure the performance of the GMO under field conditions and/or for biosafety reasons. The design of an analysis for molecular stability should take into account the natural variations on the one hand and the experimental objective with respect to product perf
39、ormance and safety on the other. Molecular stability analysis can be performed at either the structural or genomic level or at the functional or phenotypic level depending on the objective of experiment. Testing for molecular stability between generations should be carried out over an appropriate nu
40、mber of generations. Molecular stability testing could be required at various points during the development and release of a GMO into the environment. This generally starts in contained systems, like the laboratory, microcosms, growth chambers, greenhouses, animal houses and can continue during the
41、release into the environment. 4.2 Types of molecular stability Molecular stability or instability of a genomic modification can be observed at any of the following levels: the structural or genomic level; the transcriptional or RNA-level; the level of functional expression; phenotype such as morphol
42、ogy, resistance, host specificity, colour. 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 as: mutation; recombination and/or transposition; g
43、ene transfer; copy number of accessory genetic elements like plasmids; methylation. These factors influence mainly the genomic modification, the type of integration into, and/or the localization within the genome. Molecular stability of the functional expression can depend on factors which indirectl
44、y influence the genomic modification such as: recipient organism; tissue and/or organ specificity; dependence upon metabolic activity; developmental stage (e.g. germination, senescence); external factors (e.g. wounding, UV- and visible light, temperature, habitat). Factors which influence the genomi
45、c modification can act either in cis or in trans. Examples for factors acting in cis are: integration site; chromatin structure; DNA structure; copy number; methylation. Examples for factors acting in trans are: life cycle; heritability; external factors. 4.4 Analysis of molecular stability 4.4.1 Ge
46、neral The molecular stability testing is the correlation of analytical data (structure and expression) from GMOs or their genetic material between samplings taken at different time points.Page 5 EN 12683:1998 BSI 1998 4.4.2 Methods at the structural or genomic level The following is a list of method
47、s appropriate to test the molecular stability at the structural or genomic level. Not all of these methods are necessarily applicable for the analysis of every genomic modification. The method of choice and its appropriateness or combination of methods depends on the stated objectives of the analysi
48、s. As molecular biology is a rapidly evolving field of research, the listed methods are considered neither prioritized, restrictive nor exhaustive. The methods should be properly assessed with regard to their information value: restriction pattern analysis; nucleic acid hybridization methods; nuclei
49、c acid amplification methods; sequencing methods; analysis of the chromatin and/or chromosome structure such as nucleosome footprinting and FISH (Fluorescent In Situ Hybridization). 4.4.3 Methods at the functional or phenotypic level The following methods can be appropriate to test the molecular stability at the functional or phenotypic level: biological tests (e.g. resistance and/or tolerance to pests or pesticides); test of
