1、| | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | BRITISH STANDARD BS EN 12687:1998 The Euro
2、pean Standard EN 12687:1998 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 genomic modificationBS EN 12687:1998 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 ISBN 0 580 30180 X Amendment
4、s issued since publication Amd. No. Date Text affected National foreword This British Standard is the English language version of EN 12687:1998. The UK participation in its preparation was entrusted to Technical Committee CII/58, Biotechnology, which has the responsibility to: aid enquirers to under
5、stand 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 represented on this committee
6、 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 Correspondence Index”, or by using
7、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 of itself confer immunity fr
8、om legal obligations. Summary of pages This document comprises a front cover, an inside front cover, the EN title page, pages 2 to 9 and a back cover.CEN European Committee for Standardization Comite Europe en de Normalisation Europa isches Komitee fu r Normung Central Secretariat: rue de Stassart 3
9、6, 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 12687:1998 E EUROPEAN STANDARD EN 12687 NORME EUROPE ENNE EUROPA ISCHE NORM July 1998 ICS 07.080 Descriptors: biotechnology, genetics, modified organisms, envi
10、ronments, environmental protection, analysis methods, bioassay, experimental design English version Biotechnology Modified organisms for application in the environment Guidance for the characterization of the genetically modified organism by analysis of the genomic modification Biotechnologie Organi
11、smes modifie s disse mine s dans lenvironnement Guide pour la caracte risation de lorganisme ge ne tiquement modifie par lanalyse de la modification ge nomique Biotechnik Vera nderte Organismen zum Einsatz in der Umwelt Leitfaden fu r die Charakterisierung des gentechnisch vera nderten Organismus du
12、rch Untersuchung 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 Regulations which stipulate the conditions for giving this European Standard the status of a national standard without any alteration. Up-to
13、-date lists and bibliographical references concerning such national standards may be obtained on application to the Central Secretariat or to any CEN member. This European Standard exists in three official versions (English, French, German). A version in any other language made by translation under
14、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 national standards bodies of Austria, Belgium, Czech Republic, Denmark, Finland, France, Germany, Greece, Iceland, Ireland, Italy, Luxemb
15、ourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and United Kingdom.Page 2 EN 12687:1998 BSI 1998 Foreword This European Standard has been prepared by Technical Committee CEN/TC 233, Biotechnology, the Secretariat of which is held by AFNOR. This European Standard shall be given the st
16、atus 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 withdrawn at the latest by January 1999. This European Standard has been prepared under a mandate given to CEN by the European Commiss
17、ion and the European Free Trade Association. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Czech Republic, Denmark, Finland, France, Germany, Greece, Iceland, Ireland
18、, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and the United Kingdom. Contents Page Foreword 2 Introduction 3 1 Scope 3 2 Normative references 3 3 Definitions 3 4 Testing for genomic modification 4 5 Materials 4 6 Considerations for experimental procedures 5 7 Validi
19、ty of data analysis 7 8 Documentation of the results 8 Annex A (informative) Bibliography 9Page 3 EN 12687:1998 BSI 1998 Introduction This European Standard relates to part of the characterization of genetically modified organisms (GMOs). It is designed as a guideline for the adaptation of experimen
20、tal procedures to the requirements of the specific experimental design. The characterization of a GMO can include the analysis of: the genomic modification; the functional expression of the genomic modification (see EN 12682); the molecular stability of the genomic modification (see EN 12683). This
21、European Standard relates to the specific characterization of the genomic modification of GMOs. This characterization is implicit for use during environmental releases and should be applied, if required, during assessment of product quality. 1 Scope This European Standard gives guidance on the steps
22、 that should be followed during the analysis of the genetic modification of interest: to analyse and describe the genetic modification of interest as it exists in the GMO (genomic modification); to detect and/or identify the GMO accurately. This European Standard gives guidance on the factors and cr
23、iteria considered by the experimenter for the selection of the appropriate method(s) and the validity of experimental results for the analysis of the genetic modification of interest. The procedures described in this European Standard are applicable to testing the genomic modification. They include
24、techniques of biochemistry, immunology or molecular biology. 2 Normative references This 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 hereaf
25、ter. For dated references, subsequent amendments to or revisions of any of these publications 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 12682, Biotechnology Modified orga
26、nisms for application in the environment Guidance for the characterization of genetically modified organism by analysis of the functional expression of the genomic modification. EN 12683, Biotechnology Modified organisms for application in the environment Guidance for the characterization of genetic
27、ally modified organism by analysis of the molecular stability of the genomic modification. 3 Definitions 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 D
28、ata signals can be characterized: by binary decision: presence/absence (+/2); in relative terms by ordering 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 paramete
29、rs 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 certain DNA or RNA fragments by means of hybridization 3.5 genetic modification of interest co
30、nceptual design for altering the genetic material within an organism NOTE 1 The genetic modification of 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 organi
31、sm organism in which the genetic material has been altered in a way that does not occur naturally by mating and/or natural recombination NOTE Within the terms of this definition genetic modification occurs at least through the use of the techniques listed in Directive 90/220/EEC or its appropriate a
32、nnexes (see annex A 2). 3.7 genomic modification actual physical structure 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 stru
33、cture or the genetic modification of interest. NOTE 2 The certainty of identification can be affected by the types and/or number of characteristics investigated.Page 4 EN 12687:1998 BSI 1998 3.9 organism biological entity capable of replication or of transferring genetic material 3.10 phenotype sum
34、of the traits of an organism NOTE 1 The phenotype can be described with respect to one or more traits under a given set of conditions. NOTE 2 In the case of a virus, the phenotype can be described by one or more traits manifested in the infected host. 3.11 sample materials collected for analysis 3.1
35、2 trait observable and/or measurable characteristic 4 Testing for genomic modification 4.1 General considerations A genetic modification in general is intended to modify the expression of genetic traits of an organism or to produce a new gene product in a GMO, in order to modify the phenotype of tha
36、t organism. The presence of the genetic modification of interest as it exists in the GMO can be deduced from the presence or absence of an insert of specific DNA or gene product(s) such as RNA or protein, of a specific biochemical reaction, or of a specific phenotypic trait (see EN 12682). Only the
37、analysis at the level of genetic material provides information about the structure of the genetic modification of interest within the GMO. The methods described in this European Standard relate to the detection and identification of a particular GMO by determination of the presence of nucleic acid m
38、olecules which specifically characterizes the GMO. Usually the genetic modification of interest is a DNA sequence. However, in some special cases, the genetic modification of interest can be a RNA sequence (e.g. RNA-viruses). Not all of the methods described in this European Standard are necessarily
39、 applicable for every analysis of the genetic modification of interest as it exists in the GMO. This standard provides the criteria by which a suitable method or combination of methods is found for the analysis of the genomic modification, depending on the purpose of the analysis. The methods descri
40、bed in this standard can be appropriate to test GMOs provided that the genetic modification of interest is available either as cloned DNA, or as complete or partial nucleotide sequence data or other relevant data. They refer to techniques that are based on: a) restriction pattern analysis (see 4.2);
41、 b) DNA- or RNA-hybridization (see 4.3); c) DNA- or RNA-fragment amplification (see 4.4); d) DNA- or RNA-sequencing of the genomic modification (see 4.5). Examples of the application of such methods can be used to: qualitatively establish the presence of the genomic modification; estimate the copy n
42、umber; estimate the number of integration sites and their relative position in the genome of an organism; compare the genetic modification of interest with the actual genomic modification. 4.2 Restriction enzyme method Restriction pattern analysis provides the means to construct a primary physical m
43、ap of a DNA-segment. 4.3 Hybridization method Hybridization (molecular hybridization) is the sequence-dependent pairing of complementary single-stranded nucleic acid molecules resulting in a double-stranded hybrid. The detection of a genomic modification within an organism is visualized by hybridiza
44、tion with a labelled gene probe. 4.4 in-vitro DNA- or RNA-fragment amplification method The DNA- or RNA-fragment amplification method is used to obtain multiple copies (amplification) of a segment of DNA (template DNA) or RNA (template RNA) that is located between regions of known nucleotide sequenc
45、e, which are complementary with synthetic primers. 4.5 Sequencing method The methods for determination of nucleotide sequences can be used for: confirmation of sequence identity; confirmation of the sequence at the integration site; characterization of the sequence flanking the genomic modification.
46、 The determination of the sequence of the genomic modification should not be considered as a necessity. 5 Materials 5.1 Nucleic acid materials The test should be carried out on a nucleic acid target from the samples and the controls. Depending on the procedure and the experimental technique used, th
47、e requirement for quantity and purity of the extracted nucleic acids can be different. Special extraction procedures can be required for different organisms (Gram-positive and Gram-negative bacteria, yeasts, fungi, plant and animal cells). The extraction protocol should provide nucleic acid specimen
48、s of sufficient purity to ensure the reproducibility of the results. Important considerations for yield and purity of nucleic acids are the method of cellular lysis, the removal of cell debris, nucleases and hybridization inhibitors, and the gentle handling of samples to minimize mechanical shearing
49、 of long DNA molecules. The nucleic acid isolated from test samples and controls should ideally be prepared using the same procedure.Page 5 EN 12687:1998 BSI 1998 5.2 Gene probe There are different approaches for the preparation of the gene probe. For the purposes of this European Standard, it is appropriate that gene probes exist of nucleic acid fragments of known identity, for example a plasmid or a gene fragment used to define the genetic modification of interest or chemically
