1、STDmBSI BS EN 12b87-ENGL 1778 1b24bb7 0739591 747 Biotechnology - Modified organisms for application in the environment - Guidance for the characterization of the genetically modified organism by analysis of the genomic modification BRITISH STANDARD I The European Standard EN 126871998 has the statu
2、s of a British Standard ICs 07.080; NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAW STD-BSI BS EN L2b7-ENGL 1798 M LL2Libb7 U737592 883 Date BS EN 12687:1998 Textaffected National foreword This British Standard is the English language version of EN 126871998. The UK participat
3、ion in its preparation was entrusted to Technid Conunittee CIi58, Biotechnology, which has the responsibility to: - aid enquirem to understand the text; - present to the responsible European committee any enquiries on the - monitor related intedonai and European developments and promulgate interpret
4、ation, or proposals for change, and keep the UK interests informed; them in the UK. A list of organhations represented on this committee can be obtained on request to its secretary. Cross-references The British Standards which implement international or European publications refexred to in this docu
5、ment may be found in the BSI Standards Catalogue under the section entitled “Intdonal Standards Correspondence 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 coniract Users of British
6、 Standards are responsible for their correct application. Compliance with a British Standard does not of itaelf 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 9 and a back cover. This British Standa
7、rd, having Amendments issued since publication 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 16 December 1998 Amd. No. O BSI 1998 ISBN O 680 30180 X STD-BSI BS EN L2bA7-ENGL
8、 1778 I Lb24bb7 0737573 7LT = EUROPEAN STANDARD EN 12687 NO= EUR0PEE”E EUROP - the functional expression of the genomic modifidon (see EN 12682); - the molecular stability of the genomic modification (see EN 12683). This European Standard relates to the specific characterization of the genomic modif
9、cation 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 that should be followed during the analysis of the genetic modification of inter
10、est. - to analyse and describe the genetic modifcation of interest as it exists in the GMO (genomic modification); - to detect andor identify the GMO accurately. This European Standard gives guidance on the factors and criteria considered by the experimenter for the selection of the appropriate meth
11、d(s) and the validity of experimental results for the culalysis of the genetic modification of interest The procedures described in this European Standard are applicable to testing the genomic modification. They include techniques of biochemm, immunology or molecular biology 2 Normative references T
12、his European Standard incorporates by dated or undated reference, provisions from other publications. These normative references are cited at the appropriate piaces in the text and the publications ase listed hereafter. For dated references, subsequent amendments to or revisions of any of these publ
13、ications 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 organimns for applhtion in the environment - Guidance for the chamterization of genetic
14、auy moded organism by analysis of the functional wqn-msiun of the gmic modifztion. EN 12683, Bwtechmlogy - Modif? organimns for application in the environment - Guictnee for the characterization of genetically modified organism by analysis of #te molecular stubty of the genomic modification. Page 3
15、EN 12687:1998 3 Definitions For the purposes of this standard, the following definitions apply: 3.1 control prepamion of known characteristics used to standardize an culalysis 3.2 data signal output of a test system NOTE Data signals can be characterized - by binary decision: presencdabsence (+/-);
16、- in relative terms by ordering the data signal strength with respect to (a) dened contxol(s); - quantitatively by giving their output strength in absolute tem; - by position or movement; - qualitatively by describing parameters not addressed by strength or position. 3.3 detection recognition of the
17、 presence of an organism or of a molecular sh-ucture within a sample 3.4 gene probe specific nucleic acid sequence used to identify certain DNA or RNA fragments by means of hybridization 3.6 genetic modication of interest conceptual design for altering the genetic material within an organism NOTE 1
18、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 organism organism in which the genetic materiai has been altered in a way that doe
19、s not occur naturaily 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 annexes (see annex A 2). 3.7 genomic modification actual physical structure o
20、f the genetic modification of interest as it exists in the genetic* 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 identifi
21、cation can be affected by the types and/or number of characteristics investigad. O BSI 1998 Page 4 EN 126871998 3.9 organism biological entity capable of replication or of transferring genetic material 3.10 phenotype sum of the imits of an organism NOTE 1 The phenotype can be described with respect
22、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 3.12 trait observable and/or measurable cwteac 4 Testing for genomic modification 4
23、.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 that organism. The presence of the genetic modification of interest as it exists in the GMO
24、 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 analysis at the level of genetic material provides information about the stsucture of th
25、e 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 moldes which specifically characterizes theGMO. Usuaily the genetic modification of inter
26、est 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 applicable for every analysis of the genetic modification of interest as it exists in the G
27、MO. 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 described in this standard can be appropriate to test GMOs provided that the genetic modification
28、of interest is available either as cloned DNA, or as complete or partiai nucleotide sequence data or other relevant data They refer to techniques that are based on: a) restsiction pattern analysis (see 4.2); b) DNA- or RNA-hybridization (see 4.3); c) DNA- or RNA-fragment amplification (see 4.4); Exa
29、mples of the application of such methods can be used to: - qualbtively establish the presence of the genomic modification; - estimate the copy number; - esthate the number of integration sites and their relative position in the genome of an organism; - compare the genetic modification of interest wi
30、th the actual genomic modification. 4.2 Restriction enzyme method Restriction patteni analysis provides the means to construct a primary physical map of a DNA-segment. 4.3 Hybridization method Hybridization (molecular hybridization) is the sequencedependent pairing of complementary single-stranded n
31、ucleic acid molecules resulting in a double-stmnded hybrid. The detection of a genomic modification within an organism is visualized by hybridization with a labelled gene probe. 4.4 in-uitro DNA- or RNA-fragment amplification method The DNA- or RNA-fragment amplincation method is used to obtain mult
32、iple copies (amplification) of a segment of DNA (template DNA) or RNA (template RNA) that is located between regions of known nucleotide sequence, which are complementary with synthetic primers. 4.6 Sequencing method The methods for determirution of nucleotide sequences can be used for: - confinnato
33、n of sequence identity; - confinnaton of the sequence at the integration site, - characterization of the sequence flanking the genomic modification. The determination of the sequence of the genomic modification should not be considered as a necessity. 6 Materials 6.1 Nucleic acid materials The test
34、should be carried out on a nucleic acid target from the samples and the controls. Depending on the procedure and the experimental technique used, the requirement for quantity and purity of the extracted nucleic acids can be Merent. Special exhction procedures can be required for Merent organisms (Gr
35、am-positive and Gram-negative bacteria, yeasts, fungi, plant and animal cells). The extraction protocol should provide nucleic acid specimens of sufficient purity to ensure the reproducibility of the results. important consideraions for yield and purity of nucleic acids are the method of cellular ly
36、sis, the remod of cell debris, nucleases and hybridization inhibitom, and the gentle handling of samples to minimize mechanical shearing of long DNA molecules. The nucleic acid d) DNA- or RNA-sequencing of the genomic modification (see 4.6). isolated from test samples and controls should ideally be
37、prepared using the same procedure. O BSI 1998 STD-BSI BS EN 12b87-ENGL 1778 = lb24hb7 0737577 3b5 II 6.2 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
38、known identi - seif-complementari, - uniqueness of the sequence; - length of the sequence; - type of nucleic acid In general, it should be noted that the sensitivity of a method using short gene probes is lower than that of a method using longer gene probes because they incorporate fewer labelled nu
39、cleotides per hybrid. Sensitivity sometimes can be improved by using single stranded RNA- or DNA-gene probes thus reducing self-annealing. 6.3 Labelling techniques of the gene probe Guidance on choice of label and appropriate methods for incorporation of label into the gene probe can be found m any
40、current technical muai (see annexA 31). If appropriate, validated methods should be used. In determining the choice of label and the labelling technique, it is important to consider the following points - efficacy; - shelf life or half-life of the labelled gene probe; - adability and costs of approp
41、riate areas and equipment; - costs for training of personnel; - availability and costs of labelling material. 6 Considerations for experimental procedures 6.1 General The main steps of the experimental procedures for the anaysis of the genomic modification are: a) precise statement of the objective
42、for the intended analysis (see 6.2); b) experimental design according to various criteria (see 6.2); Page 5 EN 12687A998 c) execution of the analysis according to the experimental protocol (see 6.3); d) appropriate record keeping (see 6.4); e) evauation of the validity of the results (see clause 7);
43、 f) documentation of the results (see ciause 8). 6.2 Experimental design The considerations for experimental procedures to analyse the genetic modification of interest should start with the definition and statement of the objective for this analysis followed by the design of the experiment, which sh
44、ould be written down in a protocol, but keeping the flexibility needed to handle unexpected obsedons. The experimental design to analyse the genomic modification should take into consideration the following: - final objective of the analysis; - type of analysis (detection andor identincation of a GM
45、O, presence of genetic modification of interest, determination of copy number and insertion site(s); - choice of the method(s); - type of organism (micro-organism, plant, animai); - type of genetic modification (deletion, substitution, insertion); - part of the organism used for analysis; - type of
46、nucleic acid to be tested - number of samples needed for statistical confidence; - use of appropriate controis. NOTE This list should not be considered exhaustive. 6.3 Execution of the experimental protocol 6.3.1 General The following is a list of methods appropriate to specifically detect and ident
47、ify the genomic modification at the DNA or RNA level. Not all of these methods are necessarily applicable for the analycis of every genomic modification. The method of choice and its appropriateness or combination of methock depends on the stated objectives of the analysis. As molecular biology is a
48、 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: a) restriction patteni anaiysis; b) nucleic acid hybridization methods, c) nucleic acid amplificatio
49、n methods; d) sequencing methods. 6.3.2 Restriction pattern analysis The alteration in fragment length or presence or absence of inserted DNA can be detected by digestion of the isolated DNA with one or a combination of restriction enzymes, followed by fractionating DNA fragments according to size and visualizing by appropriate means. O BSI 1998 Page 6 EN 12687:1998 6.3.3 Nucleic acid hybridization methods In preparation of the hybridization assay the nucleic acid should be transferred and fixed onto a solid support (membrane or other material). Alternatively, hybridizat
