BRITISH STANDARD |

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12687:1998
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Biotechnology Ð Modified |
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organisms for application in |
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the environment Ð Guidance |
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for the characterization of the |
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genetically modified organism |
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by analysis of the genomic |
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modification |
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The European Standard EN 12687:1998 has the status of a |
British Standard |
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ICS 07.080; |
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NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAW
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Copyright British Standards Institution
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 BS EN 12687:1998

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:

Ð aid enquirers to understand the text;

Ð present to the responsible European committee any enquiries on the
interpretation, or proposals for change, and keep the UK interests informed;
Ð monitor related international and European developments and promulgate
them in the UK.

A list of organizations represented 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 ªInternational Standards Correspondence Indexº, or by using the
ªFindº 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
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 Standard, having Amendments issued since publication

been prepared under the
direction of the Sector Amd. No. Date Text affected
Committee for Materials and
Chemicals, was published under
the authority of the Standards
Committee and comes into effect
on 15 December 1998

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ISBN 0 580 30180 X

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 EUROPEAN STANDARD EN 12687
NORME EUROPEÂENNE
EUROPAÈISCHE NORM July 1998

ICS 07.080

Descriptors: biotechnology, genetics, modified organisms, environments, environmental protection, analysis methods, bioassay,
experimental design

English version

Biotechnology Ð Modified organisms for application in the

environment Ð Guidance for the characterization of the genetically
modified organism by analysis of the genomic modification

Biotechnologie Ð Organismes modifieÂs disseÂmineÂs Biotechnik Ð VeraÈnderte Organismen zum Einsatz

dans l'environnement Ð Guide pour la in der Umwelt Ð Leitfaden fuÈr die
caracteÂrisation de l'organisme geÂneÂtiquement Charakterisierung des gentechnisch veraÈnderten
modifie par l'analyse de la modification geÂnomique Organismus durch Untersuchung der
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GenomveraÈnderung

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-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 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,
Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and
United Kingdom.

CEN
European Committee for Standardization
Comite EuropeÂen de Normalisation
EuropaÈisches Komitee fuÈr 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 12687:1998 E

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 Page 2
EN 12687:1998

Foreword Contents
This European Standard has been prepared by Page
Technical Committee CEN/TC 233, Biotechnology, the
Secretariat of which is held by AFNOR. Foreword 2
This European Standard shall be given the status of a Introduction 3
national standard, either by publication of an identical 1 Scope 3
text or by endorsement, at the latest by January 1999, 2 Normative references 3
and conflicting national standards shall be withdrawn
at the latest by January 1999. 3 Definitions 3
This European Standard has been prepared under a 4 Testing for genomic modification 4
mandate given to CEN by the European Commission 5 Materials 4
and the European Free Trade Association.
6 Considerations for experimental
According to the CEN/CENELEC Internal Regulations, procedures 5
the national standards organizations of the following
countries are bound to implement this European 7 Validity of data analysis 7
Standard: Austria, Belgium, Czech Republic, Denmark, 8 Documentation of the results 8
Finland, France, Germany, Greece, Iceland, Ireland, Annex A (informative) Bibliography 9
Italy, Luxembourg, Netherlands, Norway, Portugal,
Spain, Sweden, Switzerland and the United Kingdom.

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 Page 3
EN 12687:1998

Introduction 3 Definitions
This European Standard relates to part of the For the purposes of this standard, the following
characterization of genetically modified organisms definitions apply:
(GMOs). It is designed as a guideline for the adaptation 3.1
of experimental procedures to the requirements of the
specific experimental design. The characterization of a control
GMO can include the analysis of: preparation of known characteristics used to
Ð the genomic modification; standardize an analysis
Ð the functional expression of the genomic 3.2
modification (see EN 12682); data signal
Ð the molecular stability of the genomic output of a test system
modification (see EN 12683). NOTE Data signals can be characterized:
This European Standard relates to the specific Ð by binary decision: presence/absence (+/2);
characterization of the genomic modification of GMOs. Ð in relative terms by ordering the data signal strength with
This characterization is implicit for use during respect to (a) defined control(s);
environmental releases and should be applied, if Ð quantitatively by giving their output strength in absolute
terms;
required, during assessment of product quality.
Ð by position or movement;
Ð qualitatively by describing parameters not addressed by
1 Scope strength or position.

This European Standard gives guidance on the steps 3.3

that should be followed during the analysis of the
genetic modification of interest:
Ð to analyse and describe the genetic modification
of interest as it exists in the GMO (genomic
modification);
Ð to detect and/or identify the GMO accurately.
This European Standard gives guidance on the factors
and criteria 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 techniques of biochemistry, immunology
or molecular biology.
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
conceptual 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.

2 Normative references 3.6

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 hereafter. 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 Ð Modified organisms for
application in the environment Ð Guidance for the
characterization of genetically modified organism by
analysis of the functional expression of the genomic
modification.
EN 12683, Biotechnology Ð Modified organisms for
application in the environment Ð Guidance for the
characterization of genetically modified organism by
analysis of the molecular stability of the genomic
modification.
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genetically modified organism
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 annexes (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
structure or the genetic modification of interest.
NOTE 2 The certainty of identification can be affected by the
types and/or number of characteristics investigated.

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 Page 4
EN 12687:1998
3.9
organism
biological entity capable of replication or of
transferring genetic material
3.10
phenotype
sum 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
3.12
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 that 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
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
molecules 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 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 described 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);
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).
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Examples of the application of such methods can be
used to:
Ð qualitatively establish the presence of the
genomic modification;
Ð estimate the copy number;
Ð estimate the number of integration sites and their
relative position in the genome of an organism;
Ð 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 map 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
hybridization with a labelled gene probe.
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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 sequence, which are complementary with
synthetic primers.
4.5 Sequencing method
The methods for determination of nucleotide
sequences can be used for:
Ð confirmation of sequence identity;
Ð confirmation of the sequence at the integration
site;
Ð characterization of the sequence flanking the
genomic modification.
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, the
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 specimens 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 of long DNA molecules. The nucleic acid
isolated from test samples and controls should ideally
be prepared using the same procedure.
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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
synthesized oligonucleotides. For the detection and
identification of organisms or cell-lines, gene probes
should be sufficiently specific to allow differentiation
against a background of closely related strains.
A gene probe is more specific if it is limited to a
portion of the genomic modification rather than the
entire genomic modification to reduce the potential for
cross-hybridization or non-specific hybridization.
Hybridization conditions involving short
oligonucleotides can be chosen such that a single
nucleotide mismatch prevents the pairing of the
nucleotide strands, thus increasing the specificity. In
designing a gene probe the following criteria should be
considered:
Ð guanine plus cytosine content;
Ð self-complementarity;
Ð uniqueness of the sequence;
Ð length of the sequence;
Ð 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 nucleotides per hybrid.
Sensitivity sometimes can be improved by using single
stranded RNA- or DNA-gene probes thus reducing
self-annealing.
5.3 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 in any current technical manual (see annex A [3]).
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:
Ð efficacy;
Ð shelf life or half-life of the labelled gene probe;
Ð availability and costs of appropriate areas and
equipment;
Ð costs for training of personnel;
Ð availability and costs of labelling material.
6 Considerations for experimental
procedures
6.1 General
The main steps of the experimental procedures for the
analysis of the genomic modification are:
a) precise statement of the objective for the intended
analysis (see 6.2);
b) experimental design according to various criteria
(see 6.2);
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EN 12687:1998
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 clause 7);
f) documentation of the results (see clause 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 should be written down in a protocol, but
keeping the flexibility needed to handle unexpected
observations.
The experimental design to analyse the genomic
modification should take into consideration the
following:
Ð final objective of the analysis;
Ð type of analysis (detection and/or identification of
a GMO, presence of genetic modification of interest,
determination of copy number and insertion site(s));
Ð choice of the method(s);
Ð type of organism (micro-organism, plant, animal);
Ð type of genetic modification (deletion,
substitution, insertion);
Ð part of the organism used for analysis;
Ð type of nucleic acid to be tested;
Ð number of samples needed for statistical
confidence;
Ð use of appropriate controls.
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 identify the genomic
modification at the DNA or RNA 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 analysis. 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:
a) restriction pattern analysis;
b) nucleic acid hybridization methods;
c) nucleic acid amplification 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.
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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,
hybridization assay can be performed in solution. The
different transfer procedures and starting materials are
reflected in the designation of the following relevant
techniques.
a) Slot/dot blot analysis
These techniques are suitable for screening samples
to qualitatively detect the presence or absence of the
genetic modification of interest. Quantification can
be achieved through the use of a densitometer or
liquid scintillation counting. However, false positive
data signals can result from the presence of closely
related sequences, since these techniques detect
nucleic acid in batch and closely related sequences
can react. If these sequences are size fractionated
[see 6.3.3c], the false positive data signal often can
no longer be detected.
b) Colony, plaque and cytological hybridization
This hybridization technique allows the screening of
individual clones. It should be used when
conventional culturing techniques are insufficient to
identify the GMO.
The conditions for hybridization and stringency of
washing should be adapted to the gene probe used.
The specificity of the technique depends on the
ability of the gene probe to discriminate between the
target sequence, cross-reacting sequences and
material. Because of carry-over of components of
culture media on the hybridization membrane such
as salts and impurities from cell-lysis, artifactual data
signals can arise which can be misinterpreted as
positive. To identify artifacts it is often necessary to
hybridize duplicate filters.
The sensitivity of the technique is limited to the need
to cultivate the organisms.
The advantage of the technique is that large numbers
of colonies can be screened simultaneously, thus
saving time. Only a small amount of sample
preparation is necessary without the need to purify
the nucleic acid.
c) Southern and Northern hybridization
Southern and Northern blot analysis is usually used
for verification and diagnosis rather than for large
scale screening. By this technique, single strains or
varieties can be identified, as well as
rearrangements, deletions, gene transfer and the
copy number of the genetic modification of interest
in the genome. The conditions for hybridization and
stringency of washing should be adapted to the gene
probe used and the nature of hybridization
membranes.
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The specificity of the technique depends mainly on
the gene probe used and the nature of target nucleic
acid. The limit of detection is determined by the
amount of sample DNA or RNA and the specific
activity of label of the gene probe.
False positive hybridization data signals 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 the genomic modification had
undergone a rearrangement.
If the DNA or RNA to be detected in the positive
control is originally derived from a genetically
modified micro-organism (i.e. phage vectors,
conjugating bacteria or viruses), this DNA/RNA
should ideally be isolated and analysed by the same
procedure and evaluated by the same criteria as
nucleic acid of the final GMO.
A positive control for hybridization will be obtained
by restriction of the DNA fragment containing the
genetic modification of interest with a restriction
enzyme or set of enzymes having at least two cutting
sites in this fragment. The patterns obtained after
separation by gel electrophoresis should be
compared with DNA marker fragments of known
size and/or by comparison with a restriction map of
the original donor DNA.
A negative control should be obtained by using the
nucleic acid of an organism which is identical to the
GMO except for the lack of the sequence of interest,
(e.g. DNA/RNA of recipient or parental organism).
d) Solution hybridization
Hybridization proceeds in solution before the gene
probe-target duplex is captured for identification
and/or quantification. Because the target is not fixed
and thus is completely accessible to the gene probe,
hybridization in solution has the advantage of a
higher sensitivity.
Care should be taken because the gene probe-DNA
can reassociate (see annex A [3]). In this case, it may
be difficult to separate gene probe-target hybrids
from the self-annealed gene probe. Also, non-specific
binding of the gene probe to sequences with partial
homology can generate false positive data signals.
This technique permits the use of large amounts of
DNA which can be necessary if the concentration of
the target DNA in the sample is low.
e) In situ hybridization
This technique allows the detection and
identification of individual whole cells in samples
containing mixed population(s) and the analysis of
their spatial distribution. Whole cells, which are
made permeable to labelled gene probes, can be
visualized after hybridization. This technique can
also be applied to chromosomal spreads (FISH:
fluorescent in situ hybridization).
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6.3.4 Nucleic acid amplification methods
Nucleic acid amplification methods (e.g. PCR:
polymerase chain reaction, LCR: ligase chain
reaction, 3SR: self-sustained sequence replication
reaction) are based on multiple cycles of enzyme
driven amplification of DNA or RNA. The number of
amplification cycles necessary depends on the number
of template nucleic acid molecules starting the reaction
and the number of nucleic acid molecules needed for
the specific detection procedure.
Amplification of nucleic acids is a very sensitive
technique. Controls should be included that exclude
non-specific priming. Care should be taken in the
design of the primer pair used for the amplification
(uniqueness and length of the sequence) and to the
temperature of the annealing (PCR) to obtain
unambiguous results. Detection should be done in such
a way that the false positive reactions originating from
sample contamination or closely related sequences are
minimized. Identification of false positive events could
include sequencing, restriction enzyme digestion or
Southern blot analysis of the amplified DNA and/or
increase the stringency of the amplification reaction.
Amplification techniques are dependent on the proper
activity of the enzymes (e.g. thermostable DNA
polymerase, ligase, reverse transcriptase, RNA
polymerase). Crude nucleic acid samples sometimes
contain compounds that inhibit the activity of such
enzymes. To exclude such types of false negatives,
control amplification reactions should be performed to
ensure the purity of the nucleic acid sample and the
proper working conditions for the enzymes (see 7.2.2).
6.3.5 Sequencing methods
Sequence data of the genetic modification of interest
can be necessary to identify most accurately the
transformation event. These can include the nucleotide
sequence of the modified sequence and/or flanking
sequences (see 7.2.3).
6.4 Record keeping
A record should be kept of the execution of the
experimental procedures. This should include:
Ð identification of the experiment;
Ð person(s) performing the experiment;
Ð date of the experiment;
Ð method chosen for the analysis;
Ð origin of samples;
Ð number of samples analysed;
Ð controls used;
Ð deviations from the protocol;
Ð duration of the experiment;
Ð key equipment used.
NOTE This should not be considered an exhaustive list.
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EN 12687:1998
7 Validity of data analysis
7.1 General considerations
The results, regardless of the method(s) applied,
should be regarded as valid if:
a) the experimental design has been laid down in a
written protocol;
b) the experiment has been performed according to
the written protocol, indicating deviations from the
protocol;
c) samples and controls are analysed simultaneously
using the same method;
d) appropriate controls give the expected results;
e) limits of detection and reproducibility of the
applied method(s) are appropriate;
f) results are documented and realistically assessed
with regard to the information value of the applied
method; and
g) contamination can be clearly excluded.
7.2 Specific considerations
7.2.1 Validity of data analysis based on nucleic
acid hybridization methods
The experimental conditions used for hybridization
should ensure appropriate sensitivity and specificity for
the purposes of experimental design.
Using slot/dot blot or colony hybridization methods the
sample(s) to be analysed and both positive and
negative controls should be fixed onto the same filter
to allow direct comparison. The comparison between
positive and negative controls indicates the specificity
of the technique to detect the presence of a genetic
modification of interest.
Using cytological or plaque hybridization methods, it
might not be possible to fix positive and negative
controls to the same hybridization support. Therefore,
controls should be hybridized simultaneously under
identical experimental conditions.
Using Southern or Northern hybridization methods,
sample(s) and both positive and negative controls
should be run in the same gel to allow direct
comparison.
The comparison between positive and negative
controls indicates the ability of the technique to
specifically detect and analyse the genomic
modification and sequences, which are not in the
genetic modification but which hybridize to the gene
probe.
Southern blot analysis used in combination with
restriction analysis or pulsed field gel electrophoresis
and in situ hybridization on chromosomes can provide
sufficient information for the estimation of the number
of copies of the genomic modification.
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EN 12687:1998
A positive control with respect to hybridization
analysis consists of a mixed sample combining both
total nucleic acid extracted from non-modified
organism(s) and the appropriate fragments of the
modified nucleic acid of interest. As a result, positive
hybridization data signals should show up at positions
of the expected relative molecular mass. If additional
bands are visualized by this protocol, this could mean
that non-specific hybridization exists between total
nucleic acid and the gene probes, or that
contamination has occurred during sample preparation.
A negative control with respect to hybridization
analysis consists of nucleic acid extracted from a
non-modified organism. A positive hybridization data
signal demonstrates that non-specific hybridization can
occur with total non-modified nucleic acid, or that
contamination of the sample has occurred.
7.2.2 Validity of data analysis based on nucleic
acid fragment amplification methods
Using the amplification technique (e.g. PCR) the
sample(s) and both positive and negative controls
should be amplified in parallel, and the amplification
reaction should be verified in order to avoid false
results.
Verification of false positive results can be done by
testing a specimen without the target nucleic acid.
Verification of false negative results and the success of
the reaction can be done by simultaneous amplification
of a second nucleic acid fragment of different size in
the same sample which acts as an internal marker.
Such an internal marker can be a sequence of the
genome of the recipient organism, or a foreign
sequence added to the nucleic acid to be analysed, as
well as the positive and negative controls. However,
this type of PCR control can alter the limit of
detection. The primers used to amplify the internal
marker should not give any amplification of the
genomic modification. If size separation techniques are
used for detection, the length of the amplified fragment
of the internal marker should be similar to, but clearly
distinguishable from the length of the amplified
fragment of the modified sequence.
The comparison between positive and negative
controls and between sample(s) and controls indicates
the specificity of the analysis and whether the genetic
modification of interest is present in the representative
samples.
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A positive control with respect to the amplification
procedure consists of a nucleic acid sample of the
recipient organism to which the modified sequence of
interest is added as purified nucleic acid.
A negative control with respect to the amplification
procedure consists of a nucleic acid sample of the
recipient organism, which does not contain the
sequence involved in genomic modification.
7.2.3 Validity of data analysis based on the
sequencing methods
The sequence should be clearly readable from the
visualized picture of the results of sequencing reaction.
The use of a positive control should be included in the
analysis.
The determination of the sequence of only one strand
of the nucleic acid of the sequence of interest is
sufficient if the aim is to confirm sequence identity.
The differences of nucleotides should not exceed
normal sequencing artifacts of the enzymes used. If the
sequence is determined from an amplified nucleic acid,
the template DNA used for sequencing should be
derived directly from amplification reaction.
The position of the sequenced region should be
adequately determined by reference to well-known
restriction maps or by unequivocal identification of
sequence overlaps.
If the sequence is determined at integration sites to
verify the location of the fragment, the determined
sequence should be as anticipated.
The positive control consists of an already sequenced
nucleic acid fragment.
8 Documentation of the results
The results should be documented in a comprehensible
form by films (e.g. autoradiographic films), photos of
filters or gels, and by text indicating the results
obtained (conformity of the DNA intended, modified
sequence present or not, estimated number of copies
or range of number of copies per genome or cell
number) with reference to this European Standard,
and indicating at least which technique was used. The
documentation of results should also include a
description of the genetic modification of interest as it
exists in the GMO and a short written evaluation of the
tests by the experimenter. The report should be kept in
an identified location.
 BSI 1998
Not for Resale
 Page 9
EN 12687:1998

Annex A (informative)
Bibliography
[1] Council Directive 90/219/EEC of 23 April 1990 on
the contained use of genetically modified
micro-organisms. OJEC 08.05.1990, no. L 117, p 1.
[2] Council Directive 90/220/EEC of 23 April 1990 on
the deliberate release into the environment of
genetically modified organisms. OJEC 08.05.1990,
no. L 117, p 15.
[3] Ausubel, F.A. et al. (eds. Greene). Current
Protocols in Molecular Biology. Publ. Wiley &
Sons.
[4] EN 12305, Biotechnology Ð Modified organisms
for application in the environment Ð Guidance
for the sampling strategies for deliberate releases
of genetically modified plants.
[5] EN 12686, Biotechnology Ð Modified organisms
for application in the environment Ð Guidance
for the sampling strategies for deliberate releases
of genetically modified micro-organisms,
including viruses.

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[6] Sambrook J., Fritsch E.F. and Maniatis T. (1989).
Molecular Cloning. A Laboratory Manual, Second
Edition, Cold Spring Harbor Laboratory Press.

Copyright British Standards Institution

 BSI 1998
Provided by IHS under license with BSI
No reproduction or networking permitted without license from IHS Not for Resale
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