3.2 Various nucleotide-based amplification methods and their
Most of the methods mentioned in this section have generally not
yet been used widely for the identification of genetically engineered
food or food stuffs. This survey, therefore, very much restricts
itself to survey review articles that may simplify access to additional
readings. Some of the techniques may, under certain circumstances,
be appropriate for food analyses. Ongoing research projects (see
later sections) include the evaluation of the applicability of
some of these methods for the detection of genetically engineered
3.2.1 Ligase Chain Reaction (LCR)
The ligase chain reaction is a DNA amplification method based
on repeated cycles of oligonucleotide hybridisation and ligation
(Backman and Young, 1989; Carrino and Lee, 1995). The method employs
sets of oligonucleotides specific to stretches of the target sequence
that are in close proximity to each other, as well as another
set of oligonucleotides that is complementary to the first set.
The protocol is very similar to PCR, except that LCR uses a heat-stable
ligase. Polymerase activity is not needed since the primers basically
constitute virtually the entire length of the target sequence.
Therefore, the length of the amplicon will generally be limited
by the availability of longer oligonucleotides. Although known
for years now, LCR or variations of this technique (e.g. Gap-LCR)
is by far not as significant in routine diagnostics as is PCR
(Carrino and Lee, 1995; Pfeffer et al., 1995).
3.2.2 Nucleic Acid Sequence-Based Amplification (NASBA)
This technique mimics the process of retroviral replication (Compton,
1991) and has been used until now primarily for the amplification
of RNA molecules (Carrino and Lee, 1995). The method might be
applicable for the detection of expressed transgenes and/or viable
microorganisms (Blais et al., 1997). Because RNA molecules are
present in much higher copy numbers than the respective gene (provided
the gene is expressed), NASBA may demonstrate a greater degree
of sensitivity compared to PCR for certain applications (Lunel
et al., 1995). However, RNA is much more sensitive to degradation
than DNA; therefore, the probe material must necessarily be very
fresh and appropriately handled. For heat-treated and other processed
foods the applicability of NASBA seems very limited. As PCR assays
of fresh foods are normally sufficiently sensitive, it seems unlikely
that NASBA will find broad application in food analysis.
3.2.3 'Self-sustained sequence replication' (3SR) and 'Q replicase
Methods for the identification of pathogenic microorganisms have
already been developed based on the isothermal 3SR and Q replicase
amplification techniques (Carrino and Lee, 1995; Pfeffer et al.,
1995). Despite a high amplification rate, these techniques are
of less significance in diagnostics as compared to PCR (Pfeffer
et al., 1995). Moreover, the alleged technical advantage of an
isothermal reaction (Pfeffer et al., 1995), with fast amplification
that is not limited by defined temperature and time-cycles and
requiring less special equipment, can actually be a disadvantage
when compared to methods such as PCR and LCR, which employ pre-set
cycles: discrete obligatory temperature cycles have been considered
to be a main cause for the relatively minor tendency of PCR for
certain experimental artefacts ('in vitro evolution', i.e. amplifying
artificially small DNA fragments), whereas isothermal techniques
favour fast replicators (Bull and Pease, 1995) and thus short
3.2.4 Fingerprinting techniques (RFLP, AFLP, RAPD, etc.)
Fingerprinting techniques such as RFLP (Restriction Fragment Length
Polymorphism), AFLP (Amplified Fragment Length Polymorphism) or
RAPD (Random Amplified Polymorphic DNA) are used in forensic analysis
and for the classification of organisms. They have been successfully
used in combination with PCR amplification to classify microorganisms
(Tichy and Simon, 1994) and for other applications (Welsh et al.,
1995). Fingerprint techniques are applicable for the analysis
of complex mixtures of microorganisms used as starter cultures.
In this context, fingerprinting may allow to confirm, if a given
genetic modification is indeed present in the expected genetic
background of a given microorganism. These techniques are based
on the comparison of the genomes of related organisms but they
may not be sensitive enough to resolve the difference between
the DNA of transgenic organisms and their conventional counterparts.
The genetic differences among varieties of the same crop are by
far greater than differences between a genetically engineered
crop and its conventional counterpart. Therefore, with fingerprinting
methods it is essential that the DNA compared be derived from
exactly the same crop variety before and after transformation.
If more than one transgenic product of a certain species (e.g.
corn) exists, the DNA of all the respective hosts will be required.
Such conditions are difficult to satisfy. Furthermore, fingerprint
techniques apparently cannot be used for analysing complex food
mixtures or processed foods.
3.2.5 Probe hybridisation
Hybridisations using DNA probes have been frequently used for
the detection of pathogens in food (Jones, 1991). One model system
for the detection of genetically modified bacteria in milk has
been published (Casey et al., 1993). The degree of sensitivity
and specificity of probe hybridisation is significantly lower
than that achieved through the previously described amplification
techniques. Since plants have particularly large genomes but transgenes
are present only one or a few copies (thus the relative concentration
of target sequence to total DNA is low), the application of probe
hybridisation for detecting GMO crops does not seem very promising.
However, provided that the target sequence is present in sufficient
concentrations (multiple copies of the transgenes, small genome
size [e.g. bacteria]), probe quantity is not significantly limited,
and highly specific oligonucleotide probes are available, probe
hybridisation may provide a simple technique worth considering
for screening purposes.