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Biomonitoring – New methods for detecting transgenic DNA in soil and plant samples

(2001 – 2004) University of Oldenburg, Department of Biology, Genetics Working Group

Topic

The available methods for detecting genetic material (DNA) allow sensitive detection of transgenic DNA in e.g. food and plants, but they are not so well suited to routine use in e.g. monitoring tasks that involve large sample volumes.

The polymerase chain reaction (PCR) method is often used to detect DNA. However, a number of methodological problems make it difficult to use in a routine manner, e.g. in soil tests. For instance, the method requires highly purified extracts. In addition, the sensitivity of the PCR method can vary considerably when individual parameters are modified slightly. What is needed, therefore, are detection methods that are both sensitive and easy to use and give reproducible results.

For this reason, the project aimed to develop a non-PCR method for the detection of transgenic sequences of genetically modified plants. The detection principle is based on identification of the transgene through natural transformation of living bacteria.

A specially constructed bacterium of the species Acinetobacter sp. serves as a quasi living indicator for the presence or absence of a transgene in the plant or soil sample to be investigated.

The procedure is also called biomonitoring for this reason.

Summary

The aim of the project was to develop a simple yet sensitive procedure for detecting transgenic DNA in plant and soil samples. In the biomonitoring method carried out here, special recipient bacteria of the species Acinetobacter sp. were constructed for the transgene under investigation. The bacteria can survive only by taking up the transgene and integrating it into their genome.

Using this principle, to detect transgenes with the antibiotic resistance gene, bacterial cells were used that had an incomplete antibiotic resistance gene and required the transgene DNA to repair it. The detection of transgenes without the antibiotic resistance gene was also possible using a ‘suicide gene’ introduced into the recipient cells.

The high sensitivity of biomonitoring for detecting small traces of DNA was demonstrated. An advantage is that, unlike PCR, these methods do not require highly purified DNA.

Biomonitoring systems were applied to over 500 samples of transgenic plants from release experiments. A release of recombinant DNA from pollen and roots of living transgenic plants was observed.

Experiment description

DNA detection in plant samples usually involves the PCR method. With this method, DNA traces are amplified (in vitro amplification) until they can be made visible using e.g. suitable fluorescent dye. The amplification of specific gene sequences is triggered by special start sequences (primers) that combine only with the DNA under investigation.

As with PCR, in biomonitoring the detection of the transgenes under investigation involves amplifying them, but in this case the amplification takes place in indicator bacteria (in vivo amplification). Suitable recipient bacteria are constructed specially for the transgene under investigation. If the transgene to be detected is present in the sample, the recipient bacteria exchange their own (marked) transgene copy with the transgene from the plant (see diagram).

The recipient bacteria are constructed in such a way that only those bacterial cells survive which are successfully transformed, i.e. are able to exchange their own (marked) transgene copy with the transgene from the plant. If the transgene is not present in the sample to be examined then the exchange cannot take place. The bacterial cells are then killed off during the subsequent selection. Growth under selection conditions is then evidence of the presence of the transgene in the sample.

Results

Detection of antibiotic resistance genes

Several biomonitoring systems were successfully developed. Bacterial cells were constructed, each with an incomplete antibiotic resistance gene. By taking up the relevant transgene, this gene is completed and the bacteria are capable of growing in the presence of the antibiotic in question. If the bacteria are capable of reproducing in the medium containing the antibiotic, this demonstrates the presence of the transgene in an examined soil sample. So far, this method has been used to demonstrate transgene sequences with the Kanamycin resistance gene nptII (in the nuclear genome) and with the Spectinomycin resistance gene aadA (in the plastid genome) with a high level of sensitivity.

Detecting transgenes without the antibiotic resistance gene

Effective biomonitoring systems were also developed for transgenes without the antibiotic resistance gene. For this, the bacterial cells were equipped with a ‘suicide gene’ capable of being switched on. In the absence of the transgene under investigation, the bacterial cells die off. However, if the transgene under investigation is present, it is integrated into the bacterial genome in such a way that it replaces the ‘suicide gene’. Surviving cells are therefore evidence of the presence of the transgene in an examined sample.

Using biomonitoring it was possible to demonstrate the release of recombinant DNA from the pollen and roots of living transgenic potato plants.

It was possible to confirm that the biomonitoring method is about as sensitive as PCR in detecting small traces of DNA. An advantage is that, unlike PCR, these methods do not require highly purified DNA.

The biomonitoring systems were applied to over 500 samples from release experiments of transgenic plants. The results of these studies show that transgenic plants release recombinant DNA during normal growth (and not only when they decay). This occurs both via the roots and via pollen.