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The introduction of genetically engineered (GE) crops has resulted in the
need to address concerns that were of considerably lower significance in
strictly conventional crop production systems. They include concerns over
food safety, environmental safety, and biodiversity in agroecosystems, May
2005 by Robert Gulden*, S Lerat, M Hart, R Campbell, J Powell, P Pauls, J
Klironomos, C Swanton, and J Trevors.

In Ontario, Canada, for example, where corn?soybean crop rotations are
common, the most abundant GE varieties of these crops, i.e., those resistant
to the herbicide glyphosate, comprise about 12% and 55% of the annual
acreage of these crops, respectively1 in 2004.

The body of research examining transgenic DNA in the food supply and in the
ecosystem has grown substantially in recent years. In the environment,
transgene escape through intra- and interspecific hybridization during all
stages of field production has been observed2. Although we are beginning to
understand the extent of transgenic DNA above the soil surface, very little
is known about the fate of transgenic plant DNA in the soil ecosystem.

Technical challenges of working in the soil environment, including high
spatial variability and difficulties of extracting DNA without co-extraction
of polymerase chain reaction (PCR) inhibitors, have been significant
limitations to this area of research. However, recent advances in DNA
extraction methodology and molecular techniques allow for the development of
routine high-throughput techniques to examine comprehensively the fate of
plant DNA in the soil environment.

Recently, Lerat et al.3 developed molecular tools for the detection of plant
DNA in soil for glyphosate tolerant (Roundup Ready) corn and soybean. In
these crops, the CP4 EPSPS gene (5-enolpyruvylshikimate-3-phosphate
synthase), which confers resistance to glyphosate, is identical, while other
elements of the inserted gene cassettes of RR corn (event NK603)4 and RR
soybean (event 40-3-2)5 are similar.

The challenge lies in DNA recovery

Before transgenic plant DNA can be quantified, it must be recovered from
soil. A number of DNA extraction protocols applicable to soil exist;
however, most are time and labor intensive and therefore are not suitable
for routine extraction of high sample numbers required to estimate DNA
content accurately. Thus, a high-throughput method for plant target DNA
recovery from soil was developed.

The method is based on a commercially available soil DNA extraction kit. The
UltraClean-htp kit (Mo Bio Laboratories, Solana Beach, CA) uses the 96-well
plate format for high sample throughput and is designed to extract the total
soil DNA content from 0.25 to 1.0 gm soil per sample. DNA is extracted with
a direct extraction method based on physical and chemical cell disruption
and is recovered on a silica membrane.

The manufacturer's instructions were modified for maximum plant target DNA
recovery. Specifically, glass beads, aurintricarboxylic acid, and
AlNH4(SO4)2 were added prior to bead beating. A major limitation of DNA
recovery from soil is that PCR inhibitors such as humic and fulvic acids are
co-extracted with DNA in many purification protocols. Although humic and
fulvic acids are removed by the kit, the addition of AlNH4(SO4)2, a
flocculent that precipitates these compounds, improved plant target DNA
recovery. Adding one large glass bead to each well also improved plant DNA
recovery by increasing the mechanical disruption of cells. DNAses and
nucleases are present in soil that can quickly degrade free DNA. The
addition of aurintricarboxylic acid, a DNAse and nuclease inhibitor,
improved plant DNA recovery as well. The remainder of the DNA extraction
method followed the manufacturer's instructions.

Detection of plant transgenes

Specific primers and a molecular beacon for real-time PCR analysis were
designed to detect recombinant plant DNA in soil extracts. Detection of the
recombinant CP4 EPSPS gene in soil extracts presents a challenge as this
gene originated from a strain of Agrobacterium, which is a common soil
bacteria. This gene is also present in many other soil bacteria.

A second challenge was to discern between the corn and soybean CP4 EPSPS, as
the sequences of the recombinant EPSPSs are identical in both species. To
overcome these challenges, the junction between the beginning of the EPSPS
gene and the adjacent chloroplast transit peptide (CTP) gene was targeted
for PCR amplification. The CTP gene adjacent to the CP4 EPSPS is different
in corn and soybean and therefore targeting this junction provided the
desired species specificity. This approach required only a single probe
(molecular beacon), one reverse primer, and two different forward primers.
The probe and the reverse primer annealed at the same positions on the CP4
EPSPS gene in corn and soybean. The unique forward primers ensure
amplification was initiated either on corn or soybean template DNA, without
falsely identifying other EPSPS genes, including those from soil bacteria.

Real time PCR was used to quantify plant DNA in soil extracts because this
technique is highly specific, extremely sensitive, and can accurately
quantify DNA over a large concentration range. In fact, it was possible to
detect as few as a single copy of plant target DNA in one microlitre of DNA
extract that was added to the PCR reaction. Unfortunately, using a single
probe to identify two separate target sequences does not lend itself to
multiplexing these PCR reactions. Multiplexing real-time PCR allows for the
quantitation of more than one target sequence in the same PCR reaction, but
requires separate probes for each target sequence.

What's next?

New molecular techniques and DNA extraction method for recovery of DNA from
challenging media have made it possible to develop tools that can be used to
routinely detect low quantities of plant DNA in the soil environment. This
high-throughput soil DNA extraction method can now be employed to address
questions that require analysis of large numbers of samples, including
questions such as the persistence and spatial distribution of plant DNA as
well as the influence of cropping system on the DNA cycle in the soil.

This technique can also be adapted to address questions regarding soil
microbial DNA diversity and soil microbe functionality. The introduction of
GM crops has provided the impetus, and modern molecular methods and DNA
extraction techniques have provided the means, to investigate basic
questions about the DNA cycle in the soil environment that have not yet been
addressed.

References
1. Anonymous (2004) Update summer 04. Agcare 14(3), 1-4. [Online]
[www.agcare.org]

2. Marvier & Van Acker (2005) Can crop transgenes be kept on a leash? Front.
Ecol. Environ. 3, 99-106

3. Lerat et al. (2005) Real-time polymerase chain reaction quantification of
the transgenes for Roundup Ready corn and Roundup Ready soybean in soil
samples. J. Agric. Food. Chem. 53, 1337-1342

4. See [www.agbios.com]

5. See [www.agbios.com]


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