Procedures For ELISA

-Relevant antibody bound to a solid phrase (polystyrene microtiter plates)
-Heat-treated broth culture (from food) added to the well
-Unbound antigen washed away
-Enzyme labeled antibody (conjugate) is added
-Incubated
-After incubation, unbound conjugate washed away
-Specific substrate added – to show the reaction via enzymatic reactions
-Incubation at room temperature
-Reaction ceased by addition of sulfuric acid
-Results read through photometer at specific wavelength

Toxins Found In Flour/Starch

Natural Toxins
-Amines and monoamine oxidase inhibitors with chemicals neurotransmitters like tyramine, tryptamine, serotonin, adrenaline, noradrenaline, dopamine
-Proteinase inhibitor like alpha amylase and trypsin inhibitors
-Lectins
-Coeliac sprue (gluten enteropathy)
-Solanum alkloids - esp glycoalkaloid
-Isoflavone glycosides
-Coumestans, lignans

Polycyclic Aromatic Hydrocarbons
-PAHs - present in low level in cereals mostly due to aerial deposition

Heavy Metals
-Cadmium
-Chromium
-Nickel

Pesticides

Mold Toxin
-Citreoviridin
-Ochratoxin A
-Ergot Alkaloids
-Aflatoxins
-Fusaria toxins (Including T2 toxin [type A trichothecene], Diacetoxyscirpenol [type A trichothecene], Deoxynivalenol [type B trichothecene], zearalenone, fumonisins, moniliformin, fasaric acid)

Baterial Toxins
-Bacillus spp.
-Miscellaneous Enterobacteriaceae
-Listeria
-Clostridium botulinum
-Clostridium perfringens

Transgenic Crops

How are Transgenic Crops Made?

For many years plant breeding entailed the selection of the finest plants to get the best crops. In those days, variation occurred through induced mutation or hybridization where two or more plants were crossed. Selection occurred through nature, using a “selection of the fittest” concept, where only the seeds best adapted to that environment succeeded. For example, farmers selected only the biggest seeds with non-shattering seed heads, assuming these to be the best. Today, scientists can not only select, but also create crops by inserting genes to make a seeds bare any trait desired.

In order to make a transgenic crop, there are five main steps: extracting DNA, cloning a gene of interest, designing the gene for plant infiltration, transformation, and finally plant breeding (see Figure 1).



To understand this process, one must first known a bit about DNA (deoxyribonucleic acids). DNA is the universal programming language of all cells and stores their genetic information. It contains thousands of genes, which are discrete segments of DNA that encode the information necessary to produce and assemble specific proteins. All genes require specific regions in order to be utilized (or expressed) by a cell. These regions include (see Figure 2):

1. A promoter region, which signals where a gene begins and it used to express the gene;

2. A termination sequence, which signals the end of a gene;

3. And the coding region, which contains the actual gene to be expressed.

All these regions together allow a gene to create a protein. Once a gene is transcribed into a protein, it can then function as an enzyme to catalyze biochemical reactions or as a structural unit of a cell, both of which will contribute to the appearance of a particular trait in that organism.

All species are capable of turning DNA into protein through a process known as translation. This capability makes it possible to artificially put genes from one organism into another-a process generally termed transgenics. But just isolating random DNA and inserting it into another organism is not practical. We must first know what particular segments of DNA, and in particular what genes, to insert. Unfortunately, with reference to producing new crops, not much is known about which genes are responsible for increased plant yield, tolerance to different stresses and insects, color, or various other plant characteristics. Much of the research in transgenics is now focused on how to identify and sequence genes contributing to these characteristics.

Genes that are determined to contribute to certain traits then need to be obtained in a significant amount before they can be inserted into another organism. In order to obtain the DNA comprising a gene, DNA is first extracted from cells and put into a bacterial plasmid. A plasmid is a molecular biological tool that allows any segment of DNA in be put into a carrier cell (usually a bacterial cell) and replicated to produce more of it. A bacterial cell (i.e. E. coli) that contains a plasmid can put aside and used over and over again to produce copies of the gene the researcher is interested in, a process that is generally referred to as “cloning” the gene. The word “cloning” referring to how many identical copies of the original gene can now be produced at will. Plasmids containing this gene can be used to modify the gene in any way the researcher sees fit, allowing novel effects on the gene trait to be produced (see Figure 1).

Once the gene of interest has been amplified, it is time to introduce it into the plant species we are interested in. The nucleus of the plant cell is the target for the new transgenic DNA. There are many methods of doing this but the two most common methods include the “Gene Gun” and Agrobacterium method.

The “Gene Gun” method, also known as the micro-projectile bombardment method, is most commonly used in species such as corn and rice. As its name implies, this procedure involves high velocity micro-projectiles to deliver DNA into living cells using a gun [1]. It involves sticking DNA to small micro-projectiles and then firing these into a cell. This technique is clean and safe. It enables scientists to transform organized tissue of plant species and has a universal delivery system common to many tissue types from many different species1. It can give rise to un-wanted side effects, such as the gene of interest being rearranged upon entry [1] or the target cell sustaining damage upon bombardment. Nevertheless, it has been quite useful for getting transgenes into organisms when no other options are available.

The Agrobacterium method involves the use of a soil-dwelling bacteria known as Agrobacterium tumefaciens, which has the ability to infect plant cells with a piece of its DNA. The piece of DNA that infects a plant is integrated into a plants chromosome through a tumor-inducing plasmid (Ti plasmid), which can take control of the plant’s cellular machinery and use it to make many copies of its own bacterial DNA. The Ti plasmid is a large circular DNA particle that replicates independently of the bacterial chromosome [1] (see Figure 3).

The importance of this plasmid is that it contains regions of transfer DNA (tDNA), where a researcher can insert a gene, which can be transferred to a plant cell through a process known as a floral dip. A floral dip involves dipping flowering plants into a solution of Agrobacterium carrying the gene of interest, followed by the transgenic seeds being collected directly from the plant [1]. This process is useful in that it is a natural method of transfer and therefore thought of as a more acceptable technique. In addition, Agrobacterium is capable of transferring large fragments of DNA very efficiently without substantial rearrangements, followed by maintaining high stability of the gene that was transferred [1]. One of the biggest limitations of Agrobacterium is that not all important food crops can be infected by this bacteria [1].

http://www.scq.ubc.ca/?p=518

Economic Advantages of GM Foods

Greater Profits, Lower Cost Input because of:
* Resistance to pests or weeds with a lower need for pesticides or herbicides
* Improved characteristics of the plant such as being more nutritious, less sensitive to transport or storage
* Less labour and application of machinery in agriculture
* Enlarged areas cultivated with plants adapted to extreme conditions, floods or droughts.
* The higher cost for seeds and licences are offset by higher yields.
* improved yields with lower need of expensive pesticides
* lower crop losses due to pests and weeds
* less labour and machinery
* plants which are adapted to extreme conditions, floods or droughts.
* Bioremediation is the use of bacteria to clean up contaminated land. Micro-organisms and plants can be modified to enhance their capacity to, for example, absorb heavy metals or radioactive elements and leave the soil safe. Reducing the need to clean up contaminated lands which can be very difficult and costly.

Safety Issues For GM Foods

Allergenicity - The allergenicity potential of the new protein expressed on the transgene inserted into the plant is a major food safety concern. Most traits introduced into GM crops result from the expression of one or more protein that may possess allergenic properties. Crops modified for insect resistance have been shown to have the potential for allergic responses. The allergenicity potential of GM food has often been difficult to establish with existing methods as the transgenes transferred are frequently from sources not eaten before, many have unknown allergenicity or there may be a potential for genetic modification process to result in increase of an allergen already present in the food. If the GM food contains genes, which are known to have a relation to allergenicity, a series of special chemical and immunological tests are required to identify the proteins, which cause the allergenic reaction. These in vitro tests have to be confirmed by in vivo tests. In any case, the new food should be labelled to inform the consumer that traces of an allergenic protein could be included.

Antibiotic Resistance - Potential for Gene transfer: Concern has been expressed on the possibility of transfer of GM DNA from the plant to gut microflora of humans and animals. Of importance have been the antibiotic resistant genes that are frequently used as selection markers, in the genetic modification process. Such genes have the potential to adversely affect the therapeutic efficacy of orally administered antibiotics.

Nutritional composition - Genetic modification of plants may result in alteration in nutritional composition which in turn may affect the nutritional status of the consumer or population groups. Currently developed plants with improved nutritive value include
GM rice with enriched vitamin A and GM soyabean and rapeseed with modified fatty acid. The impact of such intended modification in nutrient level in crop plants can affect nutritional status of the individual. There is also the potential for unexpected alteration in nutrients as it was observed in the case of GM rice (accumulation of xanthophylls, increase in prolamines). Such changes can affect nutrient profiles resulting in nutritional imbalances in the consumer.

Levels of antinutrients in GM Food
An antinutrient is a substance that interferes the uptake of nutrients. Antinutrients found in conventional foods include trypsin inhibitors and phytic acid. High levels of trypsin inhibitors are often found in raw cereals and legumes, especially soybean. Trypsin is an enzyme produced in the gut and digests proteins. Trypsin inhibitors interfere with the action of trypsin, affecting the digestion of proteins in the gut and subsequent absorption of nutrients, resulting in malnutrition. They are easily inactivated by heat and so may not be of concern as long as foods containing them are heat treated. Phytic acid is naturally present in plants such as soybeans and canola. It reduces the uptake of phosphorous, calcium, magnesium and zinc from food. However, phytic acid also appears to protect against some forms of cancer, so this beneficial effect needs to be weighed against the antinutritional effect.

Toxicity Potential - Various toxicants are known to be inherently present in different plants. Genetic engineering has the potential to alter such constituents or produce newer toxicants. Crops developed for pest resistance and herbicide resistance are particularly focussed for toxicity concern. The case of GM potatoes experiencing Galanthus nivalis
lectin gene for insecticidal properties is an example of the potential of GM foods to cause toxicity. In a group of rats fed with GM potato damage to immune systems and stunted growth was observed and the experiment had generated considerable controversy.

The Stability of Inserted Gene -

Other Unintended Effects -

Safety Issues for GM Foods

i) the inserted gene may itself have adverse effects;
ii) the inserted gene may code for a protein that is toxic to human beings or produces an allergic reaction
iii) the inserted gene may alter the way existing genes in a plant or animal express themselves, which may in turn increase the production of existing toxins or switch on the production of previously silent genes;
iv) the inserted gene may alter the behaviour of a micro-organism, which is carrying it to make it potentially harmful;
v) the inserted gene may be transferred from a micro-organism which is carrying it to other micro-organisms, in the human gut or respiratory tract or to animals or humans;
vi) the consumption of a GM micro-organism may alter the balance of existing
micro-organisms in the human gut.

Process of Gene Modification (GM Animals)

Process of GM:
1. identification of the gene interest;
2. isolation of the gene of interest;
3. transfer and modification of gene
3. amplifying the gene to produce many copies;
4. associating the gene with an appropriate promoter and poly A sequence and insertion into plasmids
5. multiplying the plasmid in bacteria and recovering the cloned construct for injection (aka recombinant-DNA formation, or gene splicing);
6. transference of the construct into the recipient tissue, usually fertilized eggs;
7. integration of gene into recipient genome;
8. expression of gene in recipient genome; and
9. inheritance of gene through further generations.

(adapted from http://www.fao.org/docrep/006/Y4955E/y4955e06.htm)