A Simple Guide to GMO Testing Chris Thomas, 2004 Milton Contact Limited at http://www.miltoncontact.com

Introducing DNA and where it is found Plants (and other organisms) are made of cells Each cell has a nucleus containing DNA Sometimes the DNA is visible as chromosomes The DNA contains genes, the basic instructions for a cell and ultimately an organism Bean seedling A cell The nucleus of the cell Visible chromosomes in a cell

What is the difference between Conventional breeding and GMO based breeding? Parent A is shown with blue chromosomes in a cell. Parent B is shown with red chromosomes and has a desirable trait. A is crossed with B. The progeny contain a mix of chromosomes/genes. Further crosses of the progeny with Parent A are needed so that only the small amount of B’s DNA with the desired trait remains in the new variety. GMO based breeding: A piece of DNA with the specific trait is introduced directly into the parent, creating the desired variety Conventional breeding GMO based breeding Parent DNA with specific trait Desired variety Parent A Parent B progeny New variety

GM DNA inserted into the DNA of a bacterium typically equals ~ 1/200 of the total Material to be inserted Bacterial DNA with an insert

The GM material inserted into a potato’s DNA, for example, is typically ~ 1/100,000 of the total

What is inserted to create a GMO? Using plants as an example GM DNA inserted into host DNA Host DNA Border fragment Selectable marker Gene with Desired trait Border fragment Host DNA Host DNA – the DNA of the plant into which the GM material has been introduced Border fragment – an element needed to aid the insertion the GM DNA Selectable marker – an element used in the process of making a GM plant Gene with the desired trait – the actual property or trait, such as a disease resistance

Testing for GMOs GMO testing can be done in two ways: based on the novel protein produced from the inserted gene; based on the inserted GM DNA itself Protein based testing Advantages Simple tests (similar to pregnancy tests) Rapid (minutes to hours) Can be used in the field Specific for a particular protein Disadvantages Only works if tissue sampled contains protein for example, an extract from shoots would not give a result if the protein is only produced in the roots Detection requires high levels of protein Will not detect protein in heated or processed samples DNA based testing Advantages Very sensitive Can be designed to be specific for one GMO Can be designed to detect a range of GMOs Can detect DNA if present in processed samples Can be used to look for and locate the inserted elements in a GMO’s DNA Disadvantages Requires specialist laboratories Can take several days Prone to false positives

A photograph of a simple positive GMO test Simple DNA testing for presence or absence of a specific GMO DNA insert (PCR) A photograph of a simple positive GMO test with a false +ve The DNA is purified from a specific GMO or sample containing that GMO A part of the purified GM DNA in the GMO is amplified up to a hundred million times The GM DNA is then made visible and recorded The method is qualitative not quantitative, i.e. Yes or No Even miniscule contamination of solutions and non-GM samples can result in false positives

Quantitative DNA testing for presence of specific GMO DNA (Real-time PCR) Samples = 1, 2, 3 a = high concentration standard b = medium conc. standard c = low conc. standard 1 2 3 concentration a b c 4 Threshold value time DNA is isolated and amplified as in the previous slide. The increase of amplified GM DNA is monitored over time, e.g. for samples 1, 2 & 3. The time at which the amplified DNA level exceeds a threshold level is determined. The level of GM DNA in sample 1, 2, and 3 is determined by comparison with standards of known GM DNA concentration, a, b, c. If c is at a level of 0.9% GM DNA, then sample 3 contains less than 0.9% GM DNA, whilst samples 1 and 2 contain more Sample 4 shows a result seen where the DNA might not have been purified sufficiently and the efficiency of the amplification is reduced leading to a false result

Sample size and the Sample’s size Looking for the presence of low levels of GM material in non-GM grain is also affected by the size of the grain (examples for illustration only – no GM material used or implied). The larger the grain, the greater the weight of material required for testing. Poppy Mustard Rice Maize/corn

The importance of adequate sampling To ensure that a test gives meaningful results, great care has to be taken. Some factors to consider are: Is the material you are collecting samples from Mixed sufficiently to ensure even distribution? Likely to be contaminated with remnants of a previous storage or processing run – e.g. in grain stores or mills? Do you need a quick on-site assay looking for presence/absence of GMOs at high level concentrations? Do you need a precision assay looking for GMOs present above a required low level, that may take several days? What is the lowest level of GM presence that you need to detect (0.9% or 0.1%)? How much should be collected for each sample to be representative? How many samples should be collected each time from a batch of material to give a high probability that any results are statistically significant? Do you and the testing laboratory have sufficient controls in place to detect false positive or false negative results?

In Conclusion With the introduction of new regulations governing the labelling of products containing GMOs, the need for testing is likely to increase in the future. Chris Thomas of Milton Contact Limited http://www.miltoncontact.com