Detection of genetically modified organisms
Encyclopedia
The detection of genetically modified organisms in food or feed is possible by biochemical means. It can either be qualitative, showing which genetically modified organism
(GMO) is present, or quantitative, measuring in which amount a certain GMO is present. Being able to detect a GMO is an important part of food safety, as without detection methods the traceability of GMOs
would rely solely on documentation.
(PCR) is a biochemistry and molecular biology technique for isolating and exponentially amplifying a fragment of DNA
, via enzymatic replication, without using a living organism. It enables the detection of specific strands of DNA by making millions of copies of a target genetic sequence. The target sequence is essentially photocopied at an exponential rate, and simple visualisation techniques can make the millions of copies easy to see.
The method works by pairing the targeted genetic sequence with custom designed complementary bits of DNA called primers
. In the presence of the target sequence, the primers match with it and trigger a chain reaction. DNA replication enzymes use the primers as docking points and start doubling the target sequences. The process is repeated over and over again by sequential heating and cooling until doubling and redoubling has multiplied the target sequence several million-fold. The millions of identical fragments are then purified in a slab of gel, dyed, and can be seen with UV light.It is not prone to contamination.
DNA in a food or feed sample. Q-PCR is commonly used to determine whether a DNA sequence is present in a sample and the number of its copies in the sample. The method with currently the highest level of accuracy is quantitative real-time PCR. QRT-PCR methods use fluorescent dyes, such as Sybr Green, or fluorophore-containing DNA probes, such as TaqMan
, to measure the amount of amplified product in real time. If the targeted genetic sequence is unique to a certain GMO, a positive PCR test proves that the GMO is present in the sample.
length, should be different enough to form distinct bands when visualized by gel electrophoresis
.
based. DNA based detection looks for a part of the foreign DNA inserted in a GMO. For technical reasons, certain DNA sequences are shared by several GMOs. Protein-based methods detect the product of the transgene, for example the Bt toxin. Since different GMOs may produce the same protein, construct-specific detection can test a sample for several GMOs in one step, but is unable to tell precisely, which of the similar GMOs are present. Especially in the USA, protein-based detection is used for the construct-specific approach.
are developing improved and alternative testing methods, for example DNA microarray
s.
. This refers to transgenic cultivars derived from crosses between transgenic parent lines, combining the transgenic traits of both parents. One GM maize variety now awaiting a decision by the European Commission, MON863
x MON810 x NK603, has three stacked traits. It is resistant to an herbicide and to two different kinds of insect pests. Some combined testing methods could give results that would triple the actual GM content of a sample containing this GMO.
. Researchers now compile a set of genetic sequences characteristic of GMOs. After genetic elements characteristic of GMOs are selected, methods and tools are developed for detecting them in test samples. Approaches being considered include microarrays and anchor PCR profiling.
(NIR) detection is a method that can reveal what kinds of chemicals are present in a sample based on their physical properties. By hitting a sample with near infrared light, chemical bond
s in the sample vibrate and re-release the light energy at a wavelength
characteristic for a specific molecule or chemical bond. It is not yet known if the differences between GMOs and conventional plants are large enough to detect with NIR imaging. Although the technique would require advanced machinery and data processing tools, a non-chemical approach could have some advantages such as lower costs and enhanced speed and mobility.
Genetically modified organism
A genetically modified organism or genetically engineered organism is an organism whose genetic material has been altered using genetic engineering techniques. These techniques, generally known as recombinant DNA technology, use DNA molecules from different sources, which are combined into one...
(GMO) is present, or quantitative, measuring in which amount a certain GMO is present. Being able to detect a GMO is an important part of food safety, as without detection methods the traceability of GMOs
Traceability of genetically modified organisms
The traceability of genetically modified organisms describes a system that ensures the forwarding of the identity of a GMO from its production to its final buyer. Traceability is an essential prerequisite for the co-existence of GM and non-GM foods, and for the freedom of choice for consumers.-Why...
would rely solely on documentation.
Polymerase chain reaction (PCR)
The polymerase chain reactionPolymerase chain reaction
The polymerase chain reaction is a scientific technique in molecular biology to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence....
(PCR) is a biochemistry and molecular biology technique for isolating and exponentially amplifying a fragment of DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
, via enzymatic replication, without using a living organism. It enables the detection of specific strands of DNA by making millions of copies of a target genetic sequence. The target sequence is essentially photocopied at an exponential rate, and simple visualisation techniques can make the millions of copies easy to see.
The method works by pairing the targeted genetic sequence with custom designed complementary bits of DNA called primers
Primer (molecular biology)
A primer is a strand of nucleic acid that serves as a starting point for DNA synthesis. They are required for DNA replication because the enzymes that catalyze this process, DNA polymerases, can only add new nucleotides to an existing strand of DNA...
. In the presence of the target sequence, the primers match with it and trigger a chain reaction. DNA replication enzymes use the primers as docking points and start doubling the target sequences. The process is repeated over and over again by sequential heating and cooling until doubling and redoubling has multiplied the target sequence several million-fold. The millions of identical fragments are then purified in a slab of gel, dyed, and can be seen with UV light.It is not prone to contamination.
Quantitative detection
Quantitative PCR (Q-PCR) is used to measure the quantity of a PCR product (preferably real-time, QRT-PCR). It is the method of choice to quantitatively measure amounts of transgeneTransgene
A transgene is a gene or genetic material that has been transferred naturally or by any of a number of genetic engineering techniques from one organism to another....
DNA in a food or feed sample. Q-PCR is commonly used to determine whether a DNA sequence is present in a sample and the number of its copies in the sample. The method with currently the highest level of accuracy is quantitative real-time PCR. QRT-PCR methods use fluorescent dyes, such as Sybr Green, or fluorophore-containing DNA probes, such as TaqMan
TaqMan
TaqMan probes are hydrolysis probes that are designed to increase the specificity of real-time PCR assays. The method was first reported in 1991 by researchers at Cetus Corporation, and the technology was subsequently developed by Roche Molecular Diagnostics for diagnostic assays and by Applied...
, to measure the amount of amplified product in real time. If the targeted genetic sequence is unique to a certain GMO, a positive PCR test proves that the GMO is present in the sample.
Qualitative detection
Whether or not a GMO is present in a sample can be tested by Q-PCR, but also by multiplex PCR. Multiplex PCR uses multiple, unique primer sets within a single PCR reaction to produce amplicons of varying sizes specific to different DNA sequences, i.e. different transgenes. By targeting multiple genes at once, additional information may be gained from a single test run that otherwise would require several times the reagents and more time to perform. Annealing temperatures for each of the primer sets must be optimized to work correctly within a single reaction, and amplicon sizes, i.e., their base pairBase pair
In molecular biology and genetics, the linking between two nitrogenous bases on opposite complementary DNA or certain types of RNA strands that are connected via hydrogen bonds is called a base pair...
length, should be different enough to form distinct bands when visualized by gel electrophoresis
Gel electrophoresis
Gel electrophoresis is a method used in clinical chemistry to separate proteins by charge and or size and in biochemistry and molecular biology to separate a mixed population of DNA and RNA fragments by length, to estimate the size of DNA and RNA fragments or to separate proteins by charge...
.
Event-specific vs. construct-specific detection
When producers, importers or authorities test a sample for the unintended presence of GMOs, they usually do not know, which GMO to expect. While EU authorities prefer an event-specific approach to this problem, US authorities rely on construct-specific test schemes.Event-specific detection
An event-specific detection searches for the presence of a DNA sequence unique to a certain GMO, usually the junction between the transgene and the organism's original DNA. This approach is ideal to precisely identify a GMO, yet highly similar GMOs will pass completely unnoticed. Event-specific detection is PCR-based.Construct-specific detection
The construct-specific detection methods can either be DNA or proteinProtein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
based. DNA based detection looks for a part of the foreign DNA inserted in a GMO. For technical reasons, certain DNA sequences are shared by several GMOs. Protein-based methods detect the product of the transgene, for example the Bt toxin. Since different GMOs may produce the same protein, construct-specific detection can test a sample for several GMOs in one step, but is unable to tell precisely, which of the similar GMOs are present. Especially in the USA, protein-based detection is used for the construct-specific approach.
Shortcomings of current detection methods
Currently, it is highly unlikely that the presence of unexpected or even unknown GMOs will be detected, since either the DNA sequence of the transgene or its product, the protein, must be known for detection. In addition, even testing for known GMOs is time-consuming and costly, as current reliable detection methods can test for only one GMO at a time. Therefore, research programmes such as Co-ExtraCo-Extra
Co-Extra is an EU-funded research programme on co-existence and traceability of genetically modified crops and their edible derivatives. It has been granted €13.5 million under the Sixth Framework Programme of the European Union, and is conducted by more than 200 scientists in 52 organisations in...
are developing improved and alternative testing methods, for example DNA microarray
DNA microarray
A DNA microarray is a collection of microscopic DNA spots attached to a solid surface. Scientists use DNA microarrays to measure the expression levels of large numbers of genes simultaneously or to genotype multiple regions of a genome...
s.
Improving PCR based detection
Improving PCR based detection of GMOs is a further goal of the European research programme Co-Extra. Research is now underway to develop multiplex PCR methods that can simultaneously detect many different transgenic lines. Another major challenge is the increasing prevalence of transgenic crops with stacked traitsGene stacked event
A genetically modified organism and all subsequent identical clones resulting from a transformation process are called collectively a transformation event. If more than one gene from another organism has been transferred, the created GMO has stacked genes , and is called a gene stacked event.Gene...
. This refers to transgenic cultivars derived from crosses between transgenic parent lines, combining the transgenic traits of both parents. One GM maize variety now awaiting a decision by the European Commission, MON863
MON863
MON 863 is a genetically engineered variety of maize produced by Monsanto.-History:Following legal action by parties including the Swedish Board of Agriculture and Greenpeace, a Münster appeals court ruled that Monsanto would be forced to publicly reveal its research data.-Modified mechanisms:MON...
x MON810 x NK603, has three stacked traits. It is resistant to an herbicide and to two different kinds of insect pests. Some combined testing methods could give results that would triple the actual GM content of a sample containing this GMO.
Detecting unknown GMOs
Almost all transgenic plants contain a few common building blocks that make unknown GMOs easier to find. Even though detecting a novel gene in a GMO can be like finding a needle in a haystack, the fact that the needles are usually similar makes it much easier. To trigger gene expression, scientists couple the gene they want to add with what is known as a transcription promoter. The high-performing 35S promoter is a common feature to many GMOs. In addition, the stop signal for gene transcription in most GMOs is often the same: the NOS terminatorTerminator (genetics)
In genetics, a terminator, or transcription terminator is a section of genetic sequence that marks the end of gene or operon on genomic DNA for transcription.In prokaryotes, two classes of transcription terminators are known:...
. Researchers now compile a set of genetic sequences characteristic of GMOs. After genetic elements characteristic of GMOs are selected, methods and tools are developed for detecting them in test samples. Approaches being considered include microarrays and anchor PCR profiling.
Near infrared fluorescence (NIR)
Near infrared fluorescenceFluorescence
Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation of a different wavelength. It is a form of luminescence. In most cases, emitted light has a longer wavelength, and therefore lower energy, than the absorbed radiation...
(NIR) detection is a method that can reveal what kinds of chemicals are present in a sample based on their physical properties. By hitting a sample with near infrared light, chemical bond
Chemical bond
A chemical bond is an attraction between atoms that allows the formation of chemical substances that contain two or more atoms. The bond is caused by the electromagnetic force attraction between opposite charges, either between electrons and nuclei, or as the result of a dipole attraction...
s in the sample vibrate and re-release the light energy at a wavelength
Wavelength
In physics, the wavelength of a sinusoidal wave is the spatial period of the wave—the distance over which the wave's shape repeats.It is usually determined by considering the distance between consecutive corresponding points of the same phase, such as crests, troughs, or zero crossings, and is a...
characteristic for a specific molecule or chemical bond. It is not yet known if the differences between GMOs and conventional plants are large enough to detect with NIR imaging. Although the technique would require advanced machinery and data processing tools, a non-chemical approach could have some advantages such as lower costs and enhanced speed and mobility.
External links
- Co-Extra: Research on co-existence and traceability investigates new and improved detection methods
- European Network of GMO Laboratories develops and standardises detection methods
- Institute for Reference Materials and Measurements provides reference material for GMO detection
- GMO Detection Methods Database the Institute for Health and Consumer Protection (IHCP) provides validated GMO Detection Methods