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Real Time PCR - Some Basic Principles

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GMO Detection

Now that it is more apparent why we need to detect the presence of a genetically engineered crop and even in some cases the number of copies of a transgene in a sample, let’s explore how we can make these measurements. If your background knowledge about the genetic engineering process in plants is uncertain, before going further you might want to refer to two other lessons: Just the Facts and Overview of Plant Engineering.
 

Discussion Question :
Let’s say you are a field scout and are observing two corn plants, one contains a Bt gene and the other does not. They look the same otherwise -same height, color, etc. How can you distinguish them apart? What could you measure? Use this illustration as a hint for your answer.




In all instances, GMO and non-GMO varieties differ in their genetic make-up, but might contain only subtle differences at the protein level. For example, consider a variety of Roundup Ready Corn in which the GA21 event provides protection from glyphosate herbicides. In this case there are only 2 amino acid sequence differences between the native, susceptible EPSPS protein expressed in all corn plants, compared with the resistant version of corn EPSPS protein expressed in GMO plants. A protein assay that would discern between these two isoforms of the EPSPS protein would be very difficult, if not impossible, leaving DNA testing as the only option. For all other GMO plants currently available on the market, the novel protein(s) expressed do have significantly different conformational structures and can be distinguished from the native protein(s). Another example where DNA detection is the best alternative is when transgenes turn off native genes and thus prevent protein synthesis (see Antisense RNA Technology Animation below). Two formats for protein-based testing are currently available: Lateral Flow Strip (LFS) tests and Enzyme-Linked Immunosorbent Assay (ELISA) test. These tests can be sensitive, rapid and easy to use. However, due to variable protein expression levels, protein-based testing has a limited capability for producing quantitative results.

 

Antisense RNA Technology Animation

The laboratory procedure known as PCR (see lesson Polymerase Chain Reaction ), is a DNA based method that detects a target DNA sequence unique to the transgenic variety. The term 'target DNA' refers to the particular portion of the DNA sequences which are going to be copied during PCR. DNA-based testing is designed to detect unique nucleotide sequences associated with a particular event and generally falls into two categories: Conventional Polymerase Chain Reaction (PCR) and Real Time Polymerase Chain Reaction (RT-PCR) technologies. Conventional PCR can be event specific, but has limited capability for producing quantitative results. Conversely, RT-PCR, also event specific, has the practical capability to produce quantitative results.

This lesson focuses on the RT-PCR process for detection of DNA sequences in GMO varieties. Measuring proteins or measuring target DNA sequences each have their own advantages and disadvantages. First, a few thoughts. When detecting proteins, it is important to know what promoter and coding region were used in the transgenic event. This will dictate which antibodies and what tissue you sample for obtaining a reliable result. Another situation is in processed products such as taco shells, where heating may destroy protein structure and thus eliminate detection. With DNA-based testing it is important to know some information on the event’s DNA sequence, but the gene will be present in all tissues at all life cycle stages of the plant. However, DNA can be degraded in food processing.

 

Discussion Question :
When might it be possible for a GMO to test negative for the presence of a transgene?

 

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