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Real Time PCR - Some Basic Principles

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Figure 4.Three leaf color phenotypes in soybean seedlings: yellow, light green and dark green.

With GMO detection, scientists and consumers are interested in only one of the thousands of genes a plant contains. Some genes control phenotypes that are easy to measure such as leaf color (Figure 4). However, sometimes a gene may encode for a particular protein that controls a more difficult to measure trait, such as resistance to an insect (Bt corn and cotton). The challenge is that a single gene copy is not easily detectable using some molecular biology technologies. However, with the PCR process, copies of the target DNA sequence are replicated, enabling the investigator to identify the presence of a particular gene.

The concept is similar to being in an airplane at night, looking down on a friend, holding a flashlight. You will not be able to see the light emitted by the single flashlight because it is too dim. This is analogous to one copy of the Bt gene amongst the thousands of genes found in a corn seed. However, if your friend recruited an entire city of people to also hold flashlights, then you could see the emitted light. PCR is a laboratory process which generates copies of a particular segment of DNA already present, so that it can be detected. The PCR process can enable us to determine whether our sample (i.e. plant, seed or food product) contains a transgene or not.

Unfortunately, merely detecting the presence or absence of a particular gene in grains or food products does not always satisfy researchers, government regulators, processors or consumers. As an example, they may need to know what percent of transgenic seed is present in the entire sample. Is there any contamination of an unapproved event in a shipment? Back to the flashlight analogy, you would want to determine how many of your friends initially held flashlights before they had recruited more people to hold flashlights. This is how quantitative or real time PCR works. Based upon results obtained during the PCR process, the initial amount of a particular gene present in an unknown sample can be measured. In plant breeding applications, researchers will be able to tell if a plant is homozygous for the transgene or hemizygous. Alternatively, some of the transformation methodologies often result in multiple copies of a transgene inserting into plant chromosomes. Real time PCR would be able to measure these copy numbers.

Discussion Question :
Why would someone be interested in how many copies of a gene are present?


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