4 years ago

The Partnership no. 9

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PCR, the basis of

PCR, the basis of today’s research Science Developments in biotechnology are going fast, very fast. New techniques for speeding up the breeding process and making it more efficient are introduced one after another. But at the same time some techniques that were developed dozens of years ago are still being used today. In fact, their use is actually intensifying. One of those techniques is PCR. PCR, short for Polymerase Chain Reaction, was introduced around the end of the last century and is being used increasingly often today. “’Polymerase’ is the enzyme that produces DNA,” explains Gert-Jan de Boer, Manager of Molecular Biology & Biochemistry. “We need it to determine the genetic characteristics of a plant, for example its resistance to a disease or the colour of a sweet pepper or to determine whether the seeds produced are indeed from the intended variety.” This is done by studying the specific DNA fragments that contain the genetic information for those characteristics – which are known as ‘markers’. This is essential for breeders, because it enables them to make selections at young plant stage. Amplification But what’s the role of PCR in this? You need large amounts of DNA to be able to detect differences in an organism’s DNA. Hardly any of the methods and equipment that are currently available for reading DNA or spotting differences is sensitive enough to work with only DNA from a cell. PCR enables us to amplify sufficient copies of the DNA fragments in which we are interested. Copying What we need for PCR is relatively simple: the individual DNA building blocks, an enzyme that copies DNA (the DNA polymerase) and two synthetic DNA fragments marking the beginning and end of the segment that we want to copy. They serve as the starting point for the copying process and are known as ‘primers’. DNA consists of two complementary strands, which can be separated by raising the temperature to just above the temperature at which these strands separate. By then lowering the temperature to the point at which the primers specifically bind, and once again raising it to the point at which the polymerase enzyme becomes effective you can double a DNA molecule. So you then have two copies, but bear in mind that if you double one original, say, thirty times, you’ll end up with 1,073,741,824 copies. Problem It all sounds so simple today, but when this technology was developed, there was one problem that needed to be solved: boiling causes the polymerase to become inactive. That’s not surprising, because the same happens while boiling an egg for breakfast. What was needed was a thermostable polymerase, which was found in a bacterium – Thermus aquaticus – that lives in hot springs. The so-called ‘Taq’ polymerase was isolated from the bacterium and this enzyme is most effective at a temperature of 72 degrees Centigrade. So the ultimate process comprises three steps: separating the DNA strands by raising the temperature to above 90 degrees Centigrade, cooling the reaction to a temperature between 50 and 60 degrees to effect the binding of the primers serving as the starting point for the copying by the polymerase, and finally raising the temperature to 72 degrees to enable the polymerase to do its job. Then the whole cycle is repeated, starting again with the boiling. Speeding up the breeding process De Boer: “Almost all of our breeders use DNA tests to monitor hereditary traits for the purpose to enable us to develop new varieties more quickly. Those DNA tests are carried out using the PCR method. The number of DNA tests we carry out is rapidly growing. A few years ago it was still a few million tests, but that figure has since then increased at least tenfold and will increase even more in the years to come.” 14 | The Partnership The Partnership | 15

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