Light-Activated PCR Assay | Medgadget

Researchers at the Ludwig-Maximilians-Universität München in Germany have developed a light-activated form of the enzymes that drive the polymerase chain reaction (PCR) assay. The assay has recently gained popularity among the general public due to its use as a diagnostic tool for COVID-19. The technique could lead to alternatives to current heat-activated enzymes, which are difficult to design and create, and are not suitable for enzymes that are easily damaged by high temperatures. The method can help broaden the scope of what is possible with PCR.

PCR has been around for quite some time, but recently we have come to rely heavily on it as a diagnostic test for COVID-19. The assay uses various enzymes to amplify small amounts of DNA so that we can detect the presence of the SARS-CoV-2 virus. However, technology is also a cornerstone of medical research, providing invaluable data on gene expression.

The enzymes that power the PCR assay are a bit finicky. During sample preparation, enzymes can become activated and go to work, potentially affecting results or ruining the sample. To avoid this, researchers often design enzymes that will only activate once they reach a certain temperature. These are known as “hot start” enzymes.

However, hot start enzymes are difficult to design and the concept does not work for enzymes that are particularly sensitive to heat. “For PCR-based diagnostic tests, such as the[…] In the COVID-19 test, the solution is the development of a hot-start enzyme, which does not show activity until a high activation temperature is reached, ”said Andrés Vera, a researcher involved in the study. “The main drawback of these hot start approaches is that they cannot be used for heat-damaged enzymes. Also, designing a hot start enzyme is tedious and the grueling design process has to be repeated for every new enzyme we want to design. “

To facilitate the creation of enzymes for PCR assays, Vera and her colleagues turned to light-activated enzymes as an alternative. “Light-controlled enzymes have been around for quite some time, but what makes our approach unique is that it can be applied to virtually any DNA processing enzyme,” Vera said. “In the past, you always needed very detailed information about how your enzyme works, and you were never sure you could find a clever way to block the enzyme and reactivate it with light.”

To achieve light activation, the researchers attached a piece of DNA to the enzyme, resulting in an inactive enzyme that can be activated with a pulse of ultraviolet light that frees the enzyme to begin acting on its substrate. So far, the team has shown that their light-activated enzymes show similar or improved performance to hot start equivalents.

“This will definitely help produce better enzymes for biotechnology and diagnostic use,” said Philip Tinnefeld, another researcher involved in the study. “Additionally, current real-time PCR machines already incorporate light sources and could easily be modified to bring these enzymes to market in the short term.”

To study in Nucleic Acid Research: A simple and general approach to generate photoactivatable DNA processing enzymes

Via: Ludwig-Maximilians-Universität München

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