Light activated PCR assay

Researchers at Ludwig Maximilians University in Munich have developed a light-activated form of the enzymes that drive the polymerase chain reaction (PCR) assay. The assay has recently gained notoriety among the general public for its use as a COVID-19 diagnostic tool. The technique could lead to alternatives to current heat activated enzymes that are difficult to design and manufacture and are unsuitable for enzymes that are easily damaged by high temperatures. The method can help to expand the scope of the PCR possibilities.

PCR has been around for a while, but lately we have been relying heavily on it as a COVID-19 diagnostic test. The assay uses various enzymes to amplify tiny amounts of DNA so that we can detect the presence of the SARS-CoV-2 virus. However, the technology is also a cornerstone of medical research and provides invaluable data on gene expression.

The enzymes that power the PCR assay are a little picky. During sample preparation, the enzymes can become active and do their job, possibly affecting the results or destroying the sample. To get around this, researchers typically develop enzymes that only become active when they reach a certain temperature. These are called “hot start” enzymes.

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

To simplify the process of making enzymes for PCR assays, Vera and colleagues turned to light-activated enzymes as an alternative. “Light-triggered enzymes have been around for a long time, but what makes our approach unique is that it can be applied to virtually any DNA-processing enzyme,” said Vera. “In the past, you always needed very detailed information about how your enzyme was working and you were never sure whether you would find a smart way to block the enzyme and reactivate it with light.”

To achieve light activation, the researchers tied a piece of DNA to the enzyme, resulting in an inactive enzyme that can be activated with a pulse of UV light that releases the enzyme to act on its substrate. So far, the team has shown that their light-activated enzymes perform similarly or better to hot start equivalents.

“This will definitely help to produce better enzymes for biotechnological and diagnostic purposes,” says Philip Tinnefeld, another researcher involved in the study. “In addition, current real-time PCR machines already contain light sources and could easily be modified to bring these enzymes to market shortly.”

Study of nucleic acid research: A simple and general approach to making photoactivatable DNA processing enzymes

Over: Ludwig-Maximilians-University Munich

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