PCR assays such as the RT-qPCR test for COVID-19 rely on the use of DNA polymerases and other DNA processing enzymes to complete the amplification reactions, but these enzymes must be disabled prior to the start of the assay in order to prevent unwanted activity during sample preparation. Many PCR technologies utilize “hot-start” enzymes that are only activated at high temperatures, but these enzymes can be time-consuming to design and this approach does not work for enzymes that are denatured by heat. Researchers at Ludwig-Maximilians-Universität München have now designed new light-activated DNA modifying enzymes for use in PCR assays that overcome some of these limitations and could lead to more efficient diagnostic and biological tests.
The new light-start enzymes are initially bound to an oligonucleotide and photocleavable linker in order to deactivate them prior to the PCR experiment. A pulse of UV light is then used to cleave the oligonucleotide from the enzyme and start the reaction. The main advantage of this approach is that it can be used with a broader range of DNA processing enzymes than heat-based methods, the researchers said. To demonstrate this, the team produced four light-activated versions of different enzymes including Phi29 DNA polymerase, which can be used to amplify whole genomes but cannot be used with hot-start methods due to its heat sensitivity.
The experiments showed the light-start method was effective when used with different DNA modifying enzymes, including the heat-sensitive Phi29 DNA polymerase, and performed just as well or better than commercially available hot-start enzymes for PCR. The light-start enzymes are also easier to design and produce than hot-start counterparts as the process does not require extensive knowledge of the structure or mechanics of the base enzyme being used. This work was recently published in the journal Nucleic Acids Research.
“This is definitely going to help to produce better enzymes for biotechnological and diagnostic uses,” said study co-author Philip Tinnefeld. “Besides, current real-time PCR machines already incorporate light sources and they could be easily modified to bring these enzymes to the market anytime soon.”