Effects of Lyse-It on endonuclease fragmentation, function and activity

Abstract

Nucleases are enzymes that can degrade genomic DNA and RNA that decrease the accuracy of quantitative measures of those nucleic acids. Here, we study conventional heating, standard microwave irradiation, and Lyse-It, a microwave-based lysing technology, for the potential to fragment and inactivate DNA and RNA endonucleases. Lyse-It employs the use of highly focused microwave irradiation to the sample ultimately fragmenting and inactivating RNase A, RNase B, and DNase I. Nuclease size and fragmentation were determined visually and quantitatively by SDS polyacrylamide gel electrophoresis and the mini-gel Agilent 2100 Bioanalyzer system, with a weighted size calculated to depict the wide range of nuclease fragmentation. Enzyme activity assays were conducted, and the rates were calculated to determine the effect of various lysing conditions on each of the nucleases. The results shown in this paper clearly demonstrate that Lyse-It is a rapid and highly efficient way to degrade and inactivate nucleases so that nucleic acids can be retained for down-stream detection.

Applications of Microwaves in Medicine Leveraging Artificial Intelligence: Future Perspectives

“…The level of fragmentation is dependent on MW power and irradiation time. An increase in either MW power or exposure time results in an increase in fragmentation [31].…”

Applications of Microwave Energy in Medicine

“…This method of lysing DNA is much quicker than the current diagnostic DNA extraction methods and could be incorporated into point of care diagnostics. However, crucially there are currently no benefits in sensitivity as the samples are contaminated by the microwaved gold, and currently, due to the cost of the Lyse It Slides this method is not yet suitable to be used as a common DNA extraction method within diagnostic laboratories [ 64 , 65 ].…”

Sample Preparation and Diagnostic Methods for a Variety of Settings: A Comprehensive Review

“…Geddes et al have taken a similar approach by developing a lysis setup of planar metal structures on a glass substrate called microwave lysing triangles (MLTs) or Lyse-It ® devices that utilize a common radio frequency (RF)/microwave source (300 MHz–300 GHz), microwave ovens, to prepare clinical samples for downstream detection in 30–60 s, as depicted in Figure 9 [ 125 , 129 , 130 , 131 , 132 ]. The utility of this approach comes from several of the factors needed for POC sample preparation methods such as simple operation, minimal equipment requirements, and lack of cold chain requirements for samples treated with MLTs, as well as the ability to fragment common target biomarkers such as nucleic acids, enzymes, and proteins in a tunable manner for downstream analysis [ 133 , 134 , 135 , 136 ]. While similar extraction techniques, termed microwave-assisted extraction (MAE), have found use with environmental, geological, and biological samples [ 137 , 138 , 139 ], some researchers such as Ahirwar et al have reported using microwave irradiation to reduce the time needed for diagnostic ELISAs from 2 h to less than 5 min via non-thermal effects [ 140 ].…”

“…The rapid heating, focused electric field intensity, and reactive oxygen species (ROS) responsible for lysis arise from the incident microwave photons. From Geddes et al [ 129 , 130 , 131 , 132 , 133 , 134 , 135 , 136 ].…”