UCLA

Nucleic acid amplification has applications in diagnostics, sequencing, genetic fingerprinting, among others. Currently nucleic acid amplification is treated as the “gold standard” method for several diagnostics; however, because of the multi-step protocols and the large equipment, these assays are lengthy and require laboratory settings. Digital nucleic acid amplifications assays developed have greatly improved several aspects of nucleic acid amplification by creating a more robust and sensitive assay. This is due to the reduction in background noise and the ability to effectively concentrate target analytes in nano- or picoliter volumes by compartmentalization of these samples. We were able to demonstrate a 69-fold fluorescence change in an isothermal nucleic acid amplification assay, with a >60% increase in fluorescence stability with elevated temperatures over the time course of the reaction, with the use of a unique dye combination of EvaGreen and hydroxynapthol blue (HNB). Due to the improvements in signal, we were able to demonstrate comparable results using a mobile phone based fluorescence plate reader as with that of a benchtop reader. The unique dye combination was then applied to a digital system, demonstrating signal improvements that are crucial to developing a robust assay, giving a higher efficiency (percentage of “on” wells closer to the theoretical value) and a larger difference in fluorescence intensities for “on” versus “off” wells. Lastly, we examined the mechanism of the dye combination to best determine additional ways of improving the signal generation. By sequestering EvaGreen, HNB allows amplification to proceed without interference. Additionally, a F�rster resonance energy transfer (FRET) interaction between the dye molecules, when DNA is absent from the solution, acts to lower the background fluorescence such that a greater fluorescence fold change occurs with DNA amplification. The EvaGreen and HNB have a highly-tuned binding affinity such that prior to DNA amplification, they have FRET interactions, and afterwards in the presence of large amounts of DNA, EvaGreen binding to DNA becomes more favorable. All of these developed technologies and methods work in conjunction to improve upon currently developed techniques for nucleic acid amplification in point of care settings.