Upconversion nanoparticle-based ligase-assisted method for specific and sensitive detection of T790M mutation in epidermal growth factor receptor

In this paper we report a highly specific and sensitive method for the detection of T790M mutation in epidermal growth factor receptor (EGFR). This detection scheme is based on luminescent resonance energy transfer between upconversion nanoparticles and the intercalating dye, SYBR Green I. Target DNA serves as a template for two DNA probes, one of them covalently attached to upconversion nanoparticles, to be joined into a long, hairpin-forming DNA by ligase. The number of the resulting DNA strand, which brings SYBR Green I close to the upconversion nanoparticles, is amplified by thermal cycling. A number of factors affecting the detection specificity and sensitivity, including probe design, ligation temperature, type and amount of ligase, and number of thermal cycles, have been considered and investigated to optimize the performance of the method. The method can easily differentiate the T790M mutation from the wild-type sequence with a mutant-to-wild-type ratio of 1:100. The results show that 0.01 pmole of EGFR T790M mutant can be readily detected.