title: Upconversion nanoparticle-based ligase-assisted method for specific and sensitive detection of T790M mutation in epidermal growth factor receptor.
abstract: 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 0.01pmole of EGFR T790M mutant can be readily detected.
authors.affiliation: Department of Chemistry, University of Cincinnati, Cincinnati, OH 45221, USA.
authors.forename: Peng
authors.initials: P
authors.lastname: Wang
authors.affiliation: Department of Chemistry, University of Cincinnati, Cincinnati, OH 45221, USA.
authors.forename: Padmanabh
authors.initials: P
authors.lastname: Joshi
authors.affiliation: Department of Chemistry, University of Cincinnati, Cincinnati, OH 45221, USA.
authors.forename: Abdulrahman
authors.initials: A
authors.lastname: Alazemi
authors.affiliation: Department of Chemistry, University of Cincinnati, Cincinnati, OH 45221, USA. Electronic address: peng.zhang@uc.edu.
authors.forename: Peng
authors.initials: P
authors.lastname: Zhang
mesh_terms: D020137:Base Pair Mismatch; D001483:Base Sequence; D052160:Benzothiazoles; D004247:DNA / Q000032:analysis / Q000235:genetics; D015342:DNA Probes / Q000737:chemistry; D003959:Diamines; D066246:ErbB Receptors / Q000235:genetics*; D005459:Fluorides / Q000737:chemistry; D049408:Luminescent Agents / Q000737:chemistry*; D008163:Luminescent Measurements; D008969:Molecular Sequence Data; D053758:Nanoparticles / Q000737:chemistry*; D009930:Organic Chemicals / Q000737:chemistry*; D017354:Point Mutation; D011804:Quinolines; D015019:Yttrium / Q000737:chemistry
chemical_list: D052160:Benzothiazoles; D015342:DNA Probes; D003959:Diamines; D049408:Luminescent Agents; D009930:Organic Chemicals; D011804:Quinolines; C525362:sodium yttriumtetrafluoride; C098022:SYBR Green I; D015019:Yttrium; D004247:DNA; D066246:ErbB Receptors; D005459:Fluorides
keywords: Epidermal growth factor receptor (EGFR); Ligation; T790M mutation; Upconversion nanoparticle