title: Digital quantification of rolling circle amplified single DNA molecules in a resistive pulse sensing nanopore.
abstract: Novel portable, sensitive and selective DNA sensor methods for bio-sensing applications are required that can rival conventionally used non-portable and expensive fluorescence-based sensors. In this paper, rolling circle amplification (RCA) products are detected in solution and on magnetic particles using a resistive pulse sensing (RPS) nanopore. Low amounts of DNA molecules are detected by padlock probes which are circularized in a strictly target dependent ligation reaction. The DNA-padlock probe-complex is captured on magnetic particles by sequence specific capture oligonucleotides and amplified by a short RCA. Subsequent RPS analysis is used to identify individual particles with single attached RCA products from blank particles. This proof of concept opens up for a novel non-fluorescent digital DNA quantification method that can have many applications in bio-sensing and diagnostic approaches.
authors.affiliation: Science for Life Laboratory, Department of Immunology, Genetics and Pathology, Uppsala University, Box 815, 751 08 Uppsala, Sweden.
authors.forename: M
authors.initials: M
authors.lastname: Kühnemund
authors.affiliation: Science for Life Laboratory, Department of Biochemistry and Biophysics, Stockholm University, Box 1031, SE-171 21 Solna, Sweden. Electronic address: mats.nilsson@scilifelab.se.
authors.forename: M
authors.initials: M
authors.lastname: Nilsson
mesh_terms: D015374:Biosensing Techniques; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D058185:Magnetite Nanoparticles / Q000737:chemistry; D058608:Nanopores; D021141:Nucleic Acid Amplification Techniques
chemical_list: D058185:Magnetite Nanoparticles; D004247:DNA
keywords: Nanopore; Padlock probe; RCA; Resistive pulse sensing; Single molecule detection