Digital quantification of rolling circle amplified single DNA molecules in a resistive pulse sensing nanopore.
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Kühnemund M, Nilsson M
Biosens Bioelectron 67 (-) 11-17 [2015-05-15; online 2014-06-24]
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.
PubMed 25000851
DOI 10.1016/j.bios.2014.06.040
Crossref 10.1016/j.bios.2014.06.040
pii: S0956-5663(14)00453-9