Fluorometric Detection of MicroRNA Using Isothermal Gene Amplification and Graphene Oxide

We have developed a facile fluorometric system for the detection of microRNA (miRNA), using rolling circle amplification (RCA), graphene oxide (GO), and fluorescently labeled peptide nucleic acid (F-PNA). The padlock probe DNA complementary to a target miRNA was selectively ligated to form circular...

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Bibliographic Details
Published in:Analytical chemistry (Washington) 2016-03, Vol.88 (6), p.2999-3003
Main Authors: Hong, Chaesun, Baek, Ahruem, Hah, Sang Soo, Jung, Woong, Kim, Dong-Eun
Format: Article
Language:English
Subjects:
Amplification
Deoxyribonucleic acid
DNA
Fluorescence
Fluorometers
Fluorometry - methods
Gene Amplification
Genes
Graphene
Graphite - chemistry
MicroRNAs - chemistry
Oxides
Oxides - chemistry
Peptides
Quenching
Ribonucleic acid
Ribonucleic acids
RNA
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Summary:We have developed a facile fluorometric system for the detection of microRNA (miRNA), using rolling circle amplification (RCA), graphene oxide (GO), and fluorescently labeled peptide nucleic acid (F-PNA). The padlock probe DNA complementary to a target miRNA was selectively ligated to form circular DNA that was then used as the template for RCA. F-PNAs complementary to the target miRNA were annealed to multiple sites of the isothermally amplified single-stranded RCA product (RCAP) containing multiple target miRNA sequences. This F-PNA/RCAP duplex is less adsorbed onto the GO monolayer, thus attenuating the quenching of F-PNA fluorescence by GO. In the absence of target miRNA (and hence the absence of RCA and duplex formation), the free F-PNA is completely adsorbed onto the GO monolayer and fluorescence quenching ensues. Thus, GO-based fluorescence detection coupled with isothermal gene amplification would be a simple and convenient method for the quantitative detection of miRNA.
ISSN:0003-2700
1520-6882
DOI:10.1021/acs.analchem.6b00046