Isothermal circular-strand-displacement polymerization of DNA and microRNA in digital microfluidic devices
| Autor: | Roberto Gambari, Maria Chiara Giuffrida, Giuseppe Spoto, Laura Maria Zanoli, Roberta D'Agata, Alessia Finotti |
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| Jazyk: | angličtina |
| Rok vydání: | 2015 |
| Předmět: |
Materials science
Digital microfluidics Microfluidics Loop-mediated isothermal amplification amplification Biochemistry DNA sequencing Isothermal process Polymerization NO Analytical Chemistry Isothermal amplification chemistry.chemical_compound microRNA Humans Nucleic-acid amplification MicroRNA DNA Microfluidic Analytical Techniques Molecular biology MicroRNAs Circular-strand-displacement polymerization Digital microfluidics Nucleic-acid amplification MicroRNA Isothermal amplification Circular-strand-displacement polymerization chemistry K562 Cells Biological system Nucleic-acid |
| Popis: | Nucleic-acid amplification is a crucial step in nucleic-acid-sequence-detection assays. The use of digital microfluidic devices to miniaturize amplification techniques reduces the required sample volume and the analysis time and offers new possibilities for process automation and integration in a single device. The recently introduced droplet polymerase-chain-reaction (PCR) amplification methods require repeated cycles of two or three temperature-dependent steps during the amplification of the nucleic-acid target sequence. In contrast, low-temperature isothermal-amplification methods have no need for thermal cycling, thus requiring simplified microfluidic-device features. Here, the combined use of digital microfluidics and molecular-beacon (MB)-assisted isothermal circular-strand-displacement polymerization (ICSDP) to detect microRNA-210 sequences is described. MicroRNA-210 has been described as the most consistently and predominantly upregulated hypoxia-inducible factor. The nmol L(-1)-pmol L(-1) detection capabilities of the method were first tested by targeting single-stranded DNA sequences from the genetically modified Roundup Ready soybean. The ability of the droplet-ICSDP method to discriminate between full-matched, single-mismatched, and unrelated sequences was also investigated. The detection of a range of nmol L(-1)-pmol L(-1) microRNA-210 solutions compartmentalized in nanoliter-sized droplets was performed, establishing the ability of the method to detect as little as 10(-18) mol of microRNA target sequences compartmentalized in 20 nL droplets. The suitability of the method for biological samples was tested by detecting microRNA-210 from transfected K562 cells. |
| Databáze: | OpenAIRE |
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