Microfluidics-based in situ padlock/rolling circle amplification system for counting single DNA molecules in a cell.
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Kuroda A, Ishigaki Y, Nilsson M, Sato K, Sato K
Anal Sci 30 (12) 1107-1112 [2014-12-11; online 2014-12-11]
In situ padlock/rolling circle amplification (RCA) is a method used to amplify, visualize, and quantify target DNA molecules in cells. However, the multiple reaction steps involved make this technique costly and cumbersome. We developed a novel, simplified, automated microfluidic system for RCA, and demonstrated its effectiveness by counting amplified mitochondrial DNA fragments in HeLa cells. After optimizing the volume of the reaction solutions and washing buffer composition, the product yield was equal to that obtained by the conventional manual method. The required volume of reagents was reduced to 10 μL, which is less than half the volume used in the conventional method. To the best of our knowledge, this is the first report of an automated microfluidic method for in situ padlock/RCA, which can be useful for making highly efficient pathological diagnoses.
PubMed 25492458
DOI 10.2116/analsci.30.1107
Crossref 10.2116/analsci.30.1107
pii: DN/JST.JSTAGE/analsci/30.1107