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Microfluidics-based in situ Padlock/Rolling Circle Amplification System for Counting Single DNA Molecules in a Cell
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
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2014 (English)In: Analytical Sciences, ISSN 0910-6340, E-ISSN 1348-2246, Vol. 30, no 12, 1107-1112 p.Article in journal (Refereed) Published
Abstract [en]

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 mitochondria' 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 mu L, 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.

Place, publisher, year, edition, pages
2014. Vol. 30, no 12, 1107-1112 p.
Keyword [en]
Padlock probe, rolling circle amplification, microfluidics, DNA analysis
National Category
Chemical Sciences
Identifiers
URN: urn:nbn:se:su:diva-113229DOI: 10.2116/analsci.30.1107ISI: 000346892000002OAI: oai:DiVA.org:su-113229DiVA: diva2:791353
Note

AuthorCount:5;

Available from: 2015-02-27 Created: 2015-01-26 Last updated: 2017-12-04Bibliographically approved

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