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Rolling circle amplification and circle-to-circle amplification of a specific gene integrated with electrophoretic analysis on a single chip
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
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2008 (English)In: Analytical Chemistry, ISSN 0003-2700, Vol. 80, no 7, 2483-2490 p.Article in journal (Refereed) Published
Abstract [en]

We have developed an integrated platform for rolling circle amplification (RCA) and circle-to-circle amplification (C2CA) of circular probe (padlock probe) and subsequent microchip electrophoretic detection of a specific gene on a poly(methyl methacrylate) microchip. RCA and C2CA were successfully carried out at a steady temperature of 37 degrees C in the sample well of the microchip, and their respective product was detected on the same channel of the microchip, which was prefilled with a polymer separation matrix and fluorescent dye. Using a species-specific padlock probe for bacterial pathogen V. cholerae, a 25-ng bacterial genomic DNA could be detected in less than 65 min (including RCA and microchip electrophoresis) by this platform. Stable dsDNA C2CA product of genomic DNA for V. cholerae can be detected with the introduced integrated platform. Furthermore, the usefulness of this technique for the monitoring of RCA was demonstrated. This integrated platform provides a sensitive, fast, high-throughput, and reproducible method for signal amplification and detection of the padlock probes in the same microchip and is a promising tool for highly specific gene detection strategies.

Place, publisher, year, edition, pages
2008. Vol. 80, no 7, 2483-2490 p.
National Category
Medical and Health Sciences
URN: urn:nbn:se:uu:diva-101048DOI: 10.1021/ac702289jISI: 000254593500030PubMedID: 18307323OAI: oai:DiVA.org:uu-101048DiVA: diva2:211626
Available from: 2009-04-16 Created: 2009-04-16 Last updated: 2009-11-12Bibliographically approved

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