Rolling circle amplification and circle-to-circle amplification of a specific gene integrated with electrophoretic analysis on a single chip

Laili Mahmoudian, Noritada Kaji, Manabu Tokeshi, Mats Nilsson, Yoshinobu Baba

Research output: Contribution to journalArticle

55 Citations (Scopus)

Abstract

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°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.

Original languageEnglish
Pages (from-to)2483-2490
Number of pages8
JournalAnalytical chemistry
Volume80
Issue number7
DOIs
Publication statusPublished - Apr 1 2008
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Analytical Chemistry

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