Single-Molecule DNA Patterning and Detection by Padlock Probing and Rolling Circle Amplification in Microchannels for Analysis of Small Sample Volumes

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• 作者：Yo Tanaka ; Hui Xi ; Kae Sato ; Kazuma Mawatari ; Bjo虉rn Renberg ; Mats Nilsson ; Takehiko Kitamori
• 刊名：Analytical Chemistry
• 出版年：2011
• 出版时间：May 1, 2011
• 年：2011
• 卷：83
• 期：9
• 页码：3352-3357
• 全文大小：852K
• 年卷期：v.83,no.9(May 1, 2011)
• ISSN：1520-6882

文摘

The rolling circle amplification (RCA) is a versatile DNA amplification method in which a DNA molecule is amplified using a single DNA primer, allowing the product to be counted as a single dot. Circular templates for RCA can arise from padlock probes in highly specific DNA target-mediated ligation reactions. However, improvement of detection efficiency represents an important challenge. In homogeneous assays, the detection efficiency is generally only under 0.1%, mainly because the sample volume is too large compared with the detection volume. Here, we used microchannel surfaces in a glass microchip for DNA detection in small volume samples. First, DNA patterning on glass surfaces in microchannels was demonstrated using chemical surface patterning by UV light. By using a photochemical reaction, we realized DNA patterning in a closed space. Second, RCA was demonstrated using dilutions of target molecules, and a calibration curve was obtained. The highest detection efficiency was 22.5% by virtue of the reduced sample volumes from several hundred microliters to 5.0 nL. Accordingly, a countable number of DNA molecules was successfully detected. This method is suitable for analysis of very small volume samples such as single cells, especially by using extended-nanochannels with dimensions of 10鈭?000 nm.