Single-Molecule DNA Patterning and Detection by Padlock Probing and Rolling Circle Amplification in Microchannels for Analysis of Small Sample Volumes
2011 (English)In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 83, no 9, 3352-3357 p.Article in journal (Refereed) Published
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 10-1000 nm.
Place, publisher, year, edition, pages
2011. Vol. 83, no 9, 3352-3357 p.
IdentifiersURN: urn:nbn:se:uu:diva-153652DOI: 10.1021/ac103185jISI: 000289956800021PubMedID: 21462922OAI: oai:DiVA.org:uu-153652DiVA: diva2:417383