title: Single-molecule DNA patterning and detection by padlock probing and rolling circle amplification in microchannels for analysis of small sample volumes.
abstract: 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.
authors.affiliation: Department of Applied Chemistry, School of Engineering, The University of Tokyo, Hongo, Bunkyo, Japan.
authors.forename: Yo
authors.initials: Y
authors.lastname: Tanaka
authors.forename: Hui
authors.initials: H
authors.lastname: Xi
authors.forename: Kae
authors.initials: K
authors.lastname: Sato
authors.forename: Kazuma
authors.initials: K
authors.lastname: Mawatari
authors.forename: Björn
authors.initials: B
authors.lastname: Renberg
authors.forename: Mats
authors.initials: M
authors.lastname: Nilsson
authors.forename: Takehiko
authors.initials: T
authors.lastname: Kitamori
mesh_terms: D001483:Base Sequence; D002138:Calibration; D004247:DNA / Q000032:analysis* / Q000737:chemistry / Q000235:genetics*; D005898:Glass / Q000737:chemistry; D055616:Microtechnology / Q000379:methods*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D015345:Oligonucleotide Probes / Q000235:genetics*; D013499:Surface Properties; D014466:Ultraviolet Rays
chemical_list: D015345:Oligonucleotide Probes; D004247:DNA