title: On-chip nanoliter-volume multiplex TaqMan polymerase chain reaction from a single copy based on counting fluorescence released microchambers.
abstract: A novel method for multiplex TaqMan PCR in nanoliter volumes on a highly integrated silicon microchamber array is described. Three different gene targets, related to beta-actin, sex-determining region Y (SRY), and Rhesus D (RhD) were amplified and detected simultaneously on the same chip by using three different types of human genomic DNA as the templates. The lack of cross-contamination and carryover was shown using alternate dispensing of mineral oil-coated microchambers containing template and those without template. To confirm the specificity of our system to beta-actin, SRY, and RhD genes, we employed the larger volume PCR samples to a commercial real-time PCR system, SmartCycler. The samples were cycled with the same sustaining temperatures as with the microchamber array. Instead of the conventional method of DNA quantification, counting the number of the fluorescence released microchambers in consequence to TaqMan PCR was employed to our chip. This simple method of observing the end point signal had provided a dynamic quantitative range. Stochastic amplification of 0.4 copies/reaction chamber was achieved. The microfabricated PCR chip demonstrated a rapid and highly sensitive response for simultaneous multiple-target detection, which is a promising step toward the development of a fully integrated device for the "lab-on-a-chip" DNA analysis.
authors.affiliation: School of Materials Science, Japan Advanced Institute of Science and Technology, 1-1 Asahidai, Tatsunokuchi, Ishikawa, 923-1292 Japan.
authors.forename: Yasutaka
authors.initials: Y
authors.lastname: Matsubara
authors.forename: Kagan
authors.initials: K
authors.lastname: Kerman
authors.forename: Masaaki
authors.initials: M
authors.lastname: Kobayashi
authors.forename: Shouhei
authors.initials: S
authors.lastname: Yamamura
authors.forename: Yasutaka
authors.initials: Y
authors.lastname: Morita
authors.forename: Yuzuru
authors.initials: Y
authors.lastname: Takamura
authors.forename: Eiichi
authors.initials: E
authors.lastname: Tamiya
mesh_terms: D000199:Actins / Q000235:genetics; D001483:Base Sequence; D017931:DNA Primers; D036103:Nanotechnology; D016133:Polymerase Chain Reaction / Q000379:methods*; D012204:Rh-Hr Blood-Group System / Q000235:genetics; D051876:Sex-Determining Region Y Protein / Q000235:genetics; D013050:Spectrometry, Fluorescence / Q000295:instrumentation*
chemical_list: D000199:Actins; D017931:DNA Primers; D012204:Rh-Hr Blood-Group System; C026410:Rho(D) antigen; C497745:SRY protein, human; D051876:Sex-Determining Region Y Protein