title: Microchamber array based DNA quantification and specific sequence detection from a single copy via PCR in nanoliter volumes.
abstract: A novel method for DNA quantification and specific sequence detection in a highly integrated silicon microchamber array is described. Polymerase chain reaction (PCR) mixture of only 40 nL volume could be introduced precisely into each chamber of the mineral oil layer coated microarray by using a nanoliter dispensing system. The elimination of carry-over and cross-contamination between microchambers, and multiple DNA amplification and detection by TaqMan chemistry were demonstrated, for the first time, by using our system. Five different gene targets, related to Escherichia coli were amplified and detected simultaneously on the same chip by using DNA from three different serotypes as the templates. The conventional method of DNA quantification, which depends on the real-time monitoring of variations in fluorescence intensity, was not applied to our system, instead a simple method was established. Counting the number of the microchambers with a high fluorescence signal as a consequence of TaqMan PCR provided the precise quantification of trace amounts of DNA. The initial DNA concentration for Rhesus D (RhD) gene in each microchamber was ranged from 0.4 to 12 copies, and quantification was achieved by observing the changes in the released fluorescence signals of the microchambers on the chip. DNA target could be detected as small as 0.4 copies. The amplified DNA was detected with a CCD camera built-in to a fluorescence microscope, and also evaluated by a DNA microarray scanner with associated software. This simple method of counting the high fluorescence signal released in microchambers as a consequence of TaqMan PCR was further integrated with a portable miniaturized thermal cycler unit. Such a small device is surely a strong candidate for low-cost DNA amplification, and detected as little as 0.4 copies of target DNA.
authors.affiliation: School of Materials Science, Japan Advanced Institute of Science and Technology, 1-1 Asahidai, Tatsunokuchi, Nomi-gun, 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: Eiichi
authors.initials: E
authors.lastname: Tamiya
mesh_terms: D015374:Biosensing Techniques / Q000295:instrumentation* / Q000379:methods; D004269:DNA, Bacterial / Q000032:analysis / Q000737:chemistry; D004867:Equipment Design; D019544:Equipment Failure Analysis; D004926:Escherichia coli / Q000235:genetics; D005240:Feasibility Studies; D018628:Gene Dosage; D008832:Microchemistry / Q000295:instrumentation* / Q000379:methods; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation* / Q000379:methods; D008904:Miniaturization; D036103:Nanotechnology / Q000295:instrumentation* / Q000379:methods; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation* / Q000379:methods; D016133:Polymerase Chain Reaction / Q000295:instrumentation* / Q000379:methods; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000295:instrumentation* / Q000379:methods
chemical_list: D004269:DNA, Bacterial