title: A miniature quantitative PCR device for directly monitoring a sample processing on a microfluidic rapid DNA system.
abstract: We report a microfluidic device and measurement method to perform real-time PCR (or qPCR) in a miniaturized configuration for on-chip implementation using reaction volumes of less than 20 μL. The qPCR bioreactor is designed as a module to be embedded in an automated sample-in/profile-out system for rapid DNA biometrics or human identification. The PCR mixture is excited with a 505 nm diode-pumped solid-state laser (DPSSL) and the fluorescence build-up is measured using optical fibers directly embedded to the sidewalls of the microfluidic qPCR bioreactor. We discuss manufacturing and operating parameters necessary to adjust the internal surface conditions and temperature profiles of the bioreactor and to optimize the yield and quality of the PCR reaction for the amplification of 62 bp hTERT intron fragments using the commercial Quantifiler® kit (Life Technologies, Carlsbad, CA) commonly accepted for genotyping analysis. We designed a microfluidic device suitable for continuously processing a specimen by efficiently mixing the reagents from the kit to a set volume of DNA template on chip. Our approach relies on a calibration curve for the specific device using control DNA. We successfully applied this method to determine the concentration of genomic DNA extracted from a buccal swab on separate microfluidic devices which are operated upstream the qPCR device and perform buccal swab lysis and buccal DNA extraction. A precise correlation between the amount determined on chip and that obtained using a commercial cycler is demonstrated.
authors.affiliation: Center for Applied Nanobioscience & Medicine, The University of Arizona College of Medicine, 425 N 5th Street, Phoenix, AZ, 85004, USA, churthua@gmail.com.
authors.forename: Cedric
authors.initials: C
authors.lastname: Hurth
authors.forename: Jianing
authors.initials: J
authors.lastname: Yang
authors.forename: Matthew
authors.initials: M
authors.lastname: Barrett
authors.forename: Carla
authors.initials: C
authors.lastname: Brooks
authors.forename: Alan
authors.initials: A
authors.lastname: Nordquist
authors.forename: Stanley
authors.initials: S
authors.lastname: Smith
authors.forename: Frederic
authors.initials: F
authors.lastname: Zenhausern
mesh_terms: D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D005260:Female; D060005:Genotyping Techniques / Q000295:instrumentation / Q000379:methods; D006801:Humans; D056656:Lab-On-A-Chip Devices; D054023:Lasers, Semiconductor; D008297:Male; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation / Q000379:methods; D009061:Mouth Mucosa; D060888:Real-Time Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods; D019098:Telomerase / Q000235:genetics
chemical_list: D004247:DNA; C509186:TERT protein, human; D019098:Telomerase