title: Nucleic acid amplification of individual molecules in a microfluidic device.
abstract: A microfluidic device was developed that enabled rapid polymerase chain reaction (PCR) analysis of individual DNA molecules. The device combined a means for accessing samples serially from a microtiter plate, channels for assembling eight parallel PCR reactions, and integrated resistive heaters for rapid thermocycling (>5 degrees C/s heating, >7 degrees C/s cooling) of samples as they flowed continuously through PCR channels. Amplification was monitored by fluorescence detection of Taqman probes. The long, narrow channels (10 microm x 180 microm x 40 mm) allowed sufficient separation between neighboring DNA templates to enable amplification of discreet DNA molecules. The functionality of the device was demonstrated by reproducibly amplifying a 2D6.6 CYP450 template and distinguishing between wild-type and mutant sequences using Taqman probes. A comparison of the rate of individual amplification events to the expected Poisson distribution confirmed that the device could reliably analyze individual DNA molecules. This work establishes the feasibility of rapid, single-molecule interrogation of nucleic acids.
authors.affiliation: Caliper Life Sciences, 605 Fairchild Drive, Mountain View, California 94024, USA.
authors.forename: Roger
authors.initials: R
authors.lastname: Dettloff
authors.forename: Esther
authors.initials: E
authors.lastname: Yang
authors.forename: Aaron
authors.initials: A
authors.lastname: Rulison
authors.forename: Andrea
authors.initials: A
authors.lastname: Chow
authors.forename: Javier
authors.initials: J
authors.lastname: Farinas
mesh_terms: D003365:Costs and Cost Analysis; D004247:DNA / Q000032:analysis* / Q000235:genetics / Q000302:isolation & purification; D005838:Genotype; D006361:Heating; D046210:Microfluidic Analytical Techniques / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000191:economics / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013997:Time Factors
chemical_list: D004247:DNA