Nucleic acid amplification of individual molecules in a microfluidic device.
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.
Dettloff
Roger
R
Caliper Life Sciences, 605 Fairchild Drive, Mountain View, California 94024, USA.
Yang
Esther
E
Rulison
Aaron
A
Chow
Andrea
A
Farinas
Javier
J
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
D004247:DNA