Single-molecule DNA amplification and analysis in an integrated microfluidic device

Stochastic PCR amplification of single DNA template molecules followed by c apillary electrophoretic (CE) analysis of the products is demonstrated in a n integrated microfluidic device. The microdevice consists of submicroliter PCR chambers etched into a glass substrate that are directly connected to a microfabricated CE system. Valves and hydrophobic vents provide controlle d and sensorless loading of the 280-nL PCR chambers; the low volume reactor , the low thermal mass, and the use of thin-film heaters permit cycle times as fast as 30 s, The amplified product, labeled with an intercalating fluo rescent dye, is directly injected into the gel-filled capillary channel for electrophoretic analysis. Repetitive PCR analyses at the single DNA templa te molecule level exhibit quantized product peak areas; a histogram of the normalized peak areas reveals clusters of events caused by 0, 1, 2, and 3 v iable template copies in the reactor and these event clusters are shown to fit a Poisson distribution. This device demonstrates the most sensitive PCR possible in a microfabricated device. The detection of single DNA molecule s will also facilitate single-cell and single-molecule studies to expose th e genetic variation underlying ensemble sequence and expression averages.