Single molecule analysis of bacterial polymerase chain reaction products in submicrometer fluidic channels

Laser induced fluorescence in submicrometer fluidic channels was used to characterize the synthesis of polymerase chain reaction (PCR) products from a model bacterial system in order to explore the advantages and limitations of on chip real time single molecule PCR analysis. Single oligonucleotide universal bacterial primers and PCR amplicons from the 16S rDNA of Thermobifida fusca (325 bp) were directly detected at all phases of the reaction with low sample consumption and without post-amplification purification or size screening. Primers were fluorescently labeled with single Alexa Fluor 488 or Alexa Fluor 594 fluorophores, resulting in double labeled, two color amplicons. PCR products were driven electrokinetically through a fused silica channel with a 250 nm by 500 nm rectangular cross section. Lasers with 488 nm and 568 nm wavelengths were focused and overlapped on the channel for fluorescence excitation. All molecules entering the channel were rapidly and uniformly analyzed. Photon burst analysis was used to detect and identify individual primers and amplicons, and fluorescence correlation and cross-correlation spectroscopy were used to account for analyte flow speed. Conventional gel and capillary electrophoresis were also used to characterize the PCR amplification, and the results of differences in detection sensitivity and analyte discrimination were examined. Limits were imposed by the purity and labeling efficiency of the PCR reagents, which must be improved in parallel with increases in detection sensitivity.