Three-dimensional imaging of DNA fragments during electrophoresis using a confocal detector

We have measured the three-dimensional distribution of DNA fragments within an electrophoretic band confined by a glass microchannel. The measurements were made using a confocal microscope and a photon-counting photomultiplier detector. A DNA sequencing standard was loaded into glass microchannel plates containing polyacrylamide gel. The measurements were made by scanning the plates in three dimensions using a mechanical stage under computer control, while electrophoresis was taking place. We found that the distribution of DNA was the same for all the bands measured in the sequencing ladder with an approximate Gaussian distribution along all three axes. These measurements are important to understand what physical forces shape electrophoretic bands confined by a channel and they also aid in the design of high throughput DNA sequencers.