(462g) DNA Separation and Sequencing at a Stretch

We are developing a novel DNA sequencing technology based on tethering DNA strands to a solid surface through precise end-hybridization and sequentially pulling DNA strands off the surface under an electric field. This new DNA fractionation method is expected to be much more efficient than the electrophoresis-based technologies for separation of DNA strands in different lengths produced from the Sanger reaction. Previously, we have been able to use this method to separate long DNA molecules by length. For example, we have demonstrated efficient separation of lambda dsDNA (48,502 bp) from human genomic dsDNA (> 100,000 bp). However, separation of short ssDNA fragments, which is required for DNA sequencing, by this method has been challenging, because an extremely large electric field is needed to load the short ssDNA strand with a large enough stretching force that can pull the ssDNA off the surface. This large electric field may cause various problems such as Joule heating and side electrochemical reactions in the buffer solution. Besides, the solvent-induced fluctuation force on DNA fragment becomes chain length dependent. Here we present two approaches that we have developed to overcome those challenges, through which short ssDNA strands (<100 bases) are efficiently separated. The first is based on applying a potential drop across a microchannel, which effectively reduces the current to less than several microamps and the temperature change to less than 1 K during the entire separation process. The second is based on pulling the ssDNA off a noble metal surface, which uses the Gouy-Chapman-Stern screening layer to generate a strong electric field for pulling the ssDNA. By using either approach, short ssDNA stands of 60, 80, and 90 bases are efficiently separated. General mechanism for the separation of short ssDNA fragments is demonstrated by considering the fluctuation forces on the basis of Brownian dynamics simulation.