| AVS 54th International Symposium | |
| Biomaterial Interfaces | Wednesday Sessions |
| Session BI-WeA |
| Session: | Nucleic Acid Sequencing and Technology |
| Presenter: | A.E. Barron, Stanford University |
| Authors: | A.E. Barron, Stanford University C.P. Fredlake, Northwestern University J.A. Coyne, Stanford University J.S. Lin, Northwestern University R.J. Meagher, Northwestern University |
| Correspondent: | Click to Email |
High-resolution DNA separations are necessary for electrophoretic DNA sequencing and genotyping, which remains an extremely important workhorse technology even in the present, so-called "post-genomic era". In currently used capillary electrophoresis instruments, polymer networks provide the required molecular sieving of DNA fragments. Electrophoresis in sieving matrices has intrinsic physical limitations in read length, shows reduced performance under high electric fields, and requires capillary loading with viscous polymer solutions. There is an ongoing push toward performing DNA sequencing in miniaturized "lab-on-a-chip" devices, which promise higher throughput and lower cost. We are developing "End-Labeled Free-Solution Electrophoresis" (ELFSE) as way to separate DNA according to size without the need for a sieving matrix. In ELFSE, each DNA molecule in a sample is covalently modified with a unique frictional modifier or "drag-tag" that modifies DNA electrophoretic mobility in a size-dependent fashion. We have designed and synthesized a series of non-natural polypeptide and polypeptoid drag-tags, and conjugated them to DNA for free-solution separations by microchannel electrophoresis. In one approach, artificial genes encoding repetitive polypeptides are constructed by controlled cloning, expressed in E. Coli, and purified. These protein drag-tags have so far have been used to demonstrate 4-color sequencing of ~ 180 bases of DNA by capillary electrophoresis, in the absence of a sieving matrix. The obtainment of longer drag-tags, so that we can get longer reads, is ongoing, and sequencing separations are now being carried out in glass microfluidic chips. The application of hydrophilic polymer wall coatings are a critical element of making this technology work, and this will be discussed in some detail. ELFSE may be the a breakthrough that enables rapid, high-throughput sequencing in integrated microfluidic devices, with all of the accompanying advantages that chips offer.