(273b) Band Broadening During High-Throughput Mutation Detection In Microchannels

Microfluidics and lab-on-a-chip technologies promise great advances in bioanalytical chemistry, most notably for DNA analysis. While most DNA analysis involves electrophoretic sizing, it is also possible to use electrophoresis to detect mutations in identical size DNA based upon the different interactions between wild type (or mutant DNA) and a linear polymer sieving matrix. In denaturant electrophoresis, the presence of a mutation destabilizes the DNA double helix, leading to a lower melting temperature. In the ideal case, the system is operated at a temperature such that the wild type strand is fully annealed, while the mutant strand exhibits a denaturation bubble. The denaturation bubble lowers the electrophoretic mobility of the mutant relative to the wild type, permitting a simple screen for the presence of a potentially harmful mutation. While denaturant electrophoresis techniques are well developed for macroscopic gel-based separations, there are significant challenges associated with downsizing the method. In particular, temperature control becomes a major issue, since the low thermal mass of the microchannel systems and capillaries makes it difficult to ensure a homogeneous temperature, especially for parallel, high-throughput operations. One possible method for circumventing the temperature control problem is to cycle the temperature of the microchannel between a low temperature, at which both the wild type and mutant strands are annealed, and a higher temperature, at which both strands are partially denatured. This method, known as cycling temperature electrophoresis, ensures that every channel operates at some time in the ideal temperature range for detection. We consider theoretically the effect of the temperature oscillations on the efficiency of the separation. Using a multiple-time scales analysis, we compute the mean velocity and dispersivity of the DNA as a function of the mobility in the annealed and denatured states and the kinetics for the denaturation/annealing reactions. Explicit results are presented for a step change in the reaction kinetics. We find that the relaxation to the annealed state can lead to significant band-broadening, and our analysis indicates that the separations will be most efficient at weak electric fields.