Abstract
The goal of this study is to determine the feasibility of high-resolution separation of oligonucleotides in microchannels. High-resolution separation of oligos is extremely challenging because it depends on sequence-specific migration. Also, it requires high viscosity gels, an excess field of 500 V/cm, and long separation columns between 2 to 3 cm. An improved composition of low and high molecular weight of PDMA, high electric field (550 V/cm) and longer separation columns (30 mm) were used to separate cy5 labeled poly(dT). We were able to achieve single base resolution of poly(dT), between 25 and 31 bases, in less than 40 seconds; and the number of theoretical plates was found to be around 2.7 X 105. In addition, we investigated the mobility of cy5 labeled poly(dA), poly(dC) and poly(dT) in 3 percent PDMA. We found that the differential mobility of nucleotides is very small and increases in the following order: G, T, C, and A. The difference in mobility between these bases is found to be about 1 percent between A and C, and 0.1 percent between C and T. However, we found that various buffer modifications helped in increasing the differential mobility between bases and hence the resolution of separated species. This work was done on a glass microchip with a separation column 12 um deep, 30 um wide and 30 mm long.
