IMPROVED SINGLE-STRAND DNA SIZING ACCURACY IN CAPILLARY ELECTROPHORESIS

Interpolation algorithms can be developed to size unknown single-stran ded (ss) DNA fragments based on their electrophoretic mobilities, when they are compared with the mobilities of standard fragments of known sizes; however, sequence-specific anomalous electrophoretic migration can affect the accuracy and precision of the called sizes of the fragm ents. We used the anomalous migration of ssDNA fragments to optimize d enaturation conditions for capillary electrophoresis. The capillary el ectrophoretic system uses a refillable polymer that both coats the cap illary wall to suppress electro-osmotic flow and acts as the sieving m atrix. The addition of 8 M urea to the polymer solution, as in slab ge l electrophoresis, is insufficient to fully denature some anomalously migrating ssDNA fragments in this capillary electrophoresis system. Th e sizing accuracy of these fragments is significantly improved by the addition of 2-pyrrolidinone, or increased capillary temperature (60 de grees C). the effect of these two denaturing strategies is additive, a nd the best accuracy and precision in sizing results are obtained with a combination of chemical and thermal denaturation.