Improved single-strand DNA sizing accuracy in capillary electrophoresis.

摘要

Interpolation algorithms can be used to size unknown single-stranded (ss) DNA fragments based on their electrophoretic mobilities, when they are compared with the mobilities of standard fragments of known sizes. However, sequence-specific anomalouselectrophoretic migration can affect the accuracy and precision of the deduced sizes of the fragments. In this study, the anomalous migration of ssDNA fragments was used to optimize denaturation conditions for capillary electrophoresis. The capillary electrophoretic system used a refillable polymer that both coated the capillary wall to suppress electro-osmotic flow and acted as the sieving matrix. The addition of 8 M urea to the polymer solution, as in slab gel electrophoresis, was insufficient to fully denature some anomalously migrating ssDNA fragments in this capillary electrophoresis system. The sizing accuracy of these fragments was significantly improved by the addition of 2-pyrrolidinone, or increased capillary temperature (60鳦). the effect ofthese 2 denaturing strategies was additive, and the best accuracy and precision in sizing results were obtained with a combination of chemical and thermal denaturation.