Contribution of secondary structure to DNA mobility in capillary gels

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Abstract
Intramolecular base pairing at the 3′ end of single-stranded DNA molecules can cause increased electrophoretic mobility. In DNA sequencing separations this can result in compressions, where multiple oligomers differing in length by one base comigrate, which complicate sequence analysis. Using a novel approach to the application of capillary electrophoresis to DNA sequencing, incorporation of formamide into capillary gels, different separation field strengths, and external heating of the capillary are examined for their ability to resolve compressions. The identity of the 3′ terminal nucleotide has also been observed to influence oligomer mobility in cross-linked acrylamide slab gels. We utilize automated data collection from DNA sequencing separations to show that a similar effect is also present in linear acrylamide capillary gels run at high field strengths. We also demonstrate the ability of capillary electrophoresis to separate synthetic oligos of the same length but differing in composition by one base, or of the same base composition but different sequence.