Rapid separation of synthetic oligonucleotides on polymer modified capillary surfaces using short-end injection capillary electrophoresis in free solution

Abstract

Here we use short-end electrokinetic injection capillary electrophoresis (CE) to investigate the free solution mobility of short strands of double-stranded oligonucleotides (dsODNs) on polymer modified capillaries. Single base pair (bp) resolution (R_s) of dsODNs ranging from 16–20 bp was achieved in free solution on an 8 cm capillary dynamically coated with poly(ethylpyrrolidine methacrylate-co-methyl methacrylate) (PEPyM-co-PMMA) random copolymer. Interestingly, separation of a dsODN mixture containing two 16 bp strands of different sequences resulted in partial resolution (0.52) implying that the free solution mobility of dsODNs was sequence dependent. The single bp resolution achieved for the complementary sequence strands (the sequence of all strands in the mixtures contained the same 16 bp sequence) was improved by up to 37% for separation of dsODNs containing non-complementary sequences. The 16 bp peak was not additive within each mixture, indicating the presence of ODN–ODN interactions. Investigation of these interactions (and ODN–buffer interactions) showed that they can be influenced by the ionic strength and conductivity of the background electrolyte (BGE). Increasing the ionic strength reduced the ODN–ODN interactions and improved the resolution, whereas, increasing the conductivity reduced ODN-buffer interactions, increasing the mobility, at the consequence of promoting ODN–ODN interactions, and hence decreasing the resolution.