PubMed · 9473871
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P Stevenson-Moore. 1998. FluoriSHIELD.. https://pubmed.ncbi.nlm.nih.gov/9473871/
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Single-base resolution in the separation of DNA markers V and VI was achieved in the presence of electroosmotic flow (EOF), using poly(ethylene oxide) (PEO) solutions containing ethidium bromide (EtB) under isocratic conditions. Furthermore, a new approach called stepwise capillary electrophoresis (SCE) has been developed for DNA analysis, including stepwise changes in PEO concentration, EtB concentration as well as both PEO and EtB concentrations, wherein the EOF was used to introduce different PEO solutions into the capillary during the separation. DNA fragments smaller than 80 bp were both detected under isocratic conditions using 20 micrograms/ml EtB, and SCE using 1 and 20 micrograms/ml EtB, but not under isocratic conditions using 1 microgram/ml EtB. Resolution and speed of the DNA separation in SCE were different from those obtained from isocratic means, indicating that DNA underwent different concentrations of PEO and EtB in SCE. For example, DNA fragments with 458 and 504 base pairs (bp) were partially resolved in SCE, but not under isocratic conditions. The results further suggest that it is worth developing gradient techniques for widening the separation range and enhancing resolution in DNA analysis.
Using the zwitterionic buffer N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) in the presence of a high-molecular-mass hydroxypropylmethylcellulose (HPMC) as a sieving polymer and ethidium bromide double-stranded DNA (dsDNA) was separated in an untreated capillary. The HEPES buffer shielded the DNA against the capillary wall interaction and decreased the electroosmotic flow enabling a good separation of the DNA similar to that obtained in a commercially coated capillary. In addition to the low cost of the untreated capillary it can be washed after each run. Furthermore, stacking with hydrodynamic injection filling about half of the capillary volume is demonstrated.
Four acidic, isoelectric buffers, for peptide and protein separations, have been recently described and adopted in capillary zone electrophoresis: cysteic acid [Cys-A, isoelectric point (pI) 1.85], iminodiacetic acid (IDA, pI 2.23), aspartic acid (Asp, pI 2.77) and glutamic acid (Glu, pI 3.22). These four buffers allow to explore an acidic portion of the titration curves of macroions, covering about 1.6 pH units (from pH 1.85 to ca. 3.45), thus permitting resolution of compounds having coincident titration curves at a given pH value. Given the rather acidic pI values of these buffers, their long-term stability has been investigated, by monitoring pH and conductivity changes upon increasing storage times. When dissolved in plain water, all four buffers appear to give constant pH and conductivity readings up to 15 days; after that, the conductivity keeps steadily increasing in a similar fashion. The same parameters, when the same buffers are dissolved in 6 M urea, appear to be stable for only one week, with the conductivity progressively augmenting after this period. A similar behaviour is exhibited by histidine (pI 7.70), a neutral, isoelectric buffer adopted for separation of DNA fragments. By mass spectrometry, Cys-A shows minute amounts (ca. 1%) of a degradation product after ageing for 3 weeks; in the same time period, Glu is extensively degraded (20%). No degradation species could be detected in IDA and Asp solutions. It is additionally shown that the acidic buffers are not quite stationary in the electric field, but can be transported at progressively higher rates (according to the pI value) from the cathodic to the anodic vessel. This is due to the fact that, at their respective pI values, a fraction of the amphotere has to be negatively charged in order to provide counterions to the excess of protons due to bulk water dissociation. Guidelines are given for the proper use and storage of such buffers.