Recent advances in capillary zone electrophoresis of DNA.
The present minireview summarizes recent developments in the field of DNA separations by capillary zone electrophoresis (CZE), as developed by our group. Separation of antisense oligonucleotides in sieving liquid polymers in isoelectric buffers is discussed first. It is shown that the use of isoelectric buffers permits very high voltage gradients (up to 1000 V cm-1) with much reduced transit times and increased resolution of all truncated and failed sequences. Oligonucleotides can also be analysed by zone electrophoresis against a stationary pH gradient (typically a pH 6.5-10 range): if injected at the alkaline end, the sample components experience stacking and zone sharpening due to modulation of charge as the oligonucleotides move along the pH gradient. Oligonucleotides having the same length, but differing by one single nucleotide in the chain, can be separated in free solution (i.e. in the absence of a sieving matrix) at strongly acidic pH values (pH 3.0-3.3) where charge differences due to base protonation are maximized. By working in free solution, it has also been possible to measure accurately the free mobility of DNAs, shown to reach a constant value of 3.75 +/- 0.04 10(-4) cm2 V-1 s-1 at 25 degrees C and in Tris-acetate-EDTA buffer, pH 8.3, above a critical length of ca. 400 base pairs. Finally, detection of point mutations in human genomic DNA is proven to be feasible in nonisocratic CZE, by running temperature-programmed CZE. The temperature gradient is activated within the capillary lumen by voltage ramps during the run, by exploiting joule effects. This technique has been proven to work for all point mutants, from low-, to intermediate-, to high-melters and has been applied to a number of point mutants in cystic fibrosis and thalassemia.
Righetti
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PG
Department of Agricultural and Industrial Biotechnologies, University of Verona, Italy.
Gelfi
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D002021:Buffers; D004247:DNA / Q000032:analysis* / Q000235:genetics; D017931:DNA Primers / Q000737:chemistry; D019075:Electrophoresis, Capillary / Q000379:methods; D005240:Feasibility Studies; D005554:Forensic Medicine / Q000379:methods; D015894:Genome, Human; D006801:Humans; D017354:Point Mutation; D011108:Polymers / Q000737:chemistry
D002021:Buffers; D017931:DNA Primers; D011108:Polymers; D004247:DNA