Separation of single-base sequential isomers of single-stranded DNA by capillary electrophoresis and its application in the discrimination of single-base DNA mutations.

Separation of single-base substitution sequential DNA isomers remains one of the most challenging tasks in DNA separation by capillary electrophoresis. We developed a simple, versatile capillary electrophoresis technique for the separation of single-base sequential isomers of DNA having the same chain length. This technique is based on charge differences resulting from the different protonation (acid dissociation) properties of the four DNA bases. A mixture of 13 single-base sequential isomers of 12-mer single-stranded DNA was separated by using an electrophoretic buffer solution containing 20 mM phosphoric acid (pH 2.0) and 8 M urea. We demonstrated that our method could separate all possible mutation patterns under identical experimental conditions. In addition, application of our method to the separation of the polymerase chain reaction product of a 68-mer gene fragment and its single-base isomers indicates that in combination with the appropriate genomic DNA extraction techniques, the method can detect single-base gene mutations.