Temperature-programmed capillary electrophoresis for the analysis of high-melting point mutants in thalassemias.

Abstract

The behavior of different sieving polymers for unambiguous determination of point mutations in genomic DNA, based on electrophoresis in thin capillaries, is evaluated. High melters from thalassemia patients are separated by exploiting the principle of denaturing gradient gel electrophoresis, in fact, of its variant utilizing temperature gradients (TGGE), along the migration path, encompassing the melting points of both homo- and heteroduplex, polymerase chain reaction (PCR)-amplified DNA fragments. Unlike TGGE, where the temperature gradient exists along the separation space, the denaturing temperature gradient in the fused-silica capillaries is time-programmed, so as to reach the Tm's of all species under analysis prior to electrophoretic transport past the detector window. The DNA fragments are injected in a capillary maintained (by combined chemical and thermal means) just below the expected Tm values. The deltaT applied is rather minute (1-1.5 degrees C) and the temperature gradient quite shallow (e.g., 0.05 degrees C/min). The denaturing thermal gradient is generated internally, via Joule heat produced by voltage ramps. This method is applied to the analysis of the most common point mutations in thalassemias, characterized by being high melters (in the temperature range of 60-62 degrees C) in presence of 6 M urea. Point mutants are fully resolved into a spectrum of four bands only when poly(N-acryloylaminopropanol) and hydroxyethylcellulose are used. However, the former offers the best separation capability at such high temperatures.