TEMPERATURE-PROGRAMMED CAPILLARY ELECTROPHORESIS FOR THE ANALYSIS OF HIGH-MELTING POINT MUTANTS IN THALASSEMIAS

The behavior of different sieving polymers for unambiguous determinati on 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 elec trophoresis, in fact, of its variant utilizing temperature gradients ( TGGE), along the migration path, encompassing the melting points of bo th homo- and heteroduplex, polymerase chain reaction (PCR)-amplified D NA fragments. Unlike TGGE, where the temperature gradient exists along the separation space, the denaturing temperature gradient in the fuse d-silica capillaries is time-programmed, so as to reach the T-m's of a ll species under analysis prior to electrophoretic transport past the detector window. The DNA fragments are injected in a capillary maintai ned (by combined chemical and thermal means) just below the expected T , values. The Delta T applied is rather minute (1-1.5 degrees C) and t he temperature gradient quite shallow (e.g., 0.05 degrees C/min). The denaturing thermal gradient is generated internally, via Joule heat pr oduced by voltage ramps. This method is applied to the analysis of the most common point mutations in thalassemias, characterized by being h igh 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 hydroxfethylcellulos e are used. However. the former offers the best separation capability at such high temperatures.