Separation of homo- and heteroduplexes of DNA fragments with different melting temperature by capillary electrophoresis at one single temperature.

Abstract

Heteroduplex analysis is the most popular method for double-stranded DNA mutation detection thus far. Since different DNA fragments have various melting temperatures due to different base pair compositions and sizes, the PCR-amplified DNA fragments need to be denatured, generally, over 90 degrees C and reannealed to give a mixture of four duplexes, two homoduplexes, and two heteroduplexes for electrophoresis or chromatographic analysis. To separate homoduplex and heteroduplex DNA fragments, the column temperature must be controlled at the DNA melting temperature. This is tedious for DNA mutation study, since the melting point has to be measured before heteroduplex analysis. A novel heteroduplex analysis method using a capillary electrophoresis-laser-induced fluorescence (CE-LIF) detection system is described in this paper for the separation of all homo- and heteroduplex DNA fragments, which have different melting temperatures, at a single temdegrees C, 64 degrees C, and 70 degrees C--were separated and detected. The assay is simple, accurate, and sensitive, giving it potential for multiplex analysis for DNA mutation study.