Improvement of fragment and primer selection for mutation detection by denaturing gradient gel electrophoresis.

Denaturing gradient gel electrophoresis (DGGE) is one of the most powerful methods for mutation detection currently available. For successful application the appropriate selection of PCR fragments and PCR primers is crucial. The sequence of interest should always be within the domain with the lowest melting temperature. When more than one melting domain is present the fragment is generally divided into several smaller ones. This, however, is not always necessary. We found that simple modifications of PCR fragments and primer sequences may substantially reduce the number of amplicons required. Furthermore, by plotting the (natural) melting curves of fragments without a GC-clamp, we could explain why fragments theoretically perfect for DGGE in practice failed to reveal mutations. Alternative fragment selection and the use of modified primers (addition of T/A or G/C tails) result in the detection of mutations that originally remained undetected. Our studies extend the utility of DGGE by using a minimum of PCR fragments and achieving a maximum of mutation detection.