CONSTANT DENATURANT GEL-ELECTROPHORESIS, A MODIFICATION OF DENATURING GRADIENT GEL-ELECTROPHORESIS, IN MUTATION DETECTION

Denaturing gradient gel electrophoresis (DGGE) is increasingly being utilized in mutational detection, both in characterization of variations in genomic DNA and in the generation of mutational spectra after in vitro and in vivo mutagenesis. The basis for this electrophoretic separation technique is strand dissociation of DNA fragments in discrete, sequence-dependent melting domains followed by an abrupt decrease in mobility. We have modified the DGGE by using constant denaturant gels corresponding to the specific melting domains of certain DNA fragments. This leads to increased resolution of mutants as fragment differing in as little as 1 base pair migrate with a consistently different mobility through the whole gel allowing separations of several centimeters. By using a set of constant denaturant gels it is also possible to obtain a better approximation of the location of the different mutations as each denaturant concentration will correspond to specific melting domains. We have used this technique to separate 6 out of 7 exon-3 hypoxanthine phosphoribosyltransferase (HPRT) mutants while using conventional DGGE we were only able to separate 3.