Detection of single-base mutations by a competitive mobility shift assay

We have developed an assay for the rapid screening of point mutations in specific genes. Our assay is based upon competitive hybridization of differentially labeled wild-type and mutant oligonucleotide probes to a PCR- generated DNA template and a subsequent analysis of the mobility of the probe-template hybrids. The assay is referred to as a competitive mobility shift assay. Generation of a hybridization stringency gradient allows perfect-matched hybrids to be formed to a greater extent at a slightly higher stringency than the corresponding mismatched hybrids. The stringency gradient is achieved by carrying out the hybridizations at a steadily decreasing temperature (from 95 to 20°C) in a thermal cycler. This step allows the assay to be competitive while avoiding the need to establish precise hybridization conditions for each gene-specific probe, a major disadvantage associated with reverse oligonucleotide hybridization. The assay is rapid and sensitive and can selectively detect mutant DNA in the presence of a large (up to one million-fold) excess of wild-type DNA.