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 degrees 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.
Chen
M
M
Section of Cancer Biology, Radiation Oncology Center, MIR, Washington University School of Medicine, St. Louis, Missouri, 63108, USA.
Laszlo
A
A
Roti
J L
JL
Hunt
C R
CR
D004252:DNA Mutational Analysis / Q000379:methods*; D015342:DNA Probes; D004273:DNA, Neoplasm / Q000235:genetics; D004277:DNA, Single-Stranded / Q000235:genetics; D011905:Genes, ras; D006801:Humans; D008969:Molecular Sequence Data; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D013698:Templates, Genetic
D015342:DNA Probes; D004273:DNA, Neoplasm; D004277:DNA, Single-Stranded